Commission Directive 2001/59/EC of 6 August 2001 adapting to technical progress for the 28th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (Text with EEA relevance.)
OJ L 225, 21.8.2001, p. 1–333 (ES, DA, DE, EL, EN, FR, IT, NL, PT, FI, SV)
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Commission Directive 2001/59/EC
of 6 August 2001
adapting to technical progress for the 28th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances
(Text with EEA relevance)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Community,
Having regard to Council Directive 67/548/EEC of 27 June 1967 on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances(1), as last amended by Commission Directive 2000/33/EC(2), and in particular Article 28 thereof,
Whereas:
(1) Annex I to Directive 67/548/EEC contains a list of dangerous substances, together with particulars of the classification and labelling of each substance. Present scientific and technical knowledge has shown that the list of dangerous substances in that Annex should be adapted to technical progress. Specifically, Tables A and B in the Foreword to Annex I require inclusion of the Finnish and Swedish nomenclature. Certain language versions of the Directive require technical corrections in specific sections of the Foreword to Annex I. It is useful to publish an updated and recast version of the Foreword to Annex I. Furthermore, the list itself should be updated to include notified new substances and further existing substances; the identity, nomenclature, classification, labelling and/or concentration limits for certain substances should be amended to reflect increased technical knowledge; the entries for three substances should be deleted from the list, since they are covered by other entries.
(2) Annex II to Directive 67/548/EEC contains a list of symbols and indications of danger for dangerous substances and preparations. Annex III to Directive 67/548/EEC contains a list of phrases indicating the nature of special risks attributed to dangerous substances and preparations. Annex IV to Directive 67/548/EEC contains a list of the phrases indicating the safety advice concerning dangerous substances and preparations. Annexes II, III and IV require inclusion of the Finnish and Swedish wordings. Certain language versions of the Directive require technical corrections in specific sections of Annexes II, III and IV. It is useful to publish updated and recast versions of Annexes II, III and IV.
(3) Article 1 of European Parliament and Council Directive 1999/33/EC(3) permitted Sweden from 1 January 1999 until 31 December 2000 to require the use of the additional R-phrase R340, not listed in Annex III, for substances classified as carcinogenic, category 3, instead of R-phrase R40. Member State experts have agreed to revise the text of R-phrase R40 to refer to carcinogenic, category 3 substances. A new R-phrase R-68 should be added to Annex III, containing the original text of R-phrase R40 for classification and labelling of mutagenic category 3 and harmful substances listed in Annex I. The classification and labelling and concentration limit references in Annex I that include R40 should therefore be revised for such mutagenic category 3 and harmful substances.
(4) Annex V to Directive 67/548/EEC lays down the methods for the determination of the physicochemical properties, toxicity and ecotoxicity of substances and preparations. It is necessary to adapt that Annex to technical progress. It is appropriate to reduce to a minimum the number of animals used for experimental purposes, in accordance with Council Directive 86/609/EEC(4). Chapter B.1 should therefore be deleted, since alternative methods, using fewer animals, are available. Due regard should be given to methods recognised and recommended by competent international organisations. The methods for subchronic oral toxicity in Chapters B.26 and B.27 should be revised accordingly and Chapters C.14 to C.20, on environmental toxicity should be added to Annex V. Certain language versions require technical corrections to specific sections of Annex V.
(5) Annex VI to Directive 67/548/EEC contains a guide to the classification and labelling of dangerous substances and preparations. It is necessary to adapt that Annex to technical progress. Certain language versions of the Directive require technical corrections in specific sections of Annex VI. Specific sections require publication in Finnish and Swedish. It is useful to publish an updated and recast version of Annex VI, particularly including reference to Directive 1999/45/EC of the European Parliament and of the Council of 31 May 1999 concerning the approximation of the laws, regulations and administrative provisions of the Member States relating to the classification, packaging and labelling of dangerous preparations(5).
(6) In accordance with the provisions of Directive 67/548/EEC, any new substance placed on the market should be notified to the competent authorities of the Member States by means of a notification containing certain information including a technical dossier. For new substances supplied and then consumed in a chemical reaction which are strictly controlled (intermediates with limited exposure), it is technically justifiable and appropriate to define a reduced test package (RTP). Current technical progress can guarantee minimum exposure for man and the environment through rigorous containment of the process.
(7) The technical dossier should contain a test package for intermediates with limited exposure which would supply the information necessary to evaluate their foreseeable risk for man and the environment. Annex VII should specify the content of this technical dossier and Annex VIII should detail additional tests and studies that may be required for intermediates with limited exposure marketed in higher volumes.
(8) The criteria for the notification of intermediates with limited exposure may need to be revised in the light of technical progress and experience gained with such notifications made in accordance with the new specific requirements laid down in this Directive.
(9) The measures provided for in this Directive are in accordance with the opinion of the Committee on the Adaptation to Technical Progress of the Directives for the Elimination of Technical Barriers to Trade in Dangerous Substances and Preparations,
HAS ADOPTED THIS DIRECTIVE:
Article 1
Directive 67/548/EEC is hereby amended as follows:
1. Annex I is amended as follows:
(a) Tables A and B in the foreword to Annex I shall include the Finnish and Swedish nomenclature. Certain language versions of the Directive shall include technical corrections in specific sections of the Foreword and Tables A and B. The Foreword including Tables A and B is replaced by Annex 1A to this Directive.
(b) The corresponding entries are replaced by the entries in Annex 1B to this Directive.
(c) The entries in Annex 1C to this Directive are inserted.
(d) The entries in Annex 1D to this Directive are deleted.
(e) The entries shown in Annex 1E to this Directive are amended by replacing classification references to "Muta. Cat. 3; R40" by "Muta. Cat. 3; R68" and by replacing labelling references to R40 by R68.
(f) The entries shown in Annex 1F to this Directive are amended by replacing classification references to "Xn; R40" by "Xn; R68" and by replacing labelling references to R40 by R68.
(g) The entry shown in Annex 1G to this Directive is amended by replacing concentration limit references to "Xn; R40/20/21/22" by "Xn; R68/20/21/22".
(h) The entry shown in Annex 1H to this Directive is amended by replacing concentration limit references to "Xn; R20/21/22-40/20/21/22" by "Xn; R20/21/22-68/20/21/22".
(i) The entries shown in Annex 1I to this Directive are amended by replacing classification references to "Muta. Cat. 3; R40" by "Muta. Cat. 3; R68".
(j) The entries shown in Annex 1J to this Directive are amended by replacing classification references to "Muta. Cat. 3; R40" by "Muta. Cat. 3; R68" and by adding R68 to the label.
2. Annex II shall include the Swedish and Finnish versions and technical corrections to certain language versions. Annex II is therefore replaced by Annex 2 to this Directive.
3. Annex III shall include the Swedish and Finnish versions and technical corrections to certain language versions. Annex III is therefore replaced by Annex 3 to this Directive.
4. Annex IV shall include the Swedish and Finnish versions and technical corrections to certain language versions. Annex IV is therefore replaced by Annex 4 to this Directive.
5. Annex V is amended as follows:
(a) Chapter B.1 is deleted.
(b) The title of the English version of Chapter B13/14 is replaced by the text in Annex 5A.
(c) The last sentence of the French version of paragraph 1.4.2.2 of Chapter B.39 is replaced by the text in Annex 5B.
(d) The equation in the last sentence of section 1.7.1.6 of the English version of Chapter B.41 is replaced by the text in Annex 5C.
(e) The test method for subchronic oral toxicity tests in rodents is amended in accordance with Annex 5D to this Directive, which replaces Chapter B.26.
(f) The test method for subchronic oral toxicity tests in non-rodents is amended in accordance with Annex 5E to this Directive, which replaces Chapter B.27.
(g) The seven new test methods for environmental toxicity in Annex 5F to this Directive are included in Part C.
6. Annex VI shall include the Swedish and Finnish versions, technical corrections to certain language versions and further detailed technical updates. Annex VI is therefore replaced by Annex 6 to this Directive.
7. Annex VII.A shall include a technical dossier containing a test package for intermediates with limited exposure, supplying the information necessary to evaluate their foreseeable risk for man and the environment. Annex VII.A is therefore amended as follows:
(a) the text in Annex 7A to this Directive is inserted before section 0 of Annex VII.A;
(b) the text in Annex 7B to this Directive is inserted at the end of Annex VII.A.
8. Annex VIII shall include additional tests and studies that may be required for intermediates with limited exposure marketed in higher volumes. Annex VIII is therefore amended as follows:
(a) the text in Annex 8A to this Directive is inserted between "Level 1" and "Physicochemical studies" of Annex VIII;
(b) the text in Annex 8B to this Directive is inserted between "Level 2" and "Toxicological studies" of Annex VIII.
Article 2
1. Member States shall adopt and publish before 30 July 2002 the laws, regulations and administrative provisions necessary to comply with this Directive. They shall forthwith inform the Commission thereof.
2. Member States shall apply the laws, regulations and administrative provisions referred to in paragraph 1:
(a) as from 30 July 2002 or an earlier date for dangerous substances;
(b) as from 30 July 2002 for preparations not within the scope of Council Directive 91/414/EEC(6) or European Parliament and Council Directive 98/8/EC(7);
(c) as from 30 July 2004 for preparations within the scope of Directive 91/414/EEC or Directive 98/8/EC.
They shall forthwith inform the Commission thereof.
When Member States adopt those provisions, they shall contain a reference to this Directive or be accompanied by such a reference on the occasion of their official publication. Member States shall determine how such reference is to be made.
3. Member States shall communicate to the Commission the main provisions of national law which they adopt in the field covered by this Directive and a correlation table between this Directive and the national provisions adopted.
Article 3
This Directive shall enter into force on the third day following its publication in the Official Journal of the European Communities.
Article 4
This Directive is addressed to the Member States.
Done at Brussels, 6 August 2001.
For the Commission
Margot Wallström
Member of the Commission
(1) OJ 196, 16.8.1967, p. 1.
(2) OJ L 136, 8.6.2000, p. 90.
(3) OJ L 199, 30.7.1999, p. 57.
(4) OJ L 358, 18.12.1986, p. 1.
(5) OJ L 200, 30.7.1999, p. 1.
(6) OJ L 230, 19.8.1991, p. 1.
(7) OJ L 123, 24.4.1998, p. 1.
ANNEX 1A
FOREWORD TO ANNEX I
Introduction
Annex I is an index of dangerous substances for which harmonised classification and labelling have been agreed at Community level in accordance with the procedure laid down in Article 4(3) of this Directive.
Numbering of entries
Entries in Annex I are listed according to the atomic number of the element most characteristic of the substance's properties. A list of the chemical elements, arranged according to atomic number is shown in Table A. Organic substances, because of their variety, have been placed in the usual classes, as shown in Table B.
The index number for each substance is in the form of a digit sequence of the type ABC-RST-VW-Y, where:
- ABC is either the atomic number of the most characteristic chemical element (preceded by one or two zeros to make up the sequence) or the usual class number for organic substances,
- RST is the consecutive number of the substance in the series ABC,
- VW denotes the form in which the substance is produced or placed on the market,
- Y is the check-digit calculated in accordance with the ISBN (International Standard Book Number) method.
As an example, the index number for sodium chlorate is 017-005-00-9.
For dangerous substances in the European Inventory of Existing Commercial Chemical Substance (Einecs) (OJ C 146A, 15.6.1990), the Einecs number is included. This number is a seven-digit system of the type XXX-XXX-X which starts at 200-001-8.
For dangerous substances notified under the provisions of this Directive, the number of the substance in the European List of Notified Substance (Elincs) is included. This number is a seven-digit system of the type XXX-XXX-X which starts at 400-010-9.
For dangerous substances in the list of "No-longer polymers" (Document, Office for Official Publications of the European Communities, 1997. ISBN 92-827-8995-0) the "No-longer polymer" number is included. This number is a seven-digit system of the type XXX-XXX-X which starts at 500-001-0.
The Chemical Abstracts Service (CAS) number is also included to assist identification of the entry. It should be noted that the Einecs number includes both anhydrous and hydrated forms of a substance, and there are frequently different CAS numbers for anhydrous and hydrated forms. The CAS number included is for the anhydrous form only, and therefore the CAS number shown does not always describe the entry as accurately as the Einecs number.
Einecs, Elincs, "No-longer polymer" or CAS numbers are not usually included for entries which comprise more than four individual substances.
Nomenclature
Wherever possible, dangerous substances are designated by their Einecs, Elincs or "No-longer polymer" names. Other substances not listed in Einecs, Elincs or the list of "No-longer polymers" are designated using an internationally recognised chemical name (e.g. ISO, IUPAC). An additional common name is included in some cases.
Impurities, additives and minor components are normally not mentioned unless they contribute significantly to the classification of the substance.
Some substances are described as a mixture of A and B. These entries refer to one specific mixture. In some cases where it is necessary to characterise the substance put on the market, the proportions of the main substances in the mixture are specified.
Some substances are described with a specific percentage purity. Substances containing a higher content of active material (e.g. an organic peroxide) are not included in the Annex I entry and may have other hazardous properties (e.g. explosive). Where specific concentration limits are shown, these apply to the substance or substances shown in the entry. In particular, in the case of entries which are mixtures of substances or substances described with a specific percentage purity, the limits apply to the substance as described in Annex I and not the pure substance.
Article 23(2)(a) requires that for substances appearing in Annex I, the name of the substance to be used on the label should be one of the designations given in the Annex. For certain substances, additional information has been added in square brackets in order to help identify the substance. This additional information need not be included on the label.
Certain entries contain a reference to impurities. An example is index No 607-190-00-X: methyl acrylamidomethoxyacetate (containing >= 0,1 % acrylamide). In these cases the reference in brackets forms part of the name, and must be included on the label.
Certain entries refer to groups of substances. An example is index No 006-007-00-5: "hydrogen cyanide (salts of ...) with exception of complex cyanides such as ferrocyanides, ferricyanides and mercuric oxycyanide". For individual substances covered by these entries, the Einecs name or another internationally recognised name must be used.
Format of entries
The following information is given for each substance in Annex I:
(a) the classification:
(i) the process of classification consists of placing a substance in one or more categories of danger (as defined in Article 2(2) of Council Directive 92/32/EEC (OJ L 154, 5.6.1992, p. 1)) and assigning the qualifying risk phrase or phrases. The classification has consequences not only for labelling but also for other legislation and regulatory measures on dangerous substances;
(ii) the classification for each category of danger is normally presented in the form of an abbreviation representing the category of danger together with the appropriate risk phrase or phrases. However, in some cases (i.e. substances classified as flammable, sensitising and some substances classified as dangerous for the environment) the risk phrase alone is used;
(iii) the abbreviation for each of the categories of danger is shown below:
- explosive: E
- oxidising: O
- extremely Flammable: F+
- highly Flammable: F
- flammable: R10
- very toxic: T+
- toxic: T
- harmful: Xn
- corrosive: C
- irritant: Xi
- sensitising: R42 and/or R43
- carcinogenic: Carc. Cat.(1)
- mutagenic: Muta. Cat.(2)
- toxic for reproduction: Repr. Cat.(3)
- dangerous for the environment: N or/and R52, R53, R59;
(iv) additional risk phrases which have been assigned to describe other properties (see sections 2.2.6 and 3.2.8 of the labelling guide) are shown although they are not formally part of the classification;
(b) the label, including:
(i) the letter assigned to the substance in accordance with Annex II (see Article 23(2)(c)). This acts as an abbreviation for the symbol and for the indication of danger (if these are assigned);
(ii) the risk phrases, denoted as a series of numbers preceded by the letter R indicating the nature of the special risks, in accordance with Annex III (see Article 23(2)(d)). The numbers are separated by either:
- a dash (-) to denote separate statements concerning special risks (R), or
- an oblique stroke (/) to denote a combined statement, in a single sentence, of the special risks as set out in Annex III;
(iii) the safety phrases denoted as a series of numbers preceded by the letter S indicating the recommended safety precautions, in accordance with Annex IV (see Article 23(2)(e)). Again the numbers are separated by either a dash or an oblique stroke; the significance of recommended safety precautions are set out in Annex IV. The safety phrases shown apply only to substances; for preparations, phrases are selected according to the usual rules.
Note that for certain dangerous substances and preparations sold to the general public certain S-phrases are mandatory.
S1, S2 and S45 are obligatory for all very toxic, toxic and corrosive substances and preparations sold to the general public.
S2 and S46 are obligatory for all other dangerous substances and preparations sold to the general public other than those that have only been classified as dangerous for the environment.
Safety phrases S1 and S2 are shown in brackets in Annex I and can only be omitted from the label when the substance or preparation is sold for industrial use only;
(c) the concentration limits and associated classifications necessary to classify dangerous preparations containing the substance in accordance with Directive 1999/45/EC.
Unless otherwise shown, the concentration limits are a percentage by weight of the substance calculated with reference to the total weight of the preparation.
Where no concentration limits are given, the concentration limits to be used when applying the conventional method of assessing health hazards are those in Annex II, and when applying the conventional method of assessing environmental hazards are those in Annex III of European Parliament and Council Directive 1999/45/EC (OJ L 200, 30.7.1999, p. 1).
General explanatory notes
Groups of substances
A number of group entries are included in Annex I. In these cases, the classification and labelling requirements will apply to all substances covered by the description if they are placed on the market, insofar as they are listed in Einecs or Elincs. Where a substance that is covered by a group entry occurs as an impurity in another substance, the classification and labelling requirements described in the group entry shall be taken into account in the labelling of the substance.
In some cases, there are classification and labelling requirements for specific substances that would be covered by the group entry. In such cases a specific Annex I entry will be present for the substance and the group entry will be annotated with the phrase "except those specified elsewhere in this Annex".
In some cases, individual substances may be covered by more than one group entry. Lead oxalate (Einecs No 212-413-5) is for instance covered by the entry for lead compounds (index No 082-001-00-6) as well as for salts of oxalic acid (607-007-00-3). In these cases, the labelling of the substance reflects the labelling for each of the two group entries. In cases where different classifications for the same hazard are given, the classification leading to the more severe classification is used for the label of the particular substance (see section on Note A below).
Entries in Annex I for salts (under any denomination) cover both anhydrous and hydrous forms unless specifically specified otherwise.
Substances with an Elincs number
In Annex I, substances with an Elincs number have been notified under the provisions of this Directive. A producer or importer who has not previously notified these substances must refer to the provisions of this Directive if he intends to place these substances on the market.
Explanation of the notes relating to the identification, classification and labelling of substances
Note A:
The name of the substance must appear on the label in the form of one of the designations given in Annex I (see Article 23(2)(a)).
In Annex I, use is sometimes made of a general description such as "... compounds" or "... salts". In this case, the manufacturer or any other person who markets such a substance is required to state on the label the correct name, due account being taken of the chapter entitled "Nomenclature" of the Foreword:
Example: for BeCl2 (Einecs No 232-116-4): beryllium chloride.
The Directive also requires that the symbols, indications of danger, R- and S-phrases to be used for each substance shall be those shown in Annex I (Article 23(2)(c), (d) and (e)).
For substances belonging to one particular group of substances included in Annex I, the symbols, indications of danger, R- and S-phrases to be used for each substance shall be those shown in the appropriate entry in Annex I.
For substances belonging to more than one group of substances included in Annex I, the symbols, indications of danger, R- and S-phrases to be used for each substance shall be those shown in both the appropriate entries given in Annex I. In cases where two different classifications are given in the two entries for the same hazard, the classification reflecting the more severe hazard classification is used.
Example:
for substance AB - no individual entry in Annex I:
Annex I group entry for compounds of A:
Repr. Cat. 1; R61 Repr. Cat. 3; R62 Xn; R20/22 R33 N; R50-53
Annex I group entry for compounds of B:
Carc. Cat.1; R45 T; R23/25 N; R51-53
Classification of substance AB thus becomes:
Carc. Cat. 1; R45 Repr. Cat. 1; R61 Repr. Cat. 3; R62 T; R23/25 R33 N; R50-53.
Note B:
Some substances (acids, bases, etc.) are placed on the market in aqueous solutions at various concentrations and, therefore, these solutions require different labelling since the hazards vary at different concentrations.
In Annex I entries with Note B have a general designation of the following type: "nitric acid ...%".
In this case the manufacturer or any other person who markets such a substance in aqueous solution must state the percentage concentration of the solution on the label.
Example: nitric acid 45 %.
Unless otherwise stated, it is assumed that the percentage concentration is calculated on a weight/weight basis.
The use of additional data (e.g. specific gravity, degrees Baumé) or descriptive phrases (e.g. fuming or glacial) is permissible.
Note C:
Some organic substances may be marketed either in a specific isomeric form or as a mixture of several isomers.
In Annex I, a general designation of the following type is sometimes used: "xylenol".
In this case the manufacturer or any other person who markets such a substance must state on the label whether the substance is a specific isomer (a) or a mixture of isomers (b).
Example:
(a) 2,4-dimethylphenol
(b) xylenol (mixture of isomers).
Note D:
Certain substances which are susceptible to spontaneous polymerisation or decomposition are generally placed on the market in a stabilised form. It is in this form that they are listed in Annex I to this Directive.
However, such substances are sometimes placed on the market in a non-stabilised form. In this case, the manufacturer or any person who places such a substance on the market must state on the label the name of the substance followed by the words "non-stabilised".
Example: methacrylic acid (non-stabilised).
Note E:
Substances with specific effects on human health (see Chapter 4 of Annex VI) that are classified as carcinogenic, mutagenic and/or toxic for reproduction in categories 1 or 2 are ascribed Note E if they are also classified as very toxic (T+), toxic (T) or harmful (Xn). For these substances, the risk phrases R20, R21, R22, R23, R24, R25, R26, R27, R28, R39, R68 (harmful), R48 and R65 and all combinations of these risk phrases shall be preceded by the word "Also".
Examples:
R45-23 "May cause cancer. Also toxic by inhalation"
R46-27/28 "May cause heritable genetic damage. Also very toxic in contact with skin and if swallowed".
Note F:
This substance may contain a stabiliser. If the stabiliser changes the dangerous properties of the substance, as indicated by the label in Annex I, a label should be provided in accordance with the rules for the labelling of dangerous preparations.
Note G:
This substance may be marketed in an explosive form in which case it must be evaluated using the appropriate test methods and a label should be provided reflecting its explosive property.
Note H:
The classification and label shown for this substance applies to the dangerous property(ies) indicated by the risk phrase(s) in combination with the category(ies) of danger shown. The requirements of Article 6 of this Directive on manufacturers, distributors and importers of this substance apply to all other aspects of classification and labelling. The final label shall follow the requirements of section 7 of Annex VI of this Directive.
This note applies to certain coal- and oil-derived substances and to certain entries for groups of substances in Annex I.
Note J:
The classification as a carcinogen need not apply if it can be shown that the substance contains less than 0,1 % w/w benzene (Einecs No 200-753-7). This note applies only to certain complex coal- and oil-derived substances in Annex I.
Note K:
The classification as a carcinogen need not apply if it can be shown that the substance contains less than 0,1 % w/w 1,3-butadiene (Einecs No 203-450-8). If the substance is not classified as a carcinogen, at least the S-phrases (2-)9-16 should apply. This note applies only to certain complex oil-derived substances in Annex I.
Note L:
The classification as a carcinogen need not apply if it can be shown that the substance contains less than 3 % DMSO extract as measured by IP 346. This note applies only to certain complex oil-derived substances in Annex I.
Note M:
The classification as a carcinogen need not apply if it can be shown that the substance contains less than 0,005 % w/w benzo[a]-pyrene (Einecs No 200-028-5). This note applies only to certain complex coal-derived substances in Annex I.
Note N:
The classification as a carcinogen need not apply if the full refining history is known and it can be shown that the substance from which it is produced is not a carcinogen. This note applies only to certain complex oil-derived substances in Annex I.
Note P:
The classification as a carcinogen need not apply if it can be shown that the substance contains less than 0,1 % w/w benzene (Einecs No 200-753-7).
When the substance is classified as a carcinogen, Note E shall also apply.
When the substance is not classified as a carcinogen at least the S-phrases (2-)23-24-62 shall apply.
This note applies only to certain complex oil-derived substances in Annex I.
Note Q:
The classification as a carcinogen need not apply if it can be shown that the substance fulfils one of the following conditions:
- a short term biopersistence test by inhalation has shown that the fibres longer than 20 µm have a weighted half-life less than 10 days, or
- a short term biopersistence test by intratracheal instillation has shown that the fibres longer than 20 µm have a weighted half-life less than 40 days, or
- an appropriate intra-peritoneal test has shown no evidence of excess carcinogenicity, or
- absence of relevant pathogenicity or neoplastic changes in a suitable long term inhalation test.
Note R:
The classification as a carcinogen need not apply to fibres with a length weighted geometric mean diameter less two standard geometric errors greater than 6 µm.
Note S:
This substance may not require a label according to Article 23 (see section 8 of Annex VI).
Explanation of the notes relating to the labelling of preparations
The significance of the notes that appear to the right of the concentration limits is as follows:
Note 1:
The concentration stated or, in the absence of such concentrations, the general concentrations of Directive 1999/45/EC are the percentages by weight of the metallic element calculated with reference to the total weight of the preparation.
Note 2:
The concentration of isocyanate stated is the percentage by weight of the free monomer calculated with reference to the total weight of the preparation.
Note 3:
The concentration stated is the percentage by weight of chromate ions dissolved in water calculated with reference to the total weight of the preparation.
Note 4:
Preparations containing these substances have to be classified as harmful with R65 if they meet the criteria in section 3.2.3 in Annex VI.
Note 5:
The concentration limits for gaseous preparations are expressed as volume per volume percentage.
Note 6:
Preparations containing these substances have to be assigned R67 if they meet the criteria in section 3.2.8 in Annex VI.
This note will no longer apply from the date on which the criteria for the use of R67 provided for in Directive 1999/45/EC enter into force.
TABLA A/TABEL A/TABELLE A/ΠΙΝΑΚΑΣ A/TABLE A/TABLEAU A/TABELLA A/TABEL A/TABELA A/TABELL A/TAULUKKO A
Lista de los elementos químicos clasificados por su número atómico (Z)/Liste over grundstoffer, ordnet efter deres atomvægt (Z)/Liste der chemischen Elemente, geordnet nach der Ordnungszahl (Z)/Κατάλογος χημικών στοιχείων ταξινομημένων σύμφωνα με τον ατομικό τους αριθμό (Ζ)/List of chemical elements listed according to their atomic number (Z)/Liste des éléments chimiques classés selon leur numéro atomique (Z)/Elenco degli elementi chimici ordinati secondo il loro numero atomico (Z)/Lijst van chemische elementen, gerangschikt naar atoomgewicht (Z)/Lista dos elementos químicos ordenados segundo o seu número atómico (Z)/Lista över grundämnen, ordnade efter deras atomnummer (Z)/Alkuaineiden luettelo järjestysluvun mukaan (Z)
>TABLE>
TABLA B/TABEL B/TABELLE B/ΠΙΝΑΚΑΣ B/TABLE B/TABLEAU B/TABELLA B/TABEL B/TABELA B/TABELL B/TAULUKKO B
Clasificación especial para las sustancias orgánicas/Særlig inddeling af organiske stoffer/Spezielle Anordnung für die organischen Stoffe/Ειδική ταξινόμηση των οργανικών ουσιών/Special classification for organic substances/Classification particulière aux substances organiques/Classificazione speciale per le sostanze organiche/Speciale indeling voor de organische stoffen/Classificação especial para as substâncias orgânicas/Särskild indelning av organiska ämnen/Erityisryhmät orgaanisille aineille
>TABLE>
(1) The category of carcinogen, mutagen or toxic for reproduction (i.e. 1, 2 or 3) is shown as appropriate.
(2) The category of carcinogen, mutagen or toxic for reproduction (i.e. 1, 2 or 3) is shown as appropriate.
(3) The category of carcinogen, mutagen or toxic for reproduction (i.e. 1, 2 or 3) is shown as appropriate.
ANNEX 1B
>TABLE>
ANNEX 1C
>TABLE>
ANNEX 1D
Index No
601-050-00-1
609-017-00-3
613-006-00-9
ANNEX 1E
Index No
006-005-00-4
006-012-00-2
015-022-00-6
015-048-00-8
015-072-00-9
023-001-00-8
024-012-00-0
602-002-00-2
602-079-00-2
603-056-00-X
604-009-00-6
604-042-00-6
604-055-00-7
605-016-00-7
609-020-00-X
612-033-00-3
612-111-00-7
612-128-00-X
612-147-00-3
612-148-00-9
613-048-00-8
613-049-00-3
613-140-00-8
615-023-00-7
ANNEX 1F
Index No
048-003-00-6
048-004-00-1
048-005-00-7
048-007-00-8
602-025-00-8
603-029-00-2
603-066-00-4
ANNEX 1G
Index No
015-015-00-8
ANNEX 1H
Index No
603-001-00-X
ANNEX 1I
Index No
016-023-00-4
601-048-00-0
603-063-00-8
605-020-00-9
609-007-00-9
609-049-00-8
611-001-00-6
612-035-00-4
612-051-00-1
ANNEX 1J
Index No
604-005-00-4
612-145-00-2
612-146-00-8
ANNEX 2
ANEXO II/BILAG II/ANHANG II/ΠΑΡΑΡΤΗΜΑ ΙΙ/ANNEX II/ANNEXE II/ALLEGATO II/BIJLAGE II/ANEXO II/LIITE II/BILAGA II
ANEXO II
Símbolos e indicaciones de peligro de las sustancias y preparados peligrosos
BILAG II
Faresymboler og farebetegnelser for farlige stoffer og præparater
ANHANG II
Gefahrensymbole und -bezeichnungen für gefährliche Stoffe und Zubereitungen
ΠΑΡΑΡΤΗΜΑ ΙΙ
Σύμβολα και ενδείξεις κινδύνου για επικίνδυνες ουσίες και παρασκευάσματα
ANNEX II
Symbols and indications of danger for dangerous substances and preparations
ANNEXE II
Symboles et indications de danger des substances et préparations dangereuses
ALLEGATO II
Simboli e indicazioni di pericolo delle sostanze e preparati pericolosi
BIJLAGE II
Gevaarsymbolen en -aanduidingen van gevaarlijke stoffen en preparaten
ANEXO II
Símbolos e indicações de perigo das substâncias e preparações perigosas
LIITE II
Varoitusmerkit ja niiden nimet vaarallisille aineille ja valmisteille
BILAGA II
Farosymboler och farobeteckningar för farliga ämnen och beredningar
Nota:
Las letras E, O, F, F+, T, T+, C, Xn, Xi y N no forman parte del símbolo.
Bemærkning:
Bogstaverne E, O, F, F+, T, T+, C, Xn, Xi og N udgør ikke en del af symbolet.
Anmerkung:
Die Buchstaben E, O, F, F+, T, T+, C, Xn, Xi und N sind nicht Bestandteil des Gefahrensymbols.
Σημείωση:
Τα γράμματα E, O, F, F+, T, T+, C, Xn, Xi και N δεν αποτελούν μέρος του συμβόλου.
Note:
The letters E, O, F, F+, T, T+, C, Xn, Xi and N do not form part of the symbol.
Remarque:
Les lettres E, O, F, F+, T, T+, C, Xn, Xi et N ne font pas partie du symbole.
Nota:
Le lettere E, O, F, F+, T, T+, C, Xn, Xi e N non fanno parte del simbolo.
Opmerking:
De letters E, O, F, F+, T, T+, C, Xn, Xi en N maken geen deel uit van het gevaarsymbool.
Nota:
As letras E, O, F, F+, T, T+, C, Xn, Xi e N não fazem parte do símbolo.
Huomautus:
Varoitusmerkkien kirjaintunnukset E, O, F, F+, T, T+, C, Xn, Xi ja N eivät ole osa varoitusmerkkiä.
Anmärkning:
Bokstäverna E, O, F, F+, T, T+, C, Xn, Xi och N utgör inte en del av symbolen.
E
>PIC FILE= "L_2001225EN.008101.TIF">
ES: Explosivo
DA: Eksplosiv
DE: Explosionsgefährlich
EL: Εκρηκτικό
EN: Explosive
FR: Explosif
IT: Esplosivo
NL: Ontplofbaar
PT: Explosivo
FI: Räjähtävä
SV: Explosivt
O
>PIC FILE= "L_2001225EN.008102.TIF">
ES: Comburente
DA: Brandnærende
DE: Brandfördernd
EL: Οξειδωτικό
EN: Oxidising
FR: Comburant
IT: Comburente
NL: Oxiderend
PT: Comburente
FI: Hapettava
SV: Oxiderande
F
>PIC FILE= "L_2001225EN.008201.TIF">
ES: Fácilmente inflamable
DA: Meget brandfarlig
DE: Leichtentzündlich
EL: Πολύ εύφλεκτο
EN: Highly flammable
FR: Facilement inflammable
IT: Facilmente infiammabile
NL: Licht ontvlambaar
PT: Facilmente inflamável
FI: Helposti syttyvä
SV: Mycket brandfarligt
F+
>PIC FILE= "L_2001225EN.008202.TIF">
ES: Extremadamente inflamable
DA: Yderst brandfarlig
DE: Hochentzündlich
EL: Εξαιρετικά εύφλεκτο
EN: Extremely flammable
FR: Extrêmement inflammable
IT: Estremamente infiammabile
NL: Zeer licht ontvlambaar
PT: Extremamente inflamável
FI: Erittäin helposti syttyvä
SV: Extremt brandfarligt
T
>PIC FILE= "L_2001225EN.008203.TIF">
ES: Tóxico
DA: Giftig
DE: Giftig
EL: Τοξικό
EN: Toxic
FR: Toxique
IT: Tossico
NL: Vergiftig
PT: Tóxico
FI: Myrkyllinen
SV: Giftig
T+
>PIC FILE= "L_2001225EN.008301.TIF">
ES: Muy tóxico
DA: Meget giftig
DE: Sehr giftig
EL: Πολύ τοξικό
EN: Very toxic
FR: Très toxique
IT: Molto tossico
NL: Zeer vergiftig
PT: Muito tóxico
FI: Erittäin myrkyllinen
SV: Mycket giftig
C
>PIC FILE= "L_2001225EN.008302.TIF">
ES: Corrosivo
DA: Ætsende
DE: Ätzend
EL: Διαβρωτικό
EN: Corrosive
FR: Corrosif
IT: Corrosivo
NL: Bijtend
PT: Corrosivo
FI: Syövyttävä
SV: Frätande
Xn
>PIC FILE= "L_2001225EN.008303.TIF">
ES: Nocivo
DA: Sundhedsskadelig
DE: Gesundheitsschädlich
EL: Επιβλαβές
EN: Harmful
FR: Nocif
IT: Nocivo
NL: Schadelijk
PT: Nocivo
FI: Haitallinen
SV: Hälsoskadlig
Xi
>PIC FILE= "L_2001225EN.008401.TIF">
ES: Irritante
DA: Lokalirriterende
DE: Reizend
EL: Ερεθιστικό
EN: Irritant
FR: Irritant
IT: Irritante
NL: Irriterend
PT: Irritante
FI: Ärsyttävä
SV: Irriterande
N
>PIC FILE= "L_2001225EN.008402.TIF">
ES: Peligroso para el medio ambiente
DA: Miljøfarlig
DE: Umweltgefährlich
EL: Επικίνδυνο για το περιβάλλον
EN: Dangerous for the environment
FR: Dangereux pour l'environnement
IT: Pericoloso per l'ambiente
NL: Milieugevaarlijk
PT: Perigoso para o ambiente
FI: Ympäristölle vaarallinen
SV: Miljöfarlig
ANNEX 3
ANEXO III/BILAG III/ANHANG III/ΠΑΡΑΡΤΗΜΑ ΙΙΙ/ANNEX III/ANNEXE III/ALLEGATO III/BIJLAGE III/ANEXO III/LIITE III/BILAGA III
ANEXO III
Naturaleza de los riesgos específicos atribuidos a las sustancias y preparados peligrosos
BILAG III
Arten af de særlige risici, der er forbundet med de farlige stoffer og præparater
ANHANG III
Bezeichnungen der besonderen Gefahren bei gefährlichen Stoffen und Zubereitungen
ΠΑΡΑΡΤΗΜΑ ΙΙΙ
Φύση των ειδικών κινδύνων που αφορούν επικίνδυνες ουσίες και παρασκευάσματα
ANNEX III
Nature of special risks attributed to dangerous substances and preparations
ANNEXE III
Nature des risques particuliers attribués aux substances et préparations dangereuses
ALLEGATO III
Natura dei rischi specifici attribuiti alle sostanze e preparati pericolosi
BIJLAGE III
Aard der bijzondere gevaren toegeschreven aan gevaarlijke stoffen en preparaten
ANEXO III
Natureza dos riscos específicos atribuídos às substâncias e preparações perigosas
LIITE III
Erityisten vaarojen luonne liittyen vaarallisiin aineisiin ja valmisteisiin
BILAGA III
Riskfraser som tilldelas farliga ämnen och beredningar
R1
ES: Explosivo en estado seco.
DA: Eksplosiv i tør tilstand.
DE: In trockenem Zustand explosionsgefährlich.
EL: Εκρηκτικό σε ξηρή κατάσταση.
EN: Explosive when dry.
FR: Explosif à l'état sec.
IT: Esplosivo allo stato secco.
NL: In droge toestand ontplofbaar.
PT: Explosivo no estado seco.
FI: Räjähtävää kuivana.
SV: Explosivt i torrt tillstånd.
R2
ES: Riesgo de explosión por choque, fricción, fuego u otras fuentes de ignición.
DA: Eksplosionsfarlig ved stød, gnidning, ild eller andre antændelseskilder.
DE: Durch Schlag, Reibung, Feuer oder andere Zündquellen explosionsgefährlich.
EL: Κίνδυνος εκρήξεως από κρούση, τριβή, φωτιά ή άλλες πηγές αναφλέξεως.
EN: Risk of explosion by shock, friction, fire or other sources of ignition.
FR: Risque d'explosion par le choc, la friction, le feu ou d'autres sources d'ignition.
IT: Rischio di esplosione per urto, sfregamento, fuoco o altre sorgenti d'ignizione.
NL: Ontploffingsgevaar door schok, wrijving, vuur of andere ontstekingsoorzaken.
PT: Risco de explosão por choque, fricção, fogo ou outras fontes de ignição.
FI: Räjähtävää iskun, hankauksen, avotulen tai muun sytytyslähteen vaikutuksesta.
SV: Explosivt vid stöt, friktion, eld eller annan antändningsorsak.
R3
ES: Alto riesgo de explosión por choque, fricción, fuego u otras fuentes de ignición.
DA: Meget eksplosionsfarlig ved stød, gnidning, ild eller andre antændelseskilder.
DE: Durch Schlag, Reibung, Feuer oder andere Zündquellen besonders explosionsgefährlich.
EL: Πολύ μεγάλος κίνδυνος εκρήξεως από κρούση, τριβή, φωτιά ή άλλες πηγές αναφλέξεως.
EN: Extreme risk of explosion by shock, friction, fire or other sources of ignition.
FR: Grand risque d'explosion par le choc, la friction, le feu ou d'autres sources d'ignition.
IT: Elevato rischio di esplosione per urto, sfregamento, fuoco o altre sorgenti d'ignizione.
NL: Ernstig ontploffingsgevaar door schok, wrijving, vuur of andere ontstekingsoorzaken.
PT: Grande risco de explosão por choque, fricção, fogo ou outras fontes de ignição.
FI: Erittäin helposti räjähtävää iskun, hankauksen, avotulen tai muun sytytyslähteen vaikutuksesta.
SV: Mycket explosivt vid stöt, friktion, eld eller annan antändningsorsak.
R4
ES: Forma compuestos metálicos explosivos muy sensibles.
DA: Danner meget følsomme eksplosive metalforbindelser.
DE: Bildet hochempfindliche explosionsgefährliche Metallverbindungen.
EL: Σχηματίζει πολύ ευαίσθητες εκρηκτικές μεταλλικές ενώσεις.
EN: Forms very sensitive explosive metallic compounds.
FR: Forme des composés métalliques explosifs très sensibles.
IT: Forma composti metallici esplosivi molto sensibili.
NL: Vormt met metalen zeer gemakkelijk ontplofbare verbindingen.
PT: Forma compostos metálicos explosivos muito sensíveis.
FI: Muodostaa erittäin herkästi räjähtäviä metalliyhdisteitä.
SV: Bildar mycket känsliga explosiva metallföreningar.
R5
ES: Peligro de explosión en caso de calentamiento.
DA: Eksplosionsfarlig ved opvarmning.
DE: Beim Erwärmen explosionsfähig.
EL: Η θέρμανση μπορεί να προκαλέσει έκρηξη.
EN: Heating may cause an explosion.
FR: Danger d'explosion sous l'action de la chaleur.
IT: Pericolo di esplosione per riscaldamento.
NL: Ontploffingsgevaar door verwarming.
PT: Perigo de explosão sob a acção do calor.
FI: Räjähdysvaarallinen kuumennettaessa.
SV: Explosivt vid uppvärmning.
R6
ES: Peligro de explosión, en contacto o sin contacto con el aire.
DA: Eksplosiv ved og uden kontakt med luft.
DE: Mit und ohne Luft explosionsfähig.
EL: Εκρηκτικό σε επαφή ή χωρίς επαφή με τον αέρα.
EN: Explosive with or without contact with air.
FR: Danger d'explosion en contact ou sans contact avec l'air.
IT: Esplosivo a contatto o senza contatto con l'aria.
NL: Ontplofbaar met en zonder lucht.
PT: Perigo de explosão com ou sem contacto com o ar.
FI: Räjähtävää sellaisenaan tai ilman kanssa.
SV: Explosivt vid kontakt och utan kontakt med luft.
R7
ES: Puede provocar incendios.
DA: Kan forårsage brand.
DE: Kann Brand verursachen.
EL: Μπορεί να προκαλέσει πυρκαγιά.
EN: May cause fire.
FR: Peut provoquer un incendie.
IT: Può provocare un incendio.
NL: Kan brand veroorzaken.
PT: Pode provocar incêndio.
FI: Aiheuttaa tulipalon vaaran.
SV: Kan orsaka brand.
R8
ES: Peligro de fuego en contacto con materias combustibles.
DA: Brandfarlig ved kontakt med brandbare stoffer.
DE: Feuergefahr bei Berührung mit brennbaren Stoffen.
EL: Η επαφή με καύσιμο υλικό μπορεί να προκαλέσει πυρκαγιά.
EN: Contact with combustible material may cause fire.
FR: Favorise l'inflammation des matières combustibles.
IT: Può provocare l'accensione di materie combustibili.
NL: Bevordert de ontbranding van brandbare stoffen.
PT: Favorece a inflamação de matérias combustíveis.
FI: Aiheuttaa tulipalon vaaran palavien aineiden kanssa.
SV: Kontakt med brännbart material kan orsaka brand.
R9
ES: Peligro de explosión al mezclar con materias combustibles.
DA: Eksplosionsfarlig ved blanding med brandbare stoffer.
DE: Explosionsgefahr bei Mischung mit brennbaren Stoffen.
EL: Εκρηκτικό όταν αναμειχθεί με καύσιμα υλικά.
EN: Explosive when mixed with combustible material.
FR: Peut exploser en mélange avec des matières combustibles.
IT: Esplosivo in miscela con materie combustibili.
NL: Ontploffingsgevaar bij menging met brandbare stoffen.
PT: Pode explodir quando misturado com matérias combustíveis.
FI: Räjähtävää sekoitettaessa palavien aineiden kanssa.
SV: Explosivt vid blandning med brännbart material.
R10
ES: Inflamable.
DA: Brandfarlig.
DE: Entzündlich.
EL: Εύφλεκτο.
EN: Flammable.
FR: Inflammable.
IT: Infiammabile.
NL: Ontvlambaar.
PT: Inflamável.
FI: Syttyvää.
SV: Brandfarligt.
R11
ES: Fácilmente inflamable.
DA: Meget brandfarlig.
DE: Leichtentzündlich.
EL: Πολύ εύφλεκτο.
EN: Highly flammable.
FR: Facilement inflammable.
IT: Facilmente infiammabile.
NL: Licht ontvlambaar.
PT: Facilmente inflamável.
FI: Helposti syttyvää.
SV: Mycket brandfarligt.
R12
ES: Extremadamente inflamable.
DA: Yderst brandfarlig.
DE: Hochentzündlich.
EL: Εξαιρετικά εύφλεκτο.
EN: Extremely flammable.
FR: Extrêmement inflammable.
IT: Estremamente infiammabile.
NL: Zeer licht ontvlambaar.
PT: Extremamente inflamável.
FI: Erittäin helposti syttyvää.
SV: Extremt brandfarligt.
R14
ES: Reacciona violentamente con el agua.
DA: Reagerer voldsomt med vand.
DE: Reagiert heftig mit Wasser.
EL: Αντιδρά βίαια με νερό.
EN: Reacts violently with water.
FR: Réagit violemment au contact de l'eau.
IT: Reagisce violentemente con l'acqua.
NL: Reageert heftig met water.
PT: Reage violentamente em contacto com a água.
FI: Reagoi voimakkaasti veden kanssa.
SV: Reagerar häftigt med vatten.
R15
ES: Reacciona con el agua liberando gases extremadamente inflamables.
DA: Reagerer med vand under dannelse af yderst brandfarlige gasser.
DE: Reagiert mit Wasser unter Bildung hochentzündlicher Gase.
EL: Σε επαφή με το νερό εκλύει εξαιρετικά εύφλεκτα αέρια.
EN: Contact with water liberates extremely flammable gases.
FR: Au contact de l'eau, dégage des gaz extrêmement inflammables.
IT: A contatto con l'acqua libera gas estremamente infiammabili.
NL: Vormt zeer licht ontvlambaar gas in contact met water.
PT: Em contacto com a água liberta gases extremamente inflamáveis.
FI: Vapauttaa erittäin helposti syttyviä kaasuja veden kanssa.
SV: Vid kontakt med vatten bildas extremt brandfarliga gaser.
R16
ES: Puede explosionar en mezcla con substancias comburentes.
DA: Eksplosionsfarlig ved blanding med oxiderende stoffer.
DE: Explosionsgefährlich in Mischung mit brandfördernden Stoffen.
EL: Εκρηκτικό όταν αναμειχθεί με οξειδωτικές ουσίες.
EN: Explosive when mixed with oxidising substances.
FR: Peut exploser en mélange avec des substances comburantes.
IT: Pericolo di esplosione se mescolato con sostanze comburenti.
NL: Ontploffingsgevaar bij menging met oxiderende stoffen.
PT: Explosivo quando misturado com substâncias comburentes.
FI: Räjähtävää hapettavien aineiden kanssa.
SV: Explosivt vid blandning med oxiderande ämnen.
R17
ES: Se inflama espontáneamente en contacto con el aire.
DA: Selvantændelig i luft.
DE: Selbstentzündlich an der Luft.
EL: Αυτοαναφλέγεται στον αέρα.
EN: Spontaneously flammable in air.
FR: Spontanément inflammable à l'air.
IT: Spontaneamente infiammabile all'aria.
NL: Spontaan ontvlambaar in lucht.
PT: Espontaneamente inflamável ao ar.
FI: Itsestään syttyvää ilmassa.
SV: Självantänder i luft.
R18
ES: Al usarlo pueden formarse mezclas aire-vapor explosivas/inflamables.
DA: Ved brug kan brandbare dampe/eksplosive damp-luftblandinger dannes.
DE: Bei Gebrauch Bildung explosionsfähiger/leichtentzündlicher Dampf/Luft-Gemische möglich.
EL: Κατά τη χρήση μπορεί να σχηματίσει εύφλεκτα/εκρηκτικά μείγματα ατμού-αέρος.
EN: In use, may form flammable/explosive vapour-air mixture.
FR: Lors de l'utilisation, formation possible de mélange vapeur-air inflammable/explosif.
IT: Durante l'uso può formare con aria miscele esplosive/infiammabili.
NL: Kan bij gebruik een ontvlambaar/ontplofbaar damp-luchtmengsel vormen.
PT: Pode formar mistura vapor-ar explosiva/inflamável durante a utilização.
FI: Käytössä voi muodostua syttyvä/räjähtävä höyry-ilmaseos.
SV: Vid användning kan brännbara/explosiva ång-luftblandningar bildas.
R19
ES: Puede formar peróxidos explosivos.
DA: Kan danne eksplosive peroxider.
DE: Kann explosionsfähige Peroxide bilden.
EL: Μπορεί να σχηματίσει εκρηκτικά υπεροξείδια.
EN: May form explosive peroxides.
FR: Peut former des peroxydes explosifs.
IT: Può formare perossidi esplosivi.
NL: Kan ontplofbare peroxiden vormen.
PT: Pode formar peróxidos explosivos.
FI: Saattaa muodostaa räjähtäviä peroksideja.
SV: Kan bilda explosiva peroxider.
R20
ES: Nocivo por inhalación.
DA: Farlig ved indånding.
DE: Gesundheitsschädlich beim Einatmen.
EL: Επιβλαβές όταν εισπνέεται.
EN: Harmful by inhalation.
FR: Nocif par inhalation.
IT: Nocivo per inalazione.
NL: Schadelijk bij inademing.
PT: Nocivo por inalação.
FI: Terveydelle haitallista hengitettynä.
SV: Farligt vid inandning.
R21
ES: Nocivo en contacto con la piel.
DA: Farlig ved hudkontakt.
DE: Gesundheitsschädlich bei Berührung mit der Haut.
EL: Επιβλαβές σε επαφή με το δέρμα.
EN: Harmful in contact with skin.
FR: Nocif par contact avec la peau.
IT: Nocivo a contatto con la pelle.
NL: Schadelijk bij aanraking met de huid.
PT: Nocivo em contacto com a pele.
FI: Terveydelle haitallista joutuessaan iholle.
SV: Farligt vid hudkontakt.
R22
ES: Nocivo por ingestión.
DA: Farlig ved indtagelse.
DE: Gesundheitsschädlich beim Verschlucken.
EL: Επιβλαβές σε περίπτωση καταπόσεως.
EN: Harmful if swallowed.
FR: Nocif en cas d'ingestion.
IT: Nocivo per ingestione.
NL: Schadelijk bij opname door de mond.
PT: Nocivo por ingestão.
FI: Terveydelle haitallista nieltynä.
SV: Farligt vid förtäring.
R23
ES: Tóxico por inhalación.
DA: Giftig ved indånding.
DE: Giftig beim Einatmen.
EL: Τοξικό όταν εισπνέεται.
EN: Toxic by inhalation.
FR: Toxique par inhalation.
IT: Tossico per inalazione.
NL: Vergiftig bij inademing.
PT: Tóxico por inalação.
FI: Myrkyllistä hengitettynä.
SV: Giftigt vid inandning.
R24
ES: Tóxico en contacto con la piel.
DA: Giftig ved hudkontakt.
DE: Giftig bei Berührung mit der Haut.
EL: Τοξικό σε επαφή με το δέρμα.
EN: Toxic in contact with skin.
FR: Toxique par contact avec la peau.
IT: Tossico a contatto con la pelle.
NL: Vergiftig bij aanraking met de huid.
PT: Tóxico em contacto com a pele.
FI: Myrkyllistä joutuessaan iholle.
SV: Giftigt vid hudkontakt.
R25
ES: Tóxico por ingestión.
DA: Giftig ved indtagelse.
DE: Giftig beim Verschlucken.
EL: Τοξικό σε περίπτωση καταπόσεως.
EN: Toxic if swallowed.
FR: Toxique en cas d'ingestion.
IT: Tossico per ingestione.
NL: Vergiftig bij opname door de mond.
PT: Tóxico por ingestão.
FI: Myrkyllistä nieltynä.
SV: Giftigt vid förtäring.
R26
ES: Muy tóxico por inhalación.
DA: Meget giftig ved indånding.
DE: Sehr giftig beim Einatmen.
EL: Πολύ τοξικό όταν εισπνέεται.
EN: Very toxic by inhalation.
FR: Très toxique par inhalation.
IT: Molto tossico per inalazione.
NL: Zeer vergiftig bij inademing.
PT: Muito tóxico por inalação.
FI: Erittäin myrkyllistä hengitettynä.
SV: Mycket giftigt vid inandning.
R27
ES: Muy tóxico en contacto con la piel.
DA: Meget giftig ved hudkontakt.
DE: Sehr giftig bei Berührung mit der Haut.
EL: Πολύ τοξικό σε επαφή με το δέρμα.
EN: Very toxic in contact with skin.
FR: Très toxique par contact avec la peau.
IT: Molto tossico a contatto con la pelle.
NL: Zeer vergiftig bij aanraking met de huid.
PT: Muito tóxico em contacto com a pele.
FI: Erittäin myrkyllistä joutuessaan iholle.
SV: Mycket giftigt vid hudkontakt.
R28
ES: Muy tóxico por ingestión.
DA: Meget giftig ved indtagelse.
DE: Sehr giftig beim Verschlucken.
EL: Πολύ τοξικό σε περίπτωση καταπόσεως.
EN: Very toxic if swallowed.
FR: Très toxique en cas d'ingestion.
IT: Molto tossico per ingestione.
NL: Zeer vergiftig bij opname door de mond.
PT: Muito tóxico por ingestão.
FI: Erittäin myrkyllistä nieltynä.
SV: Mycket giftigt vid förtäring.
R29
ES: En contacto con agua libera gases tóxicos.
DA: Udvikler giftig gas ved kontakt med vand.
DE: Entwickelt bei Berührung mit Wasser giftige Gase.
EL: Σε επαφή με το νερό ελευθερώνονται τοξικά αέρια.
EN: Contact with water liberates toxic gas.
FR: Au contact de l'eau, dégage des gaz toxiques.
IT: A contatto con l'acqua libera gas tossici.
NL: Vormt vergiftig gas in contact met water.
PT: Em contacto com a água liberta gases tóxicos.
FI: Kehittää myrkyllistä kaasua veden kanssa.
SV: Utvecklar giftig gas vid kontakt med vatten.
R30
ES: Puede inflamarse fácilmente al usarlo.
DA: Kan blive meget brandfarlig under brug.
DE: Kann bei Gebrauch leicht entzündlich werden.
EL: Κατά τη χρήση γίνεται πολύ εύφλεκτο.
EN: Can become highly flammable in use.
FR: Peut devenir facilement inflammable pendant l'utilisation.
IT: Può divenire facilmente infiammabile durante l'uso.
NL: Kan bij gebruik licht ontvlambaar worden.
PT: Pode tornar-se facilmente inflamável durante o uso.
FI: Käytettäessä voi muuttua helposti syttyväksi.
SV: Kan bli mycket brandfarligt vid användning.
R31
ES: En contacto con ácidos libera gases tóxicos.
DA: Udvikler giftig gas ved kontakt med syre.
DE: Entwickelt bei Berührung mit Säure giftige Gase.
EL: Σε επαφή με οξέα ελευθερώνονται τοξικά αέρια.
EN: Contact with acids liberates toxic gas.
FR: Au contact d'un acide, dégage un gaz toxique.
IT: A contatto con acidi libera gas tossico.
NL: Vormt vergiftige gassen in contact met zuren.
PT: Em contacto com ácidos liberta gases tóxicos.
FI: Kehittää myrkyllistä kaasua hapon kanssa.
SV: Utvecklar giftig gas vid kontakt med syra.
R32
ES: En contacto con ácidos libera gases muy tóxicos.
DA: Udvikler meget giftig gas ved kontakt med syre.
DE: Entwickelt bei Berührung mit Säure sehr giftige Gase.
EL: Σε επαφή με οξέα ελευθερώνονται πολύ τοξικά αέρια.
EN: Contact with acids liberates very toxic gas.
FR: Au contact d'un acide, dégage un gaz très toxique.
IT: A contatto con acidi libera gas molto tossico.
NL: Vormt zeer vergiftige gassen in contact met zuren.
PT: Em contacto com ácidos liberta gases muito tóxicos.
FI: Kehittää erittäin myrkyllistä kaasua hapon kanssa.
SV: Utvecklar mycket giftig gas vid kontakt med syra.
R33
ES: Peligro de efectos acumulativos.
DA: Kan ophobes i kroppen efter gentagen brug.
DE: Gefahr kumulativer Wirkungen.
EL: Κίνδυνος αθροιστικών επιδράσεων.
EN: Danger of cumulative effects.
FR: Danger d'effets cumulatifs.
IT: Pericolo di effetti cumulativi.
NL: Gevaar voor cumulatieve effecten.
PT: Perigo de efeitos cumulativos.
FI: Terveydellisten haittojen vaara pitkäaikaisessa altistuksessa.
SV: Kan ansamlas i kroppen och ge skador.
R34
ES: Provoca quemaduras.
DA: Ætsningsfare.
DE: Verursacht Verätzungen.
EL: Προκαλεί εγκαύματα.
EN: Causes burns.
FR: Provoque des brûlures.
IT: Provoca ustioni.
NL: Veroorzaakt brandwonden.
PT: Provoca queimaduras.
FI: Syövyttävää.
SV: Frätande.
R35
ES: Provoca quemaduras graves.
DA: Alvorlig ætsningsfare.
DE: Verursacht schwere Verätzungen.
EL: Προκαλεί σοβαρά εγκαύματα.
EN: Causes severe burns.
FR: Provoque de graves brûlures.
IT: Provoca gravi ustioni.
NL: Veroorzaakt ernstige brandwonden.
PT: Provoca queimaduras graves.
FI: Voimakkaasti syövyttävää.
SV: Starkt frätande.
R36
ES: Irrita los ojos.
DA: Irriterer øjnene.
DE: Reizt die Augen.
EL: Ερεθίζει τα μάτια.
EN: Irritating to eyes.
FR: Irritant pour les yeux.
IT: Irritante per gli occhi.
NL: Irriterend voor de ogen.
PT: Irritante para os olhos.
FI: Ärsyttää silmiä.
SV: Irriterar ögonen.
R37
ES: Irrita las vías respiratorias.
DA: Irriterer åndedrætsorganerne.
DE: Reizt die Atmungsorgane.
EL: Ερεθίζει το αναπνευστικό σύστημα.
EN: Irritating to respiratory system.
FR: Irritant pour les voies respiratoires.
IT: Irritante per le vie respiratorie.
NL: Irriterend voor de ademhalingswegen.
PT: Irritante para as vias respiratórias.
FI: Ärsyttää hengityselimiä.
SV: Irriterar andningsorganen.
R38
ES: Irrita la piel.
DA: Irriterer huden.
DE: Reizt die Haut.
EL: Ερεθίζει το δέρμα.
EN: Irritating to skin.
FR: Irritant pour la peau.
IT: Irritante per la pelle.
NL: Irriterend voor de huid.
PT: Irritante para a pele.
FI: Ärsyttää ihoa.
SV: Irriterar huden.
R39
ES: Peligro de efectos irreversibles muy graves.
DA: Fare for varig alvorlig skade på helbred.
DE: Ernste Gefahr irreversiblen Schadens.
EL: Κίνδυνος πολύ σοβαρών μονίμων επιδράσεων.
EN: Danger of very serious irreversible effects.
FR: Danger d'effets irréversibles très graves.
IT: Pericolo di effetti irreversibili molto gravi.
NL: Gevaar voor ernstige onherstelbare effecten.
PT: Perigo de efeitos irreversíveis muito graves.
FI: Erittäin vakavien pysyvien vaurioiden vaara.
SV: Risk för mycket allvarliga bestående hälsoskador.
R40
ES: Posibles efectos cancerígenos.
DA: Mulighed for kræftfremkaldende effekt.
DE: Verdacht auf krebserzeugende Wirkung.
EL: Ύποπτο καρκινογένεσης.
EN: Limited evidence of a carcinogenic effect.
FR: Effet cancérogène suspecté - preuves insuffisantes.
IT: Possibilità di effetti cancerogeni - prove insufficienti.
NL: Carcinogene effecten zijn niet uitgesloten.
PT: Possibilidade de efeitos cancerígenos.
FI: Epäillään aiheuttavan syöpäsairauden vaaraa.
SV: Misstänks kunna ge cancer.
R41
ES: Riesgo de lesiones oculares graves.
DA: Risiko for alvorlig øjenskade.
DE: Gefahr ernster Augenschäden.
EL: Κίνδυνος σοβαρών οφθαλμικών βλαβών.
EN: Risk of serious damage to eyes.
FR: Risque de lésions oculaires graves.
IT: Rischio di gravi lesioni oculari.
NL: Gevaar voor ernstig oogletsel.
PT: Risco de lesões oculares graves.
FI: Vakavan silmävaurion vaara.
SV: Risk för allvarliga ögonskador.
R42
ES: Posibilidad de sensibilización por inhalación.
DA: Kan give overfølsomhed ved indånding.
DE: Sensibilisierung durch Einatmen möglich.
EL: Μπορεί να προκαλέσει ευαισθητοποίηση όταν εισπνέεται.
EN: May cause sensitisation by inhalation.
FR: Peut entraîner une sensibilisation par inhalation.
IT: Può provocare sensibilizzazione per inalazione.
NL: Kan overgevoeligheid veroorzaken bij inademing.
PT: Pode causar sensibilização por inalação.
FI: Altistuminen hengitysteitse voi aiheuttaa herkistymistä.
SV: Kan ge allergi vid inandning
R43
ES: Posibilidad de sensibilización en contacto con la piel.
DA: Kan give overfølsomhed ved kontakt med huden.
DE: Sensibilisierung durch Hautkontakt möglich.
EL: Μπορεί να προκαλέσει ευαισθητοποίηση σε επαφή με το δέρμα.
EN: May cause sensitisation by skin contact.
FR: Peut entraîner une sensibilisation par contact avec la peau.
IT: Può provocare sensibilizzazione per contatto con la pelle.
NL: Kan overgevoeligheid veroorzaken bij contact met de huid.
PT: Pode causar sensibilização em contacto com a pele.
FI: Ihokosketus voi aiheuttaa herkistymistä.
SV: Kan ge allergi vid hudkontakt.
R44
ES: Riesgo de explosión al calentarlo en ambiente confinado.
DA: Eksplosionsfarlig ved opvarmning under indeslutning.
DE: Explosionsgefahr bei Erhitzen unter Einschluss.
EL: Κίνδυνος εκρήξεως εάν θερμανθεί υπό περιορισμό.
EN: Risk of explosion if heated under confinement.
FR: Risque d'explosion si chauffé en ambiance confinée.
IT: Rischio di esplosione per riscaldamento in ambiente confinato.
NL: Ontploffingsgevaar bij verwarming in afgesloten toestand.
PT: Risco de explosão se aquecido em ambiente fechado.
FI: Räjähdysvaara kuumennettaessa suljetussa astiassa.
SV: Explosionsrisk vid uppvärmning i sluten behållare.
R45
ES: Puede causar cáncer.
DA: Kan fremkalde kræft.
DE: Kann Krebs erzeugen.
EL: Μπορεί να προκαλέσει καρκίνο.
EN: May cause cancer.
FR: Peut provoquer le cancer.
IT: Può provocare il cancro.
NL: Kan kanker veroorzaken.
PT: Pode causar cancro.
FI: Aiheuttaa syöpäsairauden vaaraa.
SV: Kan ge cancer.
R46
ES: Puede causar alteraciones genéticas hereditarias.
DA: Kan forårsage arvelige genetiske skader.
DE: Kann vererbbare Schäden verursachen.
EL: Μπορεί να προκαλέσει κληρονομικές γενετικές βλάβες.
EN: May cause heritable genetic damage.
FR: Peut provoquer des altérations génétiques héréditaires.
IT: Può provocare alterazioni genetiche ereditarie.
NL: Kan erfelijke genetische schade veroorzaken.
PT: Pode causar alterações genéticas hereditárias.
FI: Saattaa aiheuttaa periytyviä perimävaurioita.
SV: Kan ge ärftliga genetiska skador.
R48
ES: Riesgo de efectos graves para la salud en caso de exposición prolongada.
DA: Alvorlig sundhedsfare ved længere tids påvirkning.
DE: Gefahr ernster Gesundheitsschäden bei längerer Exposition.
EL: Κίνδυνος σοβαρής βλάβης της υγείας ύστερα απο παρατεταμένη έκθεση.
EN: Danger of serious damage to health by prolonged exposure.
FR: Risque d'effets graves pour la santé en cas d'exposition prolongée.
IT: Pericolo di gravi danni per la salute in caso di esposizione prolungata.
NL: Gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling.
PT: Risco de efeitos graves para a saúde em caso de exposição prolongada.
FI: Pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle.
SV: Risk för allvarliga hälsoskador vid långvarig exponering.
R49
ES: Puede causar cáncer por inhalación.
DA: Kan fremkalde kræft ved indånding.
DE: Kann Krebs erzeugen beim Einatmen.
EL: Μπορεί να προκαλέσει καρκίνο όταν εισπνέεται.
EN: May cause cancer by inhalation.
FR: Peut provoquer le cancer par inhalation.
IT: Può provocare il cancro per inalazione.
NL: Kan kanker veroorzaken bij inademing.
PT: Pode causar cancro por inalação.
FI: Aiheuttaa syöpäsairauden vaaraa hengitettynä.
SV: Kan ge cancer vid inandning.
R50
ES: Muy tóxico para los organismos acuáticos.
DA: Meget giftig for organismer, der lever i vand.
DE: Sehr giftig für Wasserorganismen.
EL: Πολύ τοξικό για τους υδρόβιους οργανισμούς.
EN: Very toxic to aquatic organisms.
FR: Très toxique pour les organismes aquatiques.
IT: Altamente tossico per gli organismi acquatici.
NL: Zeer vergiftig voor in het water levende organismen.
PT: Muito tóxico para os organismos aquáticos.
FI: Erittäin myrkyllistä vesieliöille.
SV: Mycket giftigt för vattenlevande organismer.
R51
ES: Tóxico para los organismos acuáticos.
DA: Giftig for organismer, der lever i vand.
DE: Giftig für Wasserorganismen.
EL: Τοξικό για τους υδρόβιους οργανισμούς.
EN: Toxic to aquatic organisms.
FR: Toxique pour les organismes aquatiques.
IT: Tossico per gli organismi acquatici.
NL: Vergiftig voor in het water levende organismen.
PT: Tóxico para os organismos aquáticos.
FI: Myrkyllistä vesieliöille.
SV: Giftigt för vattenlevande organismer.
R52
ES: Nocivo para los organismos acuáticos.
DA: Skadelig for organismer, der lever i vand.
DE: Schädlich für Wasserorganismen.
EL: Επιβλαβές για τους υδρόβιους οργανισμούς.
EN: Harmful to aquatic organisms.
FR: Nocif pour les organismes aquatiques.
IT: Nocivo per gli organismi acquatici.
NL: Schadelijk voor in het water levende organismen.
PT: Nocivo para os organismos aquáticos.
FI: Haitallista vesieliöille.
SV: Skadligt för vattenlevande organismer.
R53
ES: Puede provocar a largo plazo efectos negativos en el medio ambiente acuático.
DA: Kan forårsage uønskede langtidsvirkninger i vandmiljøet.
DE: Kann in Gewässern längerfristig schädliche Wirkungen haben.
EL: Μπορεί να προκαλέσει μακροχρόνιες δυσμενείς επιπτώσεις στο υδάτινο περιβάλλον.
EN: May cause long-term adverse effects in the aquatic environment.
FR: Peut entraîner des effets néfastes à long terme pour l'environnement aquatique.
IT: Può provocare a lungo termine effetti negativi per l'ambiente acquatico.
NL: Kan in het aquatisch milieu op lange termijn schadelijke effecten veroorzaken.
PT: Pode causar efeitos nefastos a longo prazo no ambiente aquático.
FI: Voi aiheuttaa pitkäaikaisia haittavaikutuksia vesiympäristössä.
SV: Kan orsaka skadliga långtidseffekter i vattenmiljön.
R54
ES: Tóxico para la flora.
DA: Giftig for planter.
DE: Giftig für Pflanzen.
EL: Τοξικό για τη χλωρίδα.
EN: Toxic to flora.
FR: Toxique pour la flore.
IT: Tossico per la flora.
NL: Vergiftig voor planten.
PT: Tóxico para a flora.
FI: Myrkyllistä kasveille.
SV: Giftigt för växter.
R55
ES: Tóxico para la fauna.
DA: Giftig for dyr.
DE: Giftig für Tiere.
EL: Τοξικό για την πανίδα.
EN: Toxic to fauna.
FR: Toxique pour la faune.
IT: Tossico per la fauna.
NL: Vergiftig voor dieren.
PT: Tóxico para a fauna.
FI: Myrkyllistä eläimille.
SV: Giftigt för djur.
R56
ES: Tóxico para los organismos del suelo.
DA: Giftig for organismer i jordbunden.
DE: Giftig für Bodenorganismen.
EL: Τοξικό για τους οργανισμούς του εδάφους.
EN: Toxic to soil organisms.
FR: Toxique pour les organismes du sol.
IT: Tossico per gli organismi del terreno.
NL: Vergiftig voor bodemorganismen.
PT: Tóxico para os organismos do solo.
FI: Myrkyllistä maaperäeliöille.
SV: Giftigt för marklevande organismer.
R57
ES: Tóxico para las abejas.
DA: Giftig for bier.
DE: Giftig für Bienen.
EL: Τοξικό για τις μέλισσες.
EN: Toxic to bees.
FR: Toxique pour les abeilles.
IT: Tossico per le api.
NL: Vergiftig voor bijen.
PT: Tóxico para as abelhas.
FI: Myrkyllistä mehiläisille.
SV: Giftigt för bin.
R58
ES: Puede provocar a largo plazo efectos negativos en el medio ambiente.
DA: Kan forårsage uønskede langtidsvirkninger i miljøet.
DE: Kann längerfristig schädliche Wirkungen auf die Umwelt haben.
EL: Μπορεί να προκαλέσει μακροχρόνιες δυσμενείς επιπτώσεις στο περιβάλλον.
EN: May cause long-term adverse effects in the environment.
FR: Peut entraîner des effets néfastes à long terme pour l'environnement.
IT: Può provocare a lungo termine effetti negativi per l'ambiente.
NL: Kan in het milieu op lange termijn schadelijke effecten veroorzaken.
PT: Pode causar efeitos nefastos a longo prazo no ambiente.
FI: Voi aiheuttaa pitkäaikaisia haittavaikutuksia ympäristössä.
SV: Kan orsaka skadliga långtidseffekter i miljön.
R59
ES: Peligroso para la capa de ozono.
DA: Farlig for ozonlaget.
DE: Gefährlich für die Ozonschicht.
EL: Επικίνδυνο για τη στιβάδα του όζοντος.
EN: Dangerous for the ozone layer.
FR: Dangereux pour la couche d'ozone.
IT: Pericoloso per lo strato di ozono.
NL: Gevaarlijk voor de ozonlaag.
PT: Perigoso para a camada de ozono.
FI: Vaarallista otsonikerrokselle.
SV: Farligt för ozonskiktet.
R60
ES: Puede perjudicar la fertilidad.
DA: Kan skade forplantningsevnen.
DE: Kann die Fortpflanzungsfähigkeit beeinträchtigen.
EL: Μπορεί να εξασθενίσει τη γονιμότητα.
EN: May impair fertility.
FR: Peut altérer la fertilité.
IT: Può ridurre la fertilità.
NL: Kan de vruchtbaarheid schaden.
PT: Pode comprometer a fertilidade.
FI: Voi heikentää hedelmällisyyttä.
SV: Kan ge nedsatt fortplantningsförmåga.
R61
ES: Riesgo durante el embarazo de efectos adversos para el feto.
DA: Kan skade barnet under graviditeten.
DE: Kann das Kind im Mutterleib schädigen.
EL: Μπορεί να βλάψει το έμβρυο κατά τη διάρκεια της κύησης.
EN: May cause harm to the unborn child.
FR: Risque pendant la grossesse d'effets néfastes pour l'enfant.
IT: Può danneggiare i bambini non ancora nati.
NL: Kan het ongeboren kind schaden.
PT: Risco durante a gravidez com efeitos adversos na descendência.
FI: Vaarallista sikiölle.
SV: Kan ge fosterskador.
R62
ES: Posible riesgo de perjudicar la fertilidad.
DA: Mulighed for skade på forplantningsevnen.
DE: Kann möglicherweise die Fortpflanzungsfähigkeit beeinträchtigen.
EL: Πιθανός κίνδυνος για εξασθένηση της γονιμότητας.
EN: Possible risk of impaired fertility.
FR: Risque possible d'altération de la fertilité.
IT: Possibile rischio di ridotta fertilità.
NL: Mogelijk gevaar voor verminderde vruchtbaarheid.
PT: Possíveis riscos de comprometer a fertilidade.
FI: Voi mahdollisesti heikentää hedelmällisyyttä.
SV: Möjlig risk för nedsatt fortplantningsförmåga.
R63
ES: Posible riesgo durante el embarazo de efectos adversos para el feto.
DA: Mulighed for skade på barnet under graviditeten.
DE: Kann das Kind im Mutterleib möglicherweise schädigen.
EL: Πιθανός κίνδυνος δυσμενών επιδράσεων στο έμβρυο κατά τη διάρκεια της κύησης.
EN: Possible risk of harm to the unborn child.
FR: Risque possible pendant la grossesse d'effets néfastes pour l'enfant.
IT: Possibile rischio di danni ai bambini non ancora nati.
NL: Mogelijk gevaar voor beschadiging van het ongeboren kind.
PT: Possíveis riscos durante a gravidez com efeitos adversos na descendência.
FI: Voi olla vaarallista sikiölle.
SV: Möjlig risk för fosterskador.
R64
ES: Puede perjudicar a los niños alimentados con leche materna.
DA: Kan skade børn i ammeperioden.
DE: Kann Säuglinge über die Muttermilch schädigen.
EL: Μπορεί να βλάψει τα βρέφη που τρέφονται με μητρικό γάλα.
EN: May cause harm to breastfed babies.
FR: Risque possible pour les bébés nourris au lait maternel.
IT: Possibile rischio per i bambini allattati al seno.
NL: Kan schadelijk zijn via de borstvoeding.
PT: Pode causar danos às crianças alimentadas com leite materno.
FI: Saattaa aiheuttaa haittaa rintaruokinnassa oleville lapsille.
SV: Kan skada spädbarn under amningsperioden.
R65
ES: Nocivo: si se ingiere puede causar daño pulmonar.
DA: Farlig: kan give lungeskade ved indtagelse.
DE: Gesundheitsschädlich: kann beim Verschlucken Lungenschäden verursachen.
EL: Επιβλαβές: μπορεί να προκαλέσει βλάβη στους πνεύμονες σε περίπτωση κατάποσης.
EN: Harmful: may cause lung damage if swallowed.
FR: Nocif: peut provoquer une atteinte des poumons en cas d'ingestion.
IT: Nocivo: può causare danni ai polmoni in caso di ingestione.
NL: Schadelijk: kan longschade veroorzaken na verslikken.
PT: Nocivo: pode causar danos nos pulmões se ingerido.
FI: Haitallista: voi aiheuttaa keuhkovaurion nieltäessä.
SV: Farligt: kan ge lungskador vid förtäring.
R66
ES: La exposición repetida puede provocar sequedad o formación de grietas en la piel.
DA: Gentagen udsættelse kan give tør eller revnet hud.
DE: Wiederholter Kontakt kann zu spröder oder rissiger Haut führen.
EL: Η παρατεταμένη έκθεση μπορεί να προκαλέσει ξηρότητα δέρματος ή σκάσιμο.
EN: Repeated exposure may cause skin dryness or cracking.
FR: L'exposition répétée peut provoquer dessèchement ou gerçures de la peau.
IT: L'esposizione ripetuta può provocare secchezza e screpolature della pelle.
NL: Herhaalde blootstelling kan een droge of een gebarsten huid veroorzaken.
PT: Pode provocar secura da pele ou fissuras, por exposição repetida.
FI: Toistuva altistus voi aiheuttaa ihon kuivumista tai halkeilua.
SV: Upprepad kontakt kan ge torr hud eller hudsprickor.
R67
ES: La inhalación de vapores puede provocar somnolencia y vértigo.
DA: Dampe kan give sløvhed og svimmelhed.
DE: Dämpfe können Schläfrigkeit und Benommenheit verursachen.
EL: H εισπνοή ατμών μπορεί να προκαλέσει υπνηλία και ζάλη.
EN: Vapours may cause drowsiness and dizziness.
FR: L'inhalation de vapeurs peut provoquer somnolence et vertiges.
IT: L'inalazione dei vapori può provocare sonnolenza e vertigini.
NL: Dampen kunnen slaperigheid en duizeligheid veroorzaken.
PT: Pode provocar sonolência e vertigens, por inalação dos vapores.
FI: Höyryt voivat aiheuttaa uneliaisuutta ja huimausta.
SV: Ångor kan göra att man blir dåsig och omtöcknad.
R68
ES: Posibilidad de efectos irreversibles.
DA: Mulighed for varig skade på helbred.
DE: Irreversibler Schaden möglich.
EL: Πιθανοί κίνδυνοι μονίμων επιδράσεων.
EN: Possible risk of irreversible effects.
FR: Possibilité d'effets irréversibles.
IT: Possibilità di effetti irreversibili.
NL: Onherstelbare effecten zijn niet uitgesloten.
PT: Possibilidade de efeitos irreversíveis.
FI: Pysyvien vaurioiden vaara.
SV: Möjlig risk för bestående hälsoskador.
Combinación de frases-R
Kombination af R-sætninger
Kombination der R-Sätze
Συνδυασμός των Ρ-φράσεων
Combination of R-phrases
Combinaison des phrases R
Combinazioni delle frasi R
Combinatie van R-zinnen
Combinação das frases R
Yhdistetyt R-lausekkeet
Sammansatta R-fraser
R14/15
ES: Reacciona violentamente con el agua, liberando gases extremadamente inflamables.
DA: Reagerer voldsomt med vand under dannelse af yderst brandfarlige gasser.
DE: Reagiert heftig mit Wasser unter Bildung hochentzündlicher Gase.
EL: Αντιδρά βίαια σε επαφή με νερό εκλύοντας αέρια εξόχως εύφλεκτα.
EN: Reacts violently with water, liberating extremely flammable gases.
FR: Réagit violemment au contact de l'eau en dégageant des gaz extrêmement inflammables.
IT: Reagisce violentemente con l'acqua liberando gas estremamente infiammabili.
NL: Reageert heftig met water en vormt daarbij zeer ontvlambaar gas.
PT: Reage violentamente com a água libertando gases extremamente inflamáveis.
FI: Reagoi voimakkaasti veden kanssa vapauttaen helposti syttyviä kaasuja.
SV: Reagerar häftigt med vatten varvid extremt brandfarliga gaser bildas.
R15/29
ES: En contacto con el agua, libera gases tóxicos y extremadamente inflamables.
DA: Reagerer med vand under dannelse af giftige og yderst brandfarlige gasser.
DE: Reagiert mit Wasser unter Bildung giftiger und hochentzündlicher Gase.
EL: Σε επαφή με νερό ελευθερώνονται τοξικά, εξόχως εύφλεκτα αέρια.
EN: Contact with water liberates toxic, extremely flammable gas.
FR: Au contact de l'eau, dégage des gaz toxiques et extrêmement inflammables.
IT: A contatto con acqua libera gas tossici ed estremamente infiammabili.
NL: Vormt vergiftig en zeer ontvlambaar gas in contact met water.
PT: Em contacto com a água liberta gases tóxicos e extremamente inflamáveis.
FI: Vapauttaa myrkyllisiä, helposti syttyviä kaasuja veden kanssa.
SV: Utvecklar giftig och extremt brandfarlig gas vid kontakt med vatten.
R20/21
ES: Nocivo por inhalación y en contacto con la piel.
DA: Farlig ved indånding og ved hudkontakt.
DE: Gesundheitsschädlich beim Einatmen und bei Berührung mit der Haut.
EL: Επιβλαβές όταν εισπνέεται και σε επαφή με το δέρμα.
EN: Harmful by inhalation and in contact with skin.
FR: Nocif par inhalation et par contact avec la peau.
IT: Nocivo per inalazione e contatto con la pelle.
NL: Schadelijk bij inademing en bij aanraking met de huid.
PT: Nocivo por inalação e em contacto com a pele.
FI: Terveydelle haitallista hengitettynä ja joutuessaan iholle.
SV: Farligt vid inandning och hudkontakt.
R20/22
ES: Nocivo por inhalación y por ingestión.
DA: Farlig ved indånding og ved indtagelse.
DE: Gesundheitsschädlich beim Einatmen und Verschlucken.
EL: Επιβλαβές όταν εισπνέεται και σε περίπτωση καταπόσεως.
EN: Harmful by inhalation and if swallowed.
FR: Nocif par inhalation et par ingestion.
IT: Nocivo per inalazione e ingestione.
NL: Schadelijk bij inademing en opname door de mond.
PT: Nocivo por inalação e ingestão.
FI: Terveydelle haitallista hengitettynä ja nieltynä.
SV: Farligt vid inandning och förtäring.
R20/21/22
ES: Nocivo por inhalación, por ingestión y en contacto con la piel.
DA: Farlig ved indånding, ved hudkontakt og ved indtagelse.
DE: Gesundheitsschädlich beim Einatmen, Verschlucken und Berührung mit der Haut.
EL: Επιβλαβές όταν εισπνέεται, σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Harmful by inhalation, in contact with skin and if swallowed.
FR: Nocif par inhalation, par contact avec la peau et par ingestion.
IT: Nocivo per inalazione, contatto con la pelle e per ingestione.
NL: Schadelijk bij inademing, opname door de mond en aanraking met de huid.
PT: Nocivo por inalação, em contacto com a pele e por ingestão.
FI: Terveydelle haitallista hengitettynä, joutuessaan iholle ja nieltynä.
SV: Farligt vid inandning, hudkontakt och förtäring.
R21/22
ES: Nocivo en contacto con la piel y por ingestión.
DA: Farlig ved hudkontakt og ved indtagelse.
DE: Gesundheitsschädlich bei Berührung mit der Haut und beim Verschlucken.
EL: Επιβλαβές σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Harmful in contact with skin and if swallowed.
FR: Nocif par contact avec la peau et par ingestion.
IT: Nocivo a contatto con la pelle e per ingestione.
NL: Schadelijk bij aanraking met de huid en bij opname door de mond.
PT: Nocivo em contacto com a pele e por ingestão.
FI: Terveydelle haitallista joutuessaan iholle ja nieltynä.
SV: Farligt vid hudkontakt och förtäring.
R23/24
ES: Tóxico por inhalación y en contacto con la piel.
DA: Giftig ved indånding og ved hudkontakt.
DE: Giftig beim Einatmen und bei Berührung mit der Haut.
EL: Τοξικό όταν εισπνέεται και σε επαφή με το δέρμα.
EN: Toxic by inhalation and in contact with skin.
FR: Toxique par inhalation et par contact avec la peau.
IT: Tossico per inalazione e contatto con la pelle.
NL: Vergiftig bij inademing en bij aanraking met de huid.
PT: Tóxico por inalação e em contacto com a pele.
FI: Myrkyllistä hengitettynä ja joutuessaan iholle.
SV: Giftigt vid inandning och hudkontakt.
R23/25
ES: Tóxico por inhalación y por ingestión.
DA: Giftig ved indånding og ved indtagelse.
DE: Giftig beim Einatmen und Verschlucken.
EL: Τοξικό όταν εισπνέεται και σε περίπτωση καταπόσεως.
EN: Toxic by inhalation and if swallowed.
FR: Toxique par inhalation et par ingestion.
IT: Tossico per inalazione e ingestione.
NL: Vergiftig bij inademing en opname door de mond.
PT: Tóxico por inalação e ingestão.
FI: Myrkyllistä hengitettynä ja nieltynä.
SV: Giftigt vid inandning och förtäring.
R23/24/25
ES: Tóxico por inhalación, por ingestión y en contacto con la piel.
DA: Giftig ved indånding, ved hudkontakt og ved indtagelse.
DE: Giftig beim Einatmen, Verschlucken und Berührung mit der Haut.
EL: Τοξικό όταν εισπνέεται, σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Toxic by inhalation, in contact with skin and if swallowed.
FR: Toxique par inhalation, par contact avec la peau et par ingestion.
IT: Tossico per inalazione, contatto con la pelle e per ingestione.
NL: Vergiftig bij inademing, opname door de mond en aanraking met de huid.
PT: Tóxico por inalação, em contacto com a pele e por ingestão.
FI: Myrkyllistä hengitettynä, joutuessaan iholle ja nieltynä.
SV: Giftigt vid inandning, hudkontakt och förtäring.
R24/25
ES: Tóxico en contacto con la piel y por ingestión.
DA: Giftig ved hudkontakt og ved indtagelse.
DE: Giftig bei Berührung mit der Haut und beim Verschlucken.
EL: Τοξικό σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Toxic in contact with skin and if swallowed.
FR: Toxique par contact avec la peau et par ingestion.
IT: Tossico a contatto con la pelle e per ingestione.
NL: Vergiftig bij aanraking met de huid en bij opname door de mond.
PT: Tóxico em contacto com a pele e por ingestão.
FI: Myrkyllistä joutuessaan iholle ja nieltynä.
SV: Giftigt vid hudkontakt och förtäring.
R26/27
ES: Muy tóxico por inhalación y en contacto con la piel.
DA: Meget giftig ved indånding og ved hudkontakt.
DE: Sehr giftig beim Einatmen und bei Berührung mit der Haut.
EL: Πολύ τοξικό όταν εισπνέεται και σε επαφή με το δέρμα.
EN: Very toxic by inhalation and in contact with skin.
FR: Très toxique par inhalation et par contact avec la peau.
IT: Molto tossico per inalazione e contatto con la pelle.
NL: Zeer vergiftig bij inademing en bij aanraking met de huid.
PT: Muito tóxico por inalação e em contacto com a pele.
FI: Erittäin myrkyllistä hengitettynä ja joutuessaan iholle.
SV: Mycket giftigt vid inandning och hudkontakt.
R26/28
ES: Muy tóxico por inhalación y por ingestión.
DA: Meget giftig ved indånding og ved indtagelse.
DE: Sehr giftig beim Einatmen und Verschlucken.
EL: Πολύ τοξικό όταν εισπνέεται και σε περίπτωση καταπόσεως.
EN: Very toxic by inhalation and if swallowed.
FR: Très toxique par inhalation et par ingestion.
IT: Molto tossico per inalazione e per ingestione.
NL: Zeer vergiftig bij inademing en opname door de mond.
PT: Muito tóxico por inalação e ingestão.
FI: Erittäin myrkyllistä hengitettynä ja nieltynä.
SV: Mycket giftigt vid inandning och förtäring.
R26/27/28
ES: Muy tóxico por inhalación, por ingestión y en contacto con la piel.
DA: Meget giftig ved indånding, ved hudkontakt og ved indtagelse.
DE: Sehr giftig beim Einatmen, Verschlucken und Berührung mit der Haut.
EL: Πολύ τοξικό όταν εισπνέεται, σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Very toxic by inhalation, in contact with skin and if swallowed.
FR: Très toxique par inhalation, par contact avec la peau et par ingestion.
IT: Molto tossico per inalazione, contatto con la pelle e per ingestione.
NL: Zeer vergiftig bij inademing, opname door de mond en aanraking met de huid.
PT: Muito tóxico por inalação, em contacto com a pele e por ingestão.
FI: Erittäin myrkyllistä hengitettynä, joutuessaan iholle ja nieltynä.
SV: Mycket giftigt vid inandning, hudkontakt och förtäring.
R27/28
ES: Muy tóxico en contacto con la piel y por ingestión.
DA: Meget giftig ved hudkontakt og ved indtagelse.
DE: Sehr giftig bei Berührung mit der Haut und beim Verschlucken.
EL: Πολύ τοξικό σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Very toxic in contact with skin and if swallowed.
FR: Très toxique par contact avec la peau et par ingestion.
IT: Molto tossico a contatto con la pelle e per ingestione.
NL: Zeer vergiftig bij aanraking met de huid en bij opname door de mond.
PT: Muito tóxico em contacto com a pele e por ingestão.
FI: Erittäin myrkyllistä joutuessaan iholle ja nieltynä.
SV: Mycket giftigt vid hudkontakt och förtäring.
R36/37
ES: Irrita los ojos y las vías respiratorias.
DA: Irriterer øjnene og åndedrætsorganerne.
DE: Reizt die Augen und die Atmungsorgane.
EL: Ερεθίζει τα μάτια και το αναπνευστικό σύστημα.
EN: Irritating to eyes and respiratory system.
FR: Irritant pour les yeux et les voies respiratoires.
IT: Irritante per gli occhi e le vie respiratorie.
NL: Irriterend voor de ogen en de ademhalingswegen.
PT: Irritante para os olhos e vias respiratórias.
FI: Ärsyttää silmiä ja hengityselimiä.
SV: Irriterar ögonen och andningsorganen.
R36/38
ES: Irrita los ojos y la piel.
DA: Irriterer øjnene og huden.
DE: Reizt die Augen und die Haut.
EL: Ερεθίζει τα μάτια και το δέρμα.
EN: Irritating to eyes and skin.
FR: Irritant pour les yeux et la peau.
IT: Irritante per gli occhi e la pelle.
NL: Irriterend voor de ogen en de huid.
PT: Irritante para os olhos e pele.
FI: Ärsyttää silmiä ja ihoa.
SV: Irriterar ögonen och huden.
R36/37/38
ES: Irrita los ojos, la piel y las vías respiratorias.
DA: Irriterer øjnene, åndedrætsorganerne og huden.
DE: Reizt die Augen, die Atmungsorgane und die Haut.
EL: Ερεθίζει τα μάτια, το αναπνευστικό σύστημα και το δέρμα.
EN: Irritating to eyes, respiratory system and skin.
FR: Irritant pour les yeux, les voies respiratoires et la peau.
IT: Irritante per gli occhi, le vie respiratorie e la pelle.
NL: Irriterend voor de ogen, de ademhalingswegen en de huid.
PT: Irritante para os olhos, vias respiratórias e pele.
FI: Ärsyttää silmiä, hengityselimiä ja ihoa.
SV: Irriterar ögonen, andningsorganen och huden.
R37/38
ES: Irrita las vías respiratorias y la piel.
DA: Irriterer åndedrætsorganerne og huden.
DE: Reizt die Atmungsorgane und die Haut.
EL: Ερεθίζει το αναπνευστικό σύστημα και το δέρμα.
EN: Irritating to respiratory system and skin.
FR: Irritant pour les voies respiratoires et la peau.
IT: Irritante per le vie respiratorie e la pelle.
NL: Irriterend voor de ademhalingswegen en de huid.
PT: Irritante para as vias respiratórias e pele.
FI: Ärsyttää hengityselimiä ja ihoa.
SV: Irriterar andningsorganen och huden.
R39/23
ES: Tóxico: peligro de efectos irreversibles muy graves por inhalación.
DA: Giftig: fare for varig alvorlig skade på helbred ved indånding.
DE: Giftig: ernste Gefahr irreversiblen Schadens durch Einatmen.
EL: Τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων όταν εισπνέεται.
EN: Toxic: danger of very serious irreversible effects through inhalation.
FR: Toxique: danger d'effets irréversibles très graves par inhalation.
IT: Tossico: pericolo di effetti irreversibili molto gravi per inalazione.
NL: Vergiftig: gevaar voor ernstige onherstelbare effecten bij inademing.
PT: Tóxico: perigo de efeitos irreversíveis muito graves por inalação.
FI: Myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara hengitettynä.
SV: Giftigt: risk för mycket allvarliga bestående hälsoskador vid inandning.
R39/24
ES: Tóxico: peligro de efectos irreversibles muy graves por contacto con la piel.
DA: Giftig: fare for varig alvorlig skade på helbred ved hudkontakt.
DE: Giftig: ernste Gefahr irreversiblen Schadens bei Berührung mit der Haut.
EL: Τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων σε επαφή με το δέρμα.
EN: Toxic: danger of very serious irreversible effects in contact with skin.
FR: Toxique: danger d'effets irréversibles très graves par contact avec la peau.
IT: Tossico: pericolo di effetti irreversibili molto gravi a contatto con la pelle.
NL: Vergiftig: gevaar voor ernstige onherstelbare effecten bij aanraking met de huid.
PT: Tóxico: perigo de efeitos irreversíveis muito graves em contacto com a pele.
FI: Myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara joutuessaan iholle.
SV: Giftigt: risk för mycket allvarliga bestående hälsoskador vid hudkontakt.
R39/25
ES: Tóxico: peligro de efectos irreversibles muy graves por ingestión.
DA: Giftig: fare for varig alvorlig skade på helbred ved indtagelse.
DE: Giftig: ernste Gefahr irreversiblen Schadens durch Verschlucken.
EL: Τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων σε περίπτωση καταπόσεως.
EN: Toxic: danger of very serious irreversible effects if swallowed.
FR: Toxique: danger d'effets irréversibles très graves par ingestion.
IT: Tossico: pericolo di effetti irreversibili molto gravi per ingestione.
NL: Vergiftig: gevaar voor ernstige onherstelbare effecten bij opname door de mond.
PT: Tóxico: perigo de efeitos irreversíveis muito graves por ingestão.
FI: Myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara nieltynä.
SV: Giftigt: risk för mycket allvarliga bestående hälsoskador vid förtäring.
R39/23/24
ES: Tóxico: peligro de efectos irreversibles muy graves por inhalación y contacto con la piel.
DA: Giftig: fare for varig alvorlig skade på helbred ved indånding og hudkontakt.
DE: Giftig: ernste Gefahr irreversiblen Schadens durch Einatmen und bei Berührung mit der Haut.
EL: Τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων όταν εισπνέεται και σε επαφή με το δέρμα.
EN: Toxic: danger of very serious irreversible effects through inhalation and in contact with skin.
FR: Toxique: danger d'effets irréversibles très graves par inhalation et par contact avec la peau.
IT: Tossico: pericolo di effetti irreversibili molto gravi per inalazione e a contatto con la pelle.
NL: Vergiftig: gevaar voor ernstige onherstelbare effecten bij inademing en aanraking met de huid.
PT: Tóxico: perigo de efeitos irreversíveis muito graves por inalação e em contacto com a pele.
FI: Myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara hengitettynä ja joutuessaan iholle.
SV: Giftigt: risk för mycket allvarliga bestående hälsoskador vid inandning och hudkontakt.
R39/23/25
ES: Tóxico: peligro de efectos irreversibles muy graves por inhalación e ingestión.
DA: Giftig: fare for varig alvorlig skade på helbred ved indånding og indtagelse.
DE: Giftig: ernste Gefahr irreversiblen Schadens durch Einatmen und durch Verschlucken.
EL: Τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων όταν εισπνέεται και σε περίπτωση καταπόσεως.
EN: Toxic: danger of very serious irreversible effects through inhalation and if swallowed.
FR: Toxique: danger d'effets irréversibles très graves par inhalation et par ingestion.
IT: Tossico: pericolo di effetti irreversibili molto gravi per inalazione e ingestione.
NL: Vergiftig: gevaar voor ernstige onherstelbare effecten bij inademing en opname door de mond.
PT: Tóxico: perigo de efeitos irreversíveis muito graves por inalação e ingestão.
FI: Myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara hengitettynä ja nieltynä.
SV: Giftigt: risk för mycket allvarliga bestående hälsoskador vid inandning och förtäring.
R39/24/25
ES: Tóxico: peligro de efectos irreversibles muy graves por contacto con la piel e ingestión.
DA: Giftig: fare for varig alvorlig skade på helbred ved hudkontakt og indtagelse.
DE: Giftig: ernste Gefahr irreversiblen Schadens bei Berührung mit der Haut und durch Verschlucken.
EL: Τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Toxic: danger of very serious irreversible effects in contact with skin and if swallowed.
FR: Toxique: danger d'effets irréversibles très graves par contact avec la peau et par ingestion.
IT: Tossico: pericolo di effetti irreversibili molto gravi a contatto con la pelle e per ingestione.
NL: Vergiftig: gevaar voor ernstige onherstelbare effecten bij aanraking met de huid en opname door de mond.
PT: Tóxico: perigo de efeitos irreversíveis muito graves em contacto com a pele e por ingestão.
FI: Myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara joutuessaan iholle ja nieltynä.
SV: Giftigt: risk för mycket allvarliga bestående hälsoskador vid hudkontakt och förtäring.
R39/23/24/25
ES: Tóxico: peligro de efectos irreversibles muy graves por inhalación, contacto con la piel e ingestión.
DA: Giftig: fare for varig alvorlig skade på helbred ved indånding, hudkontakt og indtagelse.
DE: Giftig: ernste Gefahr irreversiblen Schadens durch Einatmen, Berührung mit der Haut und durch Verschlucken.
EL: Τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων όταν εισπνέεται, σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.
FR: Toxique: danger d'effets irréversibles très graves par inhalation, par contact avec la peau et par ingestion.
IT: Tossico: pericolo di effetti irreversibili molto gravi per inalazione, a contatto con la pelle e per ingestione.
NL: Vergiftig: gevaar voor ernstige onherstelbare effecten bij inademing, aanraking met de huid en opname door de mond.
PT: Tóxico: perigo de efeitos irreversíveis muito graves por inalação, em contacto com a pele e por ingestão.
FI: Myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara hengitettynä, joutuessaan iholle ja nieltynä.
SV: Giftigt: risk för mycket allvarliga bestående hälsoskador vid inandning, hudkontakt och förtäring.
R39/26
ES: Muy tóxico: peligro de efectos irreversibles muy graves por inhalación.
DA: Meget giftig: fare for varig alvorlig skade på helbred ved indånding.
DE: Sehr giftig: ernste Gefahr irreversiblen Schadens durch Einatmen.
EL: Πολύ τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων όταν εισπνέεται.
EN: Very toxic: danger of very serious irreversible effects through inhalation.
FR: Très toxique: danger d'effets irréversibles très graves par inhalation.
IT: Molto tossico: pericolo di effetti irreversibili molto gravi per inalazione.
NL: Zeer vergiftig: gevaar voor ernstige onherstelbare effecten bij inademing.
PT: Muito tóxico: perigo de efeitos irreversíveis muito graves por inalação.
FI: Erittäin myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara hengitettynä.
SV: Mycket giftigt: risk för mycket allvarliga bestående hälsoskador vid inandning.
R39/27
ES: Muy tóxico: peligro de efectos irreversibles muy graves por contacto con la piel.
DA: Meget giftig: fare for varig alvorlig skade på helbred ved hudkontakt.
DE: Sehr giftig: ernste Gefahr irreversiblen Schadens bei Berührung mit der Haut.
EL: Πολύ τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων σε επαφή με το δέρμα.
EN: Very toxic: danger of very serious irreversible effects in contact with skin.
FR: Très toxique: danger d'effets irréversibles très graves par contact avec la peau.
IT: Molto tossico: pericolo di effetti irreversibili molto gravi a contatto con la pelle.
NL: Zeer vergiftig: gevaar voor ernstige onherstelbare effecten bij aanraking met de huid.
PT: Muito tóxico: perigo de efeitos irreversíveis muito graves em contacto com a pele.
FI: Erittäin myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara joutuessaan iholle.
SV: Mycket giftigt: risk för mycket allvarliga bestående hälsoskador vid hudkontakt.
R39/28
ES: Muy tóxico: peligro de efectos irreversibles muy graves por ingestión.
DA: Meget giftig: fare for varig alvorlig skade på helbred ved indtagelse.
DE: Sehr giftig: ernste Gefahr irreversiblen Schadens durch Verschlucken.
EL: Πολύ τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων σε περίπτωση καταπόσεως.
EN: Very toxic: danger of very serious irreversible effects if swallowed.
FR: Très toxique: danger d'effets irréversibles très graves par ingestion.
IT: Molto tossico: pericolo di effetti irreversibili molto gravi per ingestione.
NL: Zeer vergiftig: gevaar voor ernstige onherstelbare effecten bij opname door de mond.
PT: Muito tóxico: perigo de efeitos irreversíveis muito graves por ingestão.
FI: Erittäin myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara nieltynä.
SV: Mycket giftigt: risk för mycket allvarliga bestående hälsoskador vid förtäring.
R39/26/27
ES: Muy tóxico: peligro de efectos irreversibles muy graves por inhalación y contacto con la piel.
DA: Meget giftig: fare for varig alvorlig skade på helbred ved indånding og hudkontakt.
DE: Sehr giftig: ernste Gefahr irreversiblen Schadens durch Einatmen und bei Berührung mit der Haut.
EL: Πολύ τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων όταν εισπνέεται και σε επαφή με το δέρμα.
EN: Very toxic: danger of very serious irreversible effects through inhalation and in contact with skin.
FR: Très toxique: danger d'effets irréversibles très graves par inhalation et par contact avec la peau.
IT: Molto tossico: pericolo di effetti irreversibili molto gravi per inalazione e a contatto con la pelle.
NL: Zeer vergiftig: gevaar voor ernstige onherstelbare effecten bij inademing en aanraking met de huid.
PT: Muito tóxico: perigo de efeitos irreversíveis muito graves por inalação e em contacto com a pele.
FI: Erittäin myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara hengitettynä ja joutuessaan iholle.
SV: Mycket giftigt: risk för mycket allvarliga bestående hälsoskador vid inandning och hudkontakt.
R39/26/28
ES: Muy tóxico: peligro de efectos irreversibles muy graves por inhalación e ingestión.
DA: Meget giftig: fare for varig alvorlig skade på helbred ved indånding og indtagelse.
DE: Sehr giftig: ernste Gefahr irreversiblen Schadens durch Einatmen und durch Verschlucken.
EL: Πολύ τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων όταν εισπνέεται και σε περίπτωση καταπόσεως.
EN: Very toxic: danger of very serious irreversible effects through inhalation and if swallowed.
FR: Très toxique: danger d'effets irréversibles très graves par inhalation et par ingestion.
IT: Molto tossico: pericolo di effetti irreversibili molto gravi per inalazione e ingestione.
NL: Zeer vergiftig: gevaar voor ernstige onherstelbare effecten bij inademing en opname door de mond.
PT: Muito tóxico: perigo de efeitos irreversíveis muito graves por inalação e ingestão.
FI: Erittäin myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara hengitettynä ja nieltynä.
SV: Mycket giftigt: risk för mycket allvarliga bestående hälsoskador vid inandning och förtäring.
R39/27/28
ES: Muy tóxico: peligro de efectos irreversibles muy graves por contacto con la piel e ingestión.
DA: Meget giftig: fare for varig alvorlig skade på helbred ved hudkontakt og indtagelse.
DE: Sehr giftig: ernste Gefahr irreversiblen Schadens bei Berührung mit der Haut und durch Verschlucken.
EL: Πολύ τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Very toxic: danger of very serious irreversible effects in contact with skin and if swallowed.
FR: Très toxique: danger d'effets irréversibles très graves par contact avec la peau et par ingestion.
IT: Molto tossico: pericolo di effetti irreversibili molto gravi a contatto con la pelle e per ingestione.
NL: Zeer vergiftig: gevaar voor ernstige onherstelbare effecten bij aanraking met de huid en opname door de mond.
PT: Muito tóxico: perigo de efeitos irreversíveis muito graves em contacto com a pele e por ingestão.
FI: Erittäin myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara joutuessaan iholle ja nieltynä.
SV: Mycket giftigt: risk för mycket allvarliga bestående hälsoskador vid hudkontakt och förtäring.
R39/26/27/28
ES: Muy tóxico: peligro de efectos irreversibles muy graves por inhalación, contacto con la piel e ingestión.
DA: Meget giftig: fare for varig alvorlig skade på helbred ved indånding, hudkontakt og indtagelse.
DE: Sehr giftig: ernste Gefahr irreversiblen Schadens durch Einatmen, Berührung mit der Haut und durch Verschlucken.
EL: Πολύ τοξικό: κίνδυνος πολύ σοβαρών μόνιμων επιδράσεων όταν εισπνέεται, σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Very toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.
FR: Très toxique: danger d'effets irréversibles très graves par inhalation, par contact avec la peau et par ingestion.
IT: Molto tossico: pericolo di effetti irreversibili molto gravi per inalazione, a contatto con la pelle e per ingestione.
NL: Zeer vergiftig: gevaar voor ernstige onherstelbare effecten bij inademing, aanraking met de huid en opname door de mond.
PT: Muito tóxico: perigo de efeitos irreversíveis muito graves por inalação, em contacto com a pele e por ingestão.
FI: Erittäin myrkyllistä: erittäin vakavien pysyvien vaurioiden vaara hengitettynä, joutuessaan iholle ja nieltynä.
SV: Mycket giftigt: risk för mycket allvarliga bestående hälsoskador vid inandning, hudkontakt och förtäring.
R42/43
ES: Posibilidad de sensibilización por inhalación y por contacto con la piel.
DA: Kan give overfølsomhed ved indånding og ved kontakt med huden.
DE: Sensibilisierung durch Einatmen und Hautkontakt möglich.
EL: Μπορεί να προκαλέσει ευαισθητοποίηση όταν εισπνέεται και σε επαφή με το δέρμα.
EN: May cause sensitisation by inhalation and skin contact.
FR: Peut entraîner une sensibilisation par inhalation et par contact avec la peau.
IT: Può provocare sensibilizzazione per inalazione e contatto con la pelle.
NL: Kan overgevoeligheid veroorzaken bij inademing of contact met de huid.
PT: Pode causar sensibilização por inalação e em contacto com a pele.
FI: Altistuminen hengitysteitse ja ihokosketus voi aiheuttaa herkistymistä.
SV: Kan ge allergi vid inandning och hudkontakt.
R48/20
ES: Nocivo: riesgo de efectos graves para la salud en caso de exposición prolongada por inhalación.
DA: Farlig: alvorlig sundhedsfare ved længere tids påvirkning ved indånding.
DE: Gesundheitsschädlich: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Einatmen.
EL: Επιβλαβές: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση όταν εισπνέεται.
EN: Harmful: danger of serious damage to health by prolonged exposure through inhalation.
FR: Nocif: risque d'effets graves pour la santé en cas d'exposition prolongée par inhalation.
IT: Nocivo: pericolo di gravi danni per la salute in caso di esposizione prolungata per inalazione.
NL: Schadelijk: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij inademing.
PT: Nocivo: risco de efeitos graves para a saúde em caso de exposição prolongada por inalação.
FI: Terveydelle haitallista: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle hengitettynä.
SV: Farligt: risk för allvarliga hälsoskador vid långvarig exponering genom inandning.
R48/21
ES: Nocivo: riesgo de efectos graves para la salud en caso de exposición prolongada por contacto con la piel.
DA: Farlig: alvorlig sundhedsfare ved længere tids påvirkning ved hudkontakt.
DE: Gesundheitsschädlich: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Berührung mit der Haut.
EL: Επιβλαβές: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση σε επαφή με το δέρμα.
EN: Harmful: danger of serious damage to health by prolonged exposure in contact with skin.
FR: Nocif: risque d'effets graves pour la santé en cas d'exposition prolongée par contact avec la peau.
IT: Nocivo: pericolo di gravi danni alla salute in caso di esposizione prolungata a contatto con la pelle.
NL: Schadelijk: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij aanraking met de huid.
PT: Nocivo: risco de efeitos graves para a saúde em caso de exposição prolongada em contacto com a pele.
FI: Terveydelle haitallista: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle joutuessaan iholle.
SV: Farligt: risk för allvarliga hälsoskador vid långvarig exponering genom hudkontakt.
R48/22
ES: Nocivo: riesgo de efectos graves para la salud en caso de exposición prolongada por ingestión.
DA: Farlig: alvorlig sundhedsfare ved længere tids påvirkning ved indtagelse.
DE: Gesundheitsschädlich: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Verschlucken.
EL: Επιβλαβές: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση σε περίπτωση καταπόσεως.
EN: Harmful: danger of serious damage to health by prolonged exposure if swallowed.
FR: Nocif: risque d'effets graves pour la santé en cas d'exposition prolongée par ingestion.
IT: Nocivo: pericolo di gravi danni alla salute in caso di esposizione prolungata per ingestione.
NL: Schadelijk: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij opname door de mond.
PT: Nocivo: risco de efeitos graves para a saúde em caso de exposição prolongada por ingestão.
FI: Terveydelle haitallista: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle nieltynä.
SV: Farligt: risk för allvarliga hälsoskador vid långvarig exponering genom förtäring.
R48/20/21
ES: Nocivo: riesgo de efectos graves para la salud en caso de exposición prolongada por inhalación y contacto con la piel.
DA: Farlig: alvorlig sundhedsfare ved længere tids påvirkning ved indånding og hudkontakt.
DE: Gesundheitsschädlich: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Einatmen und durch Berührung mit der Haut.
EL: Επιβλαβές: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση όταν εισπνέεται και σε επαφή με το δέρμα.
EN: Harmful: danger of serious damage to health by prolonged exposure through inhalation and in contact with skin.
FR: Nocif: risque d'effets graves pour la santé en cas d'exposition prolongée par inhalation et par contact avec la peau.
IT: Nocivo: pericolo di gravi danni alla salute in caso di esposizione prolungata per inalazione e a contatto con la pelle.
NL: Schadelijk: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij inademing en aanraking met de huid.
PT: Nocivo: risco de efeitos graves para a saúde em caso de exposição prolongada por inalação e em contacto com a pele.
FI: Terveydelle haitallista: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle hengitettynä ja joutuessaan iholle.
SV: Farligt: risk för allvarliga hälsoskador vid långvarig exponering genom inandning och hudkontakt.
R48/20/22
ES: Nocivo: riesgo de efectos graves para la salud en caso de exposición prolongada por inhalación e ingestión.
DA: Farlig: alvorlig sundhedsfare ved længere tids påvirkning ved indånding og indtagelse.
DE: Gesundheitsschädlich: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Einatmen und durch Verschlucken.
EL: Επιβλαβές: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση όταν εισπνέεται και σε περίπτωση καταπόσεως.
EN: Harmful: danger of serious damage to health by prolonged exposure through inhalation and if swallowed.
FR: Nocif: risque d'effets graves pour la santé en cas d'exposition prolongée par inhalation et par ingestion.
IT: Nocivo: pericolo di gravi danni alla salute in caso di esposizione prolungata per inalazione e ingestione.
NL: Schadelijk: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij inademing en opname door de mond.
PT: Nocivo: risco de efeitos graves para a saúde em caso de exposição prolongada por inalação e ingestão.
FI: Terveydelle haitallista: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle hengitettynä ja nieltynä.
SV: Farligt: risk för allvarliga hälsoskador vid långvarig exponering genom inandning och förtäring.
R48/21/22
ES: Nocivo: riesgo de efectos graves para la salud en caso de exposición prolongada por contacto con la piel e ingestión.
DA: Farlig: alvorlig sundhedsfare ved længere tids påvirkning ved hudkontakt og indtagelse.
DE: Gesundheitsschädlich: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Berührung mit der Haut und durch Verschlucken.
EL: Επιβλαβές: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Harmful: danger of serious damage to health by prolonged exposure in contact with skin and if swallowed.
FR: Nocif: risque d'effets graves pour la santé en cas d'exposition prolongée par contact avec la peau et par ingestion.
IT: Nocivo: pericolo di gravi danni alla salute in caso di esposizione prolungata a contatto con la pelle e per ingestione.
NL: Schadelijk: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij aanraking met de huid en opname door de mond.
PT: Nocivo: risco de efeitos graves para a saúde em caso de exposição prolongada em contacto com a pele e por ingestão.
FI: Terveydelle haitallista: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle joutuessaan iholle ja nieltynä.
SV: Farligt: risk för allvarliga hälsoskador vid långvarig exponering genom hudkontakt och förtäring.
R48/20/21/22
ES: Nocivo: riesgo de efectos graves para la salud en caso de exposición prolongada por inhalación, contacto con la piel e ingestión.
DA: Farlig: alvorlig sundhedsfare ved længere tids påvirkning ved indånding, hudkontakt og indtagelse.
DE: Gesundheitsschädlich: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Einatmen, Berührung mit der Haut und durch Verschlucken.
EL: Επιβλαβές: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση όταν εισπνέεται, σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Harmful: danger of serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed.
FR: Nocif: risque d'effets graves pour la santé en cas d'exposition prolongée par inhalation, par contact avec la peau et par ingestion.
IT: Nocivo: pericolo di gravi danni alla salute in caso di esposizione prolungata per inalazione, a contatto con la pelle e per ingestione.
NL: Schadelijk: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij inademing, aanraking met de huid en opname door de mond.
PT: Nocivo: risco de efeitos graves para a saúde em caso de exposição prolongada por inalação, em contacto com a pele e por ingestão.
FI: Terveydelle haitallista: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle hengitettynä, joutuessaan iholle ja nieltynä.
SV: Farligt: risk för allvarliga hälsoskador vid långvarig exponering genom inandning, hudkontakt och förtäring.
R48/23
ES: Tóxico: riesgo de efectos graves para la salud en caso de exposición prolongada por inhalación.
DA: Giftig: alvorlig sundhedsfare ved længere tids påvirkning ved indånding.
DE: Giftig: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Einatmen.
EL: Τοξικό: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση όταν εισπνέεται.
EN: Toxic: danger of serious damage to health by prolonged exposure through inhalation.
FR: Toxique: risque d'effets graves pour la santé en cas d'exposition prolongée par inhalation.
IT: Tossico: pericolo di gravi danni alla salute in caso di esposizione prolungata per inalazione.
NL: Vergiftig: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij inademing.
PT: Tóxico: risco de efeitos graves para a saúde em caso de exposição prolongada por inalação.
FI: Myrkyllistä: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle hengitettynä.
SV: Giftigt: risk för allvarliga hälsoskador vid långvarig exponering genom inandning.
R48/24
ES: Tóxico: riesgo de efectos graves para la salud en caso de exposición prolongada por contacto con la piel.
DA: Giftig: alvorlig sundhedsfare ved længere tids påvirkning ved hudkontakt.
DE: Giftig: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Berührung mit der Haut.
EL: Τοξικό: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση σε επαφή με το δέρμα.
EN: Toxic: danger of serious damage to health by prolonged exposure in contact with skin.
FR: Toxique: risque d'effets graves pour la santé en cas d'exposition prolongée par contact avec la peau.
IT: Tossico: pericolo di gravi danni alla salute in caso di esposizione prolungata a contatto con la pelle.
NL: Vergiftig: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij aanraking met de huid.
PT: Tóxico: risco de efeitos graves para a saúde em caso de exposição prolongada em contacto com a pele.
FI: Myrkyllistä: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle joutuessaan iholle.
SV: Giftigt: risk för allvarliga hälsoskador vid långvarig exponering genom hudkontakt.
R48/25
ES: Tóxico: riesgo de efectos graves para la salud en caso de exposición prolongada por ingestión.
DA: Giftig: alvorlig sundhedsfare ved længere tids påvirkning ved indtagelse.
DE: Giftig: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Verschlucken.
EL: Τοξικό: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση σε περίπτωση καταπόσεως.
EN: Toxic: danger of serious damage to health by prolonged exposure if swallowed.
FR: Toxique: risque d'effets graves pour la santé en cas d'exposition prolongée par ingestion.
IT: Tossico: pericolo di gravi danni alla salute in caso di esposizione prolungata per ingestione.
NL: Vergiftig: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij opname door de mond.
PT: Tóxico: risco de efeitos graves para a saúde em caso de exposição prolongada por ingestão.
FI: Myrkyllistä: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle nieltynä.
SV: Giftigt: risk för allvarliga hälsoskador vid långvarig exponering genom förtäring.
R48/23/24
ES: Tóxico: riesgo de efectos graves para la salud en caso de exposición prolongada por inhalación y contacto con la piel.
DA: Giftig: alvorlig sundhedsfare ved længere tids påvirkning ved indånding og hudkontakt.
DE: Giftig: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Einatmen und durch Berührung mit der Haut.
EL: Τοξικό: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση όταν εισπνέεται και σε επαφή με το δέρμα.
EN: Toxic: danger of serious damage to health by prolonged exposure through inhalation and in contact with skin.
FR: Toxique: risque d'effets graves pour la santé en cas d'exposition prolongée par inhalation et par contact avec la peau.
IT: Tossico: pericolo di gravi danni alla salute in caso di esposizione prolungata per inalazione e a contatto con la pelle.
NL: Vergiftig: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij inademing en aanraking met de huid.
PT: Tóxico: risco de efeitos graves para a saúde em caso de exposição prolongada por inalação e em contacto com a pele.
FI: Myrkyllistä: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle hengitettynä ja joutuessaan iholle.
SV: Giftigt: risk för allvarliga hälsoskador vid långvarig exponering genom inandning och hudkontakt.
R48/23/25
ES: Tóxico: riesgo de efectos graves para la salud en caso de exposición prolongada por inhalación e ingestión.
DA: Giftig: alvorlig sundhedsfare ved længere tids påvirkning ved indånding og indtagelse.
DE: Giftig: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Einatmen und durch Verschlucken.
EL: Τοξικό: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση όταν εισπνέεται και σε περίπτωση καταπόσεως.
EN: Toxic: danger of serious damage to health by prolonged exposure through inhalation and if swallowed.
FR: Toxique: risque d'effets graves pour la santé en cas d'exposition prolongée par inhalation et par ingestion.
IT: Tossico: pericolo di gravi danni alla salute in caso di esposizione prolungata per inalazione ed ingestione.
NL: Vergiftig: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij inademing en opname door de mond.
PT: Tóxico: risco de efeitos graves para a saúde em caso de exposição prolongada por inalação e ingestão.
FI: Myrkyllistä: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle hengitettynä ja nieltynä.
SV: Giftigt: risk för allvarliga hälsoskador vid långvarig exponering genom inandning och förtäring.
R48/24/25
ES: Tóxico: riesgo de efectos graves para la salud en caso de exposición prolongada por contacto con la piel e ingestión.
DA: Giftig: alvorlig sundhedsfare ved længere tids påvirkning ved hudkontakt og indtagelse.
DE: Giftig: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Berührung mit der Haut und durch Verschlucken.
EL: Τοξικό: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Toxic: danger of serious damage to health by prolonged exposure in contact with skin and if swallowed.
FR: Toxique: risque d'effets graves pour la santé en cas d'exposition prolongée par contact avec la peau et par ingestion.
IT: Tossico: pericolo di gravi danni alla salute in caso di esposizione prolungata a contatto con la pelle e per ingestione.
NL: Vergiftig: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij aanraking met de huid en opname door de mond.
PT: Tóxico: risco de efeitos graves para a saúde em caso de exposição prolongada em contacto com a pele e por ingestão.
FI: Myrkyllistä: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle joutuessaan iholle ja nieltynä.
SV: Giftigt: risk för allvarliga hälsoskador vid långvarig exponering genom hudkontakt och förtäring.
R48/23/24/25
ES: Tóxico: riesgo de efectos graves para la salud en caso de exposición prolongada por inhalación, contacto con la piel e ingestión.
DA: Giftig: alvorlig sundhedsfare ved længere tids påvirkning ved indånding, hudkontakt og indtagelse.
DE: Giftig: Gefahr ernster Gesundheitsschäden bei längerer Exposition durch Einatmen, Berührung mit der Haut und durch Verschlucken.
EL: Τοξικό: κίνδυνος σοβαρής βλάβης της υγείας ύστερα από παρατεταμένη έκθεση όταν εισπνέεται, σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Toxic: danger of serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed.
FR: Toxique: risque d'effets graves pour la santé en cas d'exposition prolongée par inhalation, par contact avec la peau et par ingestion.
IT: Tossico: pericolo di gravi danni alla salute in caso di esposizione prolungata per inalazione, a contatto con la pelle e per ingestione.
NL: Vergiftig: gevaar voor ernstige schade aan de gezondheid bij langdurige blootstelling bij inademing, aanraking met de huid en opname door de mond.
PT: Tóxico: risco de efeitos graves para a saúde em caso de exposição prolongada por inalação, em contacto com a pele e por ingestão.
FI: Myrkyllistä: pitkäaikainen altistus voi aiheuttaa vakavaa haittaa terveydelle hengitettynä, joutuessaan iholle ja nieltynä.
SV: Giftigt: risk för allvarliga hälsoskador vid långvarig exponering genom inandning, hudkontakt och förtäring.
R50/53
ES: Muy tóxico para los organismos acuáticos, puede provocar a largo plazo efectos negativos en el medio ambiente acuático.
DA: Meget giftig for organismer, der lever i vand; kan forårsage uønskede langtidsvirkninger i vandmiljøet.
DE: Sehr giftig für Wasserorganismen, kann in Gewässern längerfristig schädliche Wirkungen haben.
EL: Πολύ τοξικό για τους υδρόβιους οργανισμούς, μπορεί να προκαλέσει μακροχρόνιες δυσμενείς επιπτώσεις στο υδάτινο περιβάλλον.
EN: Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
FR: Très toxique pour les organismes aquatiques, peut entraîner des effets néfastes à long terme pour l'environnement aquatique.
IT: Altamente tossico per gli organismi acquatici, può provocare a lungo termine effetti negativi per l'ambiente acquatico.
NL: Zeer vergiftig voor in het water levende organismen; kan in het aquatisch milieu op lange termijn schadelijke effecten veroorzaken.
PT: Muito tóxico para os organismos aquáticos, podendo causar efeitos nefastos a longo prazo no ambiente aquático.
FI: Erittäin myrkyllistä vesieliöille, voi aiheuttaa pitkäaikaisia haittavaikutuksia vesiympäristössä.
SV: Mycket giftigt för vattenlevande organismer, kan orsaka skadliga långtidseffekter i vattenmiljön.
R51/53
ES: Tóxico para los organismos acuáticos, puede provocar a largo plazo efectos negativos en el medio ambiente acuático.
DA: Giftig for organismer, der lever i vand; kan forårsage uønskede langtidsvirkninger i vandmiljøet.
DE: Giftig für Wasserorganismen, kann in Gewässern längerfristig schädliche Wirkungen haben.
EL: Τοξικό για τους υδρόβιους οργανισμούς, μπορεί να προκαλέσει μακροχρόνιες δυσμενείς επιπτώσεις στο υδάτινο περιβάλλον.
EN: Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
FR: Toxique pour les organismes aquatiques, peut entraîner des effets néfastes à long terme pour l'environnement aquatique.
IT: Tossico per gli organismi acquatici, può provocare a lungo termine effetti negativi per l'ambiente acquatico.
NL: Vergiftig voor in het water levende organismen; kan in het aquatisch milieu op lange termijn schadelijke effecten veroorzaken.
PT: Tóxico para os organismos aquáticos, podendo causar efeitos nefastos a longo prazo no ambiente aquático.
FI: Myrkyllistä vesieliöille, voi aiheuttaa pitkäaikaisia haittavaikutuksia vesiympäristössä.
SV: Giftigt för vattenlevande organismer, kan orsaka skadliga långtidseffekter i vattenmiljön.
R52/53
ES: Nocivo para los organismos acuáticos, puede provocar a largo plazo efectos negativos en el medio ambiente acuático.
DA: Skadelig for organismer, der lever i vand; kan forårsage uønskede langtidsvirkninger i vandmiljøet.
DE: Schädlich für Wasserorganismen, kann in Gewässern längerfristig schädliche Wirkungen haben.
EL: Επιβλαβές για τους υδρόβιους οργανισμούς, μπορεί να προκαλέσει μακροχρόνιες δυσμενείς επιπτώσεις στο υδάτινο περιβάλλον.
EN: Harmful to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
FR: Nocif pour les organismes aquatiques, peut entraîner des effets néfastes à long terme pour l'environnement aquatique.
IT: Nocivo per gli organismi acquatici, può provocare a lungo termine effetti negativi per l'ambiente acquatico.
NL: Schadelijk voor in het water levende organismen; kan in het aquatisch milieu op lange termijn schadelijke effecten veroorzaken.
PT: Nocivo para os organismos aquáticos, podendo causar efeitos nefastos a longo prazo no ambiente aquático.
FI: Haitallista vesieliöille, voi aiheuttaa pitkäaikaisia haittavaikutuksia vesiympäristössä.
SV: Skadligt för vattenlevande organismer, kan orsaka skadliga långtidseffekter i vattenmiljön.
R68/20
ES: Nocivo: posibilidad de efectos irreversibles por inhalación.
DA: Farlig: mulighed for varig skade på helbred ved indånding.
DE: Gesundheitsschädlich: Möglichkeit irreversiblen Schadens durch Einatmen.
EL: Επιβλαβές: πιθανοί κίνδυνοι μονίμων επιδράσεων όταν εισπνέεται.
EN: Harmful: possible risk of irreversible effects through inhalation.
FR: Nocif: possibilité d'effets irréversibles par inhalation.
IT: Nocivo: possibilità di effetti irreversibili per inalazione.
NL: Schadelijk: bij inademing zijn onherstelbare effecten niet uitgesloten.
PT: Nocivo: possibilidade de efeitos irreversíveis por inalação.
FI: Terveydelle haitallista: pysyvien vaurioiden vaara hengitettynä.
SV: Farligt: möjlig risk för bestående hälsoskador vid inandning.
R68/21
ES: Nocivo: posibilidad de efectos irreversibles por contacto con la piel
DA: Farlig: mulighed for varig skade på helbred ved hudkontakt.
DE: Gesundheitsschädlich: Möglichkeit irreversiblen Schadens bei Berührung mit der Haut.
EL: Επιβλαβές: πιθανοί κίνδυνοι μονίμων επιδράσεων σε επαφή με το δέρμα.
EN: Harmful: possible risk of irreversible effects in contact with skin.
FR: Nocif: possibilité d'effets irréversibles par contact avec la peau.
IT: Nocivo: possibilità di effetti irreversibili a contatto con la pelle.
NL: Schadelijk: bij aanraking met de huid zijn onherstelbare effecten niet uitgesloten.
PT: Nocivo: possibilidade de efeitos irreversíveis em contacto com a pele.
FI: Terveydelle haitallista: pysyvien vaurioiden vaara joutuessaan iholle.
SV: Farligt: möjlig risk för bestående hälsoskador vid hudkontakt.
R68/22
ES: Nocivo: posibilidad de efectos irreversibles por ingestión.
DA: Farlig: mulighed for varig skade på helbred ved indtagelse.
DE: Gesundheitsschädlich: Möglichkeit irreversiblen Schadens durch Verschlucken.
EL: Επιβλαβές: πιθανοί κίνδυνοι μονίμων επιδράσεων σε περίπτωση καταπόσεως.
EN: Harmful: possible risk of irreversible effects if swallowed.
FR: Nocif: possibilité d'effets irréversibles par ingestion.
IT: Nocivo: possibilità di effetti irreversibili per ingestione.
NL: Schadelijk: bij opname door de mond zijn onherstelbare effecten niet uitgesloten.
PT: Nocivo: possibilidade de efeitos irreversíveis por ingestão.
FI: Terveydelle haitallista: pysyvien vaurioiden vaara nieltynä.
SV: Farligt: möjlig risk för bestående hälsoskador vid förtäring.
R68/20/21
ES: Nocivo: posibilidad de efectos irreversibles por inhalación y contacto con la piel.
DA: Farlig: mulighed for varig skade på helbred ved indånding og hudkontakt.
DE: Gesundheitsschädlich: Möglichkeit irreversiblen Schadens durch Einatmen und bei Berührung mit der Haut.
EL: Επιβλαβές: πιθανοί κίνδυνοι μονίμων επιδράσεων όταν εισπνέεται και σε επαφή με το δέρμα.
EN: Harmful: possible risk of irreversible effects through inhalation and in contact with skin.
FR: Nocif: possibilité d'effets irréversibles par inhalation et par contact avec la peau.
IT: Nocivo: possibilità di effetti irreversibili per inalazione e a contatto con la pelle.
NL: Schadelijk: bij inademing en aanraking met de huid zijn onherstelbare effecten niet uitgesloten.
PT: Nocivo: possibilidade de efeitos irreversíveis por inalação e em contacto com a pele.
FI: Terveydelle haitallista: pysyvien vaurioiden vaara hengitettynä ja joutuessaan iholle.
SV: Farligt: möjlig risk för bestående hälsoskador vid inandning och hudkontakt.
R68/20/22
ES: Nocivo: posibilidad de efectos irreversibles por inhalación e ingestión.
DA: Farlig: mulighed for varig skade på helbred ved indånding og indtagelse.
DE: Gesundheitsschädlich: Möglichkeit irreversiblen Schadens durch Einatmen und durch Verschlucken.
EL: Επιβλαβές: πιθανοί κίνδυνοι μονίμων επιδράσεων όταν εισπνέεται και σε περίπτωση καταπόσεως.
EN: Harmful: possible risk of irreversible effects through inhalation and if swallowed.
FR: Nocif: possibilité d'effets irréversibles par inhalation et par ingestion.
IT: Nocivo: possibilità di effetti irreversibili per inalazione e ingestione.
NL: Schadelijk: bij inademing en opname door de mond zijn onherstelbare effecten niet uitgesloten.
PT: Nocivo: possibilidade de efeitos irreversíveis por inalação e ingestão.
FI: Terveydelle haitallista: pysyvien vaurioiden vaara hengitettynä ja nieltynä.
SV: Farligt: möjlig risk för bestående hälsoskador vid inandning och förtäring.
R68/21/22
ES: Nocivo: posibilidad de efectos irreversibles por contacto con la piel e ingestión.
DA: Farlig: mulighed for varig skade på helbred ved hudkontakt og indtagelse.
DE: Gesundheitsschädlich: Möglichkeit irreversiblen Schadens bei Berührung mit der Haut und durch Verschlucken.
EL: Επιβλαβές: πιθανοί κίνδυνοι μονίμων επιδράσεων σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Harmful: possible risk of irreversible effects in contact with skin and if swallowed.
FR: Nocif: possibilité d'effets irréversibles par contact avec la peau et par ingestion.
IT: Nocivo: possibilità di effetti irreversibili a contatto con la pelle e per ingestione.
NL: Schadelijk: bij aanraking met de huid en opname door de mond zijn onherstelbare effecten niet uitgesloten.
PT: Nocivo: possibilidade de efeitos irreversíveis em contacto com a pele e por ingestão.
FI: Terveydelle haitallista: pysyvien vaurioiden vaara joutuessaan iholle ja nieltynä.
SV: Farligt: möjlig risk för bestående hälsoskador vid hudkontakt och förtäring.
R68/20/21/22
ES: Nocivo: posibilidad de efectos irreversibles por inhalación, contacto con la piel e ingestión.
DA: Farlig: mulighed for varig skade på helbred ved indånding, hudkontakt og indtagelse.
DE: Gesundheitsschädlich: Möglichkeit irreversiblen Schadens durch Einatmen, Berührung mit der Haut und durch Verschlucken.
EL: Επιβλαβές: πιθανοί κίνδυνοι μονίμων επιδράσεων όταν εισπνέεται, σε επαφή με το δέρμα και σε περίπτωση καταπόσεως.
EN: Harmful: possible risk of irreversible effects through inhalation, in contact with skin and if swallowed.
FR: Nocif: possibilité d'effets irréversibles par inhalation, par contact avec la peau et par ingestion.
IT: Nocivo: possibilità di effetti irreversibili per inalazione, a contatto con la pelle e per ingestione.
NL: Schadelijk: bij inademing, aanraking met de huid en opname door de mond zijn onherstelbare effecten niet uitgesloten.
PT: Nocivo: possibilidade de efeitos irreversíveis por inalação, em contacto com a pele e por ingestão.
FI: Terveydelle haitallista: pysyvien vaurioiden vaara hengitettynä, joutuessaan iholle ja nieltynä.
SV: Farligt: möjlig risk för bestående hälsoskador vid inandning, hudkontakt och förtäring.
ANNEX 4
ANEXO IV/BILAG IV/ANHANG IV/ΠΑΡΑΡΤΗΜΑ IV/ANNEX IV/ANNEXE IV/ALLEGATO IV/BIJLAGE IV/ANEXO IV/LIITE IV/BILAGA IV
ANEXO IV
Consejos de prudencia relativos a las sustancias y preparados peligrosos
/BILAG IV
Forsigtighedsregler for farlige stoffer og præparater
/ANHANG IV
Sicherheitsratschläge für gefährliche Stoffe und Zubereitungen
/ΠΑΡΑΡΤΗΜΑ IV
Οδηγίες ασφαλούς χρήσης που αφορούν επικίνδυνες χημικές ουσίες και παρασκευάσματα
/ANNEX IV
Safety advice concerning dangerous substances and preparations
/ANNEXE IV
Conseils de prudence concernant les substances et préparations dangereuses
/ALLEGATO IV
Consigli di prudenza riguardanti le sostanze e preparati pericolosi
/BIJLAGE IV
Veiligheidsaanbevelingen met betrekking tot gevaarlijke stoffen en preparaten
/ANEXO IV
Conselhos de prudência relativos a substâncias e preparações perigosas
/LIITE IV
Vaarallisten aineiden ja valmisteiden turvallisuusohjeet
/BILAGA IV
Skyddsfraser för farliga ämnen och beredningar
S1
ES: Consérvese bajo llave.
DA: Opbevares under lås.
DE: Unter Verschluss aufbewahren.
EL: Να φυλάσσεται κλειδωμένο.
EN: Keep locked up.
FR: Conserver sous clé.
IT: Conservare sotto chiave.
NL: Achter slot bewaren.
PT: Guardar fechado à chave.
FI: Säilytettävä lukitussa tilassa.
SV: Förvaras i låst utrymme.
S2
ES: Manténgase fuera del alcance de los niños.
DA: Opbevares utilgængeligt for børn.
DE: Darf nicht in die Hände von Kindern gelangen.
EL: Μακρυά από παιδιά.
EN: Keep out of the reach of children.
FR: Conserver hors de portée des enfants.
IT: Conservare fuori della portata dei bambini.
NL: Buiten bereik van kinderen bewaren.
PT: Manter fora do alcance das crianças.
FI: Säilytettävä lasten ulottumattomissa.
SV: Förvaras oåtkomligt för barn.
S3
ES: Consérvese en lugar fresco.
DA: Opbevares køligt.
DE: Kühl aufbewahren.
EL: Να φυλάσσεται σε δροσερό μέρος.
EN: Keep in a cool place.
FR: Conserver dans un endroit frais.
IT: Conservare in luogo fresco.
NL: Op een koele plaats bewaren.
PT: Guardar em lugar fresco.
FI: Säilytettävä viileässä.
SV: Förvaras svalt.
S4
ES: Manténgase lejos de locales habitados.
DA: Må ikke opbevares i nærheden af beboelse.
DE: Von Wohnplätzen fernhalten.
EL: Μακρυά από κατοικημένους χώρους.
EN: Keep away from living quarters.
FR: Conserver à l'écart de tout local d'habitation.
IT: Conservare lontano da locali di abitazione.
NL: Verwijderd van woonruimten opbergen.
PT: Manter fora de qualquer zona de habitação.
FI: Ei saa säilyttää asuintiloissa.
SV: Förvaras avskilt från bostadsutrymmen.
S5
ES: Consérvese en ... (líquido apropiado a especificar por el fabricante).
DA: Opbevares under ... (en egnet væske, som angives af fabrikanten).
DE: Unter ... aufbewahren (geeignete Flüssigkeit vom Hersteller anzugeben).
EL: Να διατηρείται το περιεχόμενο μέσα σε ... (το είδος του κατάλληλου υγρού καθορίζεται από τον παραγωγό).
EN: Keep contents under ... (appropriate liquid to be specified by the manufacturer).
FR: Conserver sous ... (liquide approprié à spécifier par le fabricant).
IT: Conservare sotto ... (liquido appropriato da indicarsi da parte del fabbricante).
NL: Onder ... houden (geschikte vloeistof aan te geven door fabrikant).
PT: Manter sob ... (líquido apropriado a especificar pelo produtor).
FI: Sisältö säilytettävä ... (tarkoitukseen soveltuvan nesteen ilmoittaa valmistaja/maahantuoja).
SV: Förvara innehållet i ... (lämplig vätska anges av tillverkaren).
S6
ES: Consérvese en ... (gas inerte a especificar por el fabricante).
DA: Opbevares under ... (en inaktiv gas, som angives af fabrikanten).
DE: Unter ... aufbewahren (inertes Gas vom Hersteller anzugeben).
EL: Να διατηρείται σε ατμόσφαιρα ... (το είδος του αδρανούς αερίου καθορίζεται από τον παραγωγό).
EN: Keep under ... (inert gas to be specified by the manufacturer).
FR: Conserver sous ... (gaz inerte à spécifier par le fabricant).
IT: Conservare sotto ... (gas inerte da indicarsi da parte del fabbricante).
NL: Onder ... houden (inert gas aan te geven door fabrikant).
PT: Manter sob ... (gás inerte a especificar pelo produtor).
FI: Säilytettävä ... (inertin kaasun ilmoittaa valmistaja/maahantuoja).
SV: Förvaras i ... (inert gas anges av tillverkaren).
S7
ES: Manténgase el recipiente bien cerrado.
DA: Emballagen skal holdes tæt lukket.
DE: Behälter dicht geschlossen halten.
EL: Το δοχείο να διατηρείται ερμητικά κλεισμένο.
EN: Keep container tightly closed.
FR: Conserver le récipient bien fermé.
IT: Conservare il recipiente ben chiuso.
NL: In goed gesloten verpakking bewaren.
PT: Manter o recipiente bem fechado.
FI: Säilytettävä tiiviisti suljettuna.
SV: Förpackningen förvaras väl tillsluten.
S8
ES: Manténgase el recipiente en lugar seco.
DA: Emballagen skal opbevares tørt.
DE: Behälter trocken halten.
EL: Το δοχείο να προστατεύεται από την υγρασία.
EN: Keep container dry.
FR: Conserver le récipient à l'abri de l'humidité.
IT: Conservare al riparo dall'umidità.
NL: Verpakking droog houden.
PT: Manter o recipiente ao abrigo da humidade.
FI: Säilytettävä kuivana.
SV: Förpackningen förvaras torrt.
S9
ES: Consérvese el recipiente en lugar bien ventilado.
DA: Emballagen skal opbevares på et godt ventileret sted.
DE: Behälter an einem gut gelüfteten Ort aufbewahren.
EL: Το δοχείο να διατηρείται σε καλά αεριζόμενο μέρος.
EN: Keep container in a well-ventilated place.
FR: Conserver le récipient dans un endroit bien ventilé.
IT: Conservare il recipiente in luogo ben ventilato.
NL: Op een goed geventileerde plaats bewaren.
PT: Manter o recipiente num local bem ventilado.
FI: Säilytettävä paikassa, jossa on hyvä ilmanvaihto.
SV: Förpackningen förvaras på väl ventilerad plats.
S12
ES: No cerrar el recipiente herméticamente.
DA: Emballagen må ikke lukkes tæt.
DE: Behälter nicht gasdicht verschließen.
EL: Μη διατηρείτε το δοχείο ερμητικά κλεισμένο.
EN: Do not keep the container sealed.
FR: Ne pas fermer hermétiquement le récipient.
IT: Non chiudere ermeticamente il recipiente.
NL: De verpakking niet hermetisch sluiten.
PT: Não fechar o recipiente hermeticamente.
FI: Pakkausta ei saa sulkea ilmatiiviisti.
SV: Förpackningen får inte tillslutas lufttätt.
S13
ES: Manténgase lejos de alimentos, bebidas y piensos.
DA: Må ikke opbevares sammen med fødevarer, drikkevarer og foderstoffer.
DE: Von Nahrungsmitteln, Getränken und Futtermitteln fernhalten.
EL: Μακρυά από τρόφιμα, ποτά και ζωοτροφές.
EN: Keep away from food, drink and animal feedingstuffs.
FR: Conserver à l'écart des aliments et boissons, y compris ceux pour animaux.
IT: Conservare lontano da alimenti o mangimi e da bevande.
NL: Verwijderd houden van eet- en drinkwaren en van diervoeder.
PT: Manter afastado de alimentos e bebidas, incluindo os dos animais.
FI: Ei saa säilyttää yhdessä elintarvikkeiden eikä eläinravinnon kanssa.
SV: Förvaras åtskilt från livsmedel och djurfoder.
S14
ES: Consérvese lejos de ... (materiales incompatibles a especificar por el fabricante).
DA: Opbevares adskilt fra ... (uforligelige stoffer, som angives af fabrikanten).
DE: Von ... fernhalten (inkompatible Substanzen sind vom Hersteller anzugeben).
EL: Μακρυά από ... (ασύμβατες ουσίες καθορίζονται από τον παραγωγό).
EN: Keep away from ... (incompatible materials to be indicated by the manufacturer).
FR: Conserver à l'écart des ... (matières incompatibles à indiquer par le fabricant).
IT: Conservare lontano da ... (sostanze incompatibili da precisare da parte del produttore).
NL: Verwijderd houden van ... (stoffen waarmee contact vermeden dient te worden aan te geven door de fabrikant).
PT: Manter afastado de ... (matérias incompatíveis a indicar pelo produtor).
FI: Säilytettävä erillään ... (yhteensopimattomat aineet ilmoittaa valmistaja/maahantuoja).
SV: Förvaras åtskilt från ... (oförenliga ämnen anges av tillverkaren).
S15
ES: Conservar alejado del calor.
DA: Må ikke udsættes for varme.
DE: Vor Hitze schützen.
EL: Μακρυά από θερμότητα.
EN: Keep away from heat.
FR: Conserver à l'écart de la chaleur.
IT: Conservare lontano dal calore.
NL: Verwijderd houden van warmte.
PT: Manter afastado do calor.
FI: Suojattava lämmöltä.
SV: Får inte utsättas för värme.
S16
ES: Conservar alejado de toda llama o fuente de chispas - No fumar.
DA: Holdes væk fra antændelseskilder - Rygning forbudt.
DE: Von Zündquellen fernhalten - Nicht rauchen.
EL: Μακρυά από πηγές ανάφλεξης - Απαγορεύεται το κάπνισμα.
EN: Keep away from sources of ignition - No smoking.
FR: Conserver à l'écart de toute flamme ou source d'étincelles - Ne pas fumer.
IT: Conservare lontano da fiamme e scintille - Non fumare.
NL: Verwijderd houden van ontstekingsbronnen - Niet roken.
PT: Manter afastado de qualquer chama ou fonte de ignição - Não fumar.
FI: Eristettävä sytytyslähteistä - Tupakointi kielletty.
SV: Förvaras åtskilt från antändningskällor - Rökning förbjuden.
S17
ES: Manténgase lejos de materias combustibles.
DA: Holdes væk fra brandbare stoffer.
DE: Von brennbaren Stoffen fernhalten.
EL: Μακρυά από καύσιμα υλικά.
EN: Keep away from combustible material.
FR: Tenir à l'écart des matières combustibles.
IT: Tenere lontano da sostanze combustibili.
NL: Verwijderd houden van brandbare stoffen.
PT: Manter afastado de matérias combustíveis.
FI: Säilytettävä erillään syttyvistä kemikaaleista.
SV: Förvaras åtskilt från brandfarliga ämnen.
S18
ES: Manipúlese y ábrase el recipiente con prudencia.
DA: Emballagen skal behandles og åbnes med forsigtighed.
DE: Behälter mit Vorsicht öffnen und handhaben.
EL: Χειριστείτε και ανοίξτε το δοχείο προσεκτικά.
EN: Handle and open container with care.
FR: Manipuler et ouvrir le récipient avec prudence.
IT: Manipolare ed aprire il recipiente con cautela.
NL: Verpakking voorzichtig behandelen en openen.
PT: Manipular e abrir o recipiente com prudência.
FI: Pakkauksen käsittelyssä ja avaamisessa on noudatettava varovaisuutta.
SV: Förpackningen hanteras och öppnas försiktigt.
S20
ES: No comer ni beber durante su utilización.
DA: Der må ikke spises eller drikkes under brugen.
DE: Bei der Arbeit nicht essen und trinken.
EL: Μην τρώτε ή πίνετε όταν το χρησιμοποιείτε.
EN: When using do not eat or drink.
FR: Ne pas manger et ne pas boire pendant l'utilisation.
IT: Non mangiare né bere durante l'impiego.
NL: Niet eten of drinken tijdens gebruik.
PT: Não comer nem beber durante a utilização.
FI: Syöminen ja juominen kielletty kemikaalia käsiteltäessä.
SV: Ät inte eller drick inte under hanteringen
S21
ES: No fumar durante su utilización.
DA: Der må ikke ryges under brugen.
DE: Bei der Arbeit nicht rauchen.
EL: Μην καπνίζετε όταν το χρησιμοποιείτε.
EN: When using do not smoke.
FR: Ne pas fumer pendant l'utilisation.
IT: Non fumare durante l'impiego.
NL: Niet roken tijdens gebruik.
PT: Não fumar durante a utilização.
FI: Tupakointi kielletty kemikaalia käytettäessä.
SV: Rök inte under hanteringen.
S22
ES: No respirar el polvo.
DA: Undgå indånding af støv.
DE: Staub nicht einatmen.
EL: Μην αναπνέετε την σκόνη.
EN: Do not breathe dust.
FR: Ne pas respirer les poussières.
IT: Non respirare le polveri.
NL: Stof niet inademen.
PT: Não respirar as poeiras.
FI: Vältettävä pölyn hengittämistä.
SV: Undvik inandning av damm.
S23
ES: No respirar los gases/humos/vapores/aerosoles [denominación(es) adecuada(s) a especificar por el fabricante].
DA: Undgå indånding af gas/røg/dampe/aerosol-tåger (den eller de pågældende betegnelser angives af fabrikanten).
DE: Gas/Rauch/Dampf/Aerosol nicht einatmen (geeignete Bezeichnung(en) vom Hersteller anzugeben).
EL: Μην αναπνέετε αέρια/αναθυμιάσεις/ατμούς/εκνεφώματα (η κατάλληλη διατύπωση καθορίζεται από τον παραγωγό).
EN: Do not breathe gas/fumes/vapour/spray (appropriate wording to be specified by the manufacturer).
FR: Ne pas respirer les gaz/fumées/vapeurs/aérosols [terme(s) approprié(s) à indiquer par le fabricant].
IT: Non respirare i gas/fumi/vapori/aerosoli [termine(i) appropriato(i) da precisare da parte del produttore].
NL: Gas/rook/damp/spuitnevel niet inademen (toepasselijke term(en) aan te geven door de fabrikant).
PT: Não respirar os gases/vapores/fumos/aerossóis [termo(s) apropriado(s) a indicar pelo produtor].
FI: Vältettävä kaasun/huurun/höyryn/sumun hengittämistä (oikean sanamuodon valitsee valmistaja/maahantuoja).
SV: Undvik inandning av gas/rök/ånga/dimma (lämplig formulering anges av tillverkaren).
S24
ES: Evítese el contacto con la piel.
DA: Undgå kontakt med huden.
DE: Berührung mit der Haut vermeiden.
EL: Αποφεύγετε την επαφή με το δέρμα.
EN: Avoid contact with skin.
FR: Éviter le contact avec la peau.
IT: Evitare il contatto con la pelle.
NL: Aanraking met de huid vermijden.
PT: Evitar o contacto com a pele.
FI: Varottava kemikaalin joutumista iholle.
SV: Undvik kontakt med huden.
S25
ES: Evítese el contacto con los ojos.
DA: Undgå kontakt med øjnene.
DE: Berührung mit den Augen vermeiden.
EL: Αποφεύγετε την επαφή με τα μάτια.
EN: Avoid contact with eyes.
FR: Éviter le contact avec les yeux.
IT: Evitare il contatto con gli occhi.
NL: Aanraking met de ogen vermijden.
PT: Evitar o contacto com os olhos.
FI: Varottava kemikaalin joutumista silmiin.
SV: Undvik kontakt med ögonen.
S26
ES: En caso de contacto con los ojos, lávense inmediata y abundantemente con agua y acúdase a un médico.
DA: Kommer stoffet i øjnene, skylles straks grundigt med vand og læge kontaktes.
DE: Bei Berührung mit den Augen sofort gründlich mit Wasser abspülen und Arzt konsultieren.
EL: Σε περίπτωση επαφής με τα μάτια πλύνετέ τα αμέσως με άφθονο νερό και ζητήστε ιατρική συμβουλή.
EN: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
FR: En cas de contact avec les yeux, laver immédiatement et abondamment avec de l'eau et consulter un spécialiste.
IT: In caso di contatto con gli occhi, lavare immediatamente e abbondantemente con acqua e consultare un medico.
NL: Bij aanraking met de ogen onmiddellijk met overvloedig water afspoelen en deskundig medisch advies inwinnen.
PT: Em caso de contacto com os olhos, lavar imediata e abundantemente com água e consultar um especialista.
FI: Roiskeet silmistä huuhdeltava välittömästi runsaalla vedellä ja mentävä lääkäriin.
SV: Vid kontakt med ögonen, spola genast med mycket vatten och kontakta läkare.
S27
ES: Quítese inmediatamente la ropa manchada o salpicada.
DA: Tilsmudset tøj tages straks af.
DE: Beschmutzte, getränkte Kleidung sofort ausziehen.
EL: Αφαιρέστε αμέσως όλα τα ενδύματα που έχουν μολυνθεί.
EN: Take off immediately all contaminated clothing.
FR: Enlever immédiatement tout vêtement souillé ou éclaboussé.
IT: Togliersi di dosso immediatamente gli indumenti contaminati.
NL: Verontreinigde kleding onmiddellijk uittrekken.
PT: Retirar imediatamente todo o vestuário contaminado.
FI: Riisuttava välittömästi saastunut vaatetus.
SV: Tag genast av alla nedstänkta kläder.
S28
ES: En caso de contacto con la piel, lávese inmediata y abundantemente con ... (productos a especificar por el fabricante).
DA: Kommer stof på huden vaskes straks med store mængder ... (angives af fabrikanten).
DE: Bei Berührung mit der Haut sofort abwaschen mit viel ... (vom Hersteller anzugeben).
EL: Σε περίπτωση επαφής με το δέρμα, πλυθείτε αμέσως με άφθονο ... (το είδος του υγρού καθορίζεται από τον παραγωγό).
EN: After contact with skin, wash immediately with plenty of ... (to be specified by the manufacturer).
FR: Après contact avec la peau, se laver immédiatement et abondamment avec ... (produits appropriés à indiquer par le fabricant).
IT: In caso di contatto con la pelle lavarsi immediatamente ed abbondantemente con ... (prodotti idonei da indicarsi da parte del fabbricante).
NL: Na aanraking met de huid onmiddellijk wassen met veel ... (aan te geven door de fabrikant).
PT: Após contacto com a pele, lavar imediata e abundantemente com ... (produtos adequados a indicar pelo produtor).
FI: Roiskeet iholta huuhdeltava välittömästi runsaalla määrällä ... (aineen ilmoittaa valmistaja/maahantuoja).
SV: Vid kontakt med huden tvätta genast med mycket ... (anges av tillverkaren).
S29
ES: No tirar los residuos por el desagüe.
DA: Må ikke tømmes i kloakafløb.
DE: Nicht in die Kanalisation gelangen lassen.
EL: Μην αδειάζετε το υπόλοιπο του περιεχομένου στην αποχέτευση.
EN: Do not empty into drains.
FR: Ne pas jeter les résidus à l'égout.
IT: Non gettare i residui nelle fognature.
NL: Afval niet in de gootsteen werpen.
PT: Não deitar os resíduos no esgoto.
FI: Ei saa tyhjentää viemäriin.
SV: Töm ej i avloppet.
S30
ES: No echar jamás agua a este producto.
DA: Hæld aldrig vand på eller i produktet.
DE: Niemals Wasser hinzugießen.
EL: Ποτέ μην προσθέτετε νερό στο προϊόν αυτό.
EN: Never add water to this product.
FR: Ne jamais verser de l'eau dans ce produit.
IT: Non versare acqua sul prodotto.
NL: Nooit water op deze stof gieten.
PT: Nunca adicionar água a este produto.
FI: Tuotteeseen ei saa lisätä vettä.
SV: Häll aldrig vatten på eller i produkten.
S33
ES: Evítese la acumulación de cargas electroestáticas.
DA: Træf foranstaltninger mod statisk elektricitet.
DE: Maßnahmen gegen elektrostatische Aufladungen treffen.
EL: Λάβετε προστατευτικά μέτρα έναντι ηλεκτροστατικών εκκενώσεων.
EN: Take precautionary measures against static discharges.
FR: Éviter l'accumulation de charges électrostatiques.
IT: Evitare l'accumulo di cariche elettrostatiche.
NL: Maatregelen treffen tegen ontladingen van statische elektriciteit.
PT: Evitar acumulação de cargas electrostáticas.
FI: Estettävä staattisen sähkön aiheuttama kipinöinti.
SV: Vidtag åtgärder mot statisk elektricitet.
S35
ES: Elimínense los residuos del producto y sus recipientes con todas las precauciones posibles.
DA: Materialet og dets beholder skal bortskaffes på en sikker måde.
DE: Abfälle und Behälter müssen in gesicherter Weise beseitigt werden.
EL: Το υλικό και ο περιέκτης του πρέπει να διατεθεί με ασφαλή τρόπο.
EN: This material and its container must be disposed of in a safe way.
FR: Ne se débarrasser de ce produit et de son récipient qu'en prenant toutes les précautions d'usage.
IT: Non disfarsi del prodotto e del recipiente se non con le dovute precauzioni.
NL: Deze stof en de verpakking op veilige wijze afvoeren.
PT: Não se desfazer deste produto e do seu recipiente sem tomar as precauções de segurança devidas.
FI: Tämä aine ja sen pakkaus on hävitettävä turvallisesti.
SV: Produkt och förpackning skall oskadliggöras på säkert sätt.
S36
ES: Úsese indumentaria protectora adecuada.
DA: Brug særligt arbejdstøj.
DE: Bei der Arbeit geeignete Schutzkleidung tragen.
EL: Να φοράτε κατάλληλη προστατευτική ενδυμασία.
EN: Wear suitable protective clothing.
FR: Porter un vêtement de protection approprié.
IT: Usare indumenti protettivi adatti.
NL: Draag geschikte beschermende kleding.
PT: Usar vestuário de protecção adequado.
FI: Käytettävä sopivaa suojavaatetusta.
SV: Använd lämpliga skyddskläder.
S37
ES: Úsense guantes adecuados.
DA: Brug egnede beskyttelseshandsker under arbejdet.
DE: Geeignete Schutzhandschuhe tragen.
EL: Να φοράτε κατάλληλα γάντια.
EN: Wear suitable gloves.
FR: Porter des gants appropriés.
IT: Usare guanti adatti.
NL: Draag geschikte handschoenen.
PT: Usar luvas adequadas.
FI: Käytettävä sopivia suojakäsineitä.
SV: Använd lämpliga skyddshandskar.
S38
ES: En caso de ventilación insuficiente, úsese equipo respiratorio adecuado.
DA: Brug egnet åndedrætsværn, hvis effektiv ventilation ikke er mulig.
DE: Bei unzureichender Belüftung Atemschutzgerät anlegen.
EL: Σε περίπτωση ανεπαρκούς αερισμού, χρησιμοποιείτε κατάλληλη αναπνευστική συσκευή.
EN: In case of insufficient ventilation, wear suitable respiratory equipment.
FR: En cas de ventilation insuffisante, porter un appareil respiratoire approprié.
IT: In caso di ventilazione insufficiente, usare un apparecchio respiratorio adatto.
NL: Bij ontoereikende ventilatie een geschikte adembescherming dragen.
PT: Em caso de ventilação insuficiente, usar equipamento respiratório adequado.
FI: Kemikaalin käyttö edellyttää tehokasta ilmanvaihtoa tai sopivaa hengityksensuojainta.
SV: Använd lämpligt andningsskydd vid otillräcklig ventilation.
S39
ES: Úsese protección para los ojos/la cara.
DA: Brug beskyttelsesbriller/ansigtsskærm under arbejdet.
DE: Schutzbrille/Gesichtsschutz tragen.
EL: Χρησιμοποιείτε συσκευή προστασίας ματιών/προσώπου.
EN: Wear eye/face protection.
FR: Porter un appareil de protection des yeux/du visage.
IT: Proteggersi gli occhi/la faccia.
NL: Een bescherming voor de ogen/voor het gezicht dragen.
PT: Usar um equipamento protector para os olhos/face.
FI: Käytettävä silmien- tai kasvonsuojainta.
SV: Använd skyddsglasögon eller ansiktsskydd.
S40
ES: Para limpiar el suelo y los objetos contaminados por este producto, úsese ... (a especificar por el fabricante).
DA: Gulvet og tilsmudsede genstande renses med ... (midlerne angives af fabrikanten).
DE: Fußboden und verunreinigte Gegenstände mit ... reinigen (Material vom Hersteller anzugeben).
EL: Για τον καθαρισμό του δαπέδου και όλων των αντικειμένων που έχουν μολυνθεί από το υλικό αυτό χρησιμοποιείτε ... (το είδος καθορίζεται από τον παραγωγό).
EN: To clean the floor and all objects contaminated by this material, use ... (to be specified by the manufacturer).
FR: Pour nettoyer le sol ou les objets souillés par ce produit, utiliser ... (à préciser par le fabricant).
IT: Per pulire il pavimento e gli oggetti contaminati da questo prodotto, usare ... (da precisare da parte del produttore).
NL: Voor de reiniging van de vloer en alle voorwerpen verontreinigd met dit materiaal, ... gebruiken (aan te geven door de fabrikant).
PT: Para limpeza do chão e objectos contaminados por este produto, utilizar ... (a especificar pelo produtor).
FI: Kemikaali puhdistettava pinnoilta käyttäen ... (kemikaalin ilmoittaa valmistaja/maahantuoja).
SV: Golv och förorenade föremål tvättas med ... (anges av tillverkaren).
S41
ES: En caso de incendio y/o de explosión no respire los humos.
DA: Undgå at indånde røgen ved brand eller eksplosion.
DE: Explosions- und Brandgase nicht einatmen.
EL: Σε περίπτωση πυρκαγιάς ή/και εκρήξεως μην αναπνέετε τους καπνούς.
EN: In case of fire and/or explosion do not breathe fumes.
FR: En cas d'incendie et/ou d'explosion, ne pas respirer les fumées.
IT: In caso di incendio e/o esplosione non respirare i fumi.
NL: In geval van brand en/of explosie inademen van rook vermijden.
PT: Em caso de incêndio e/ou explosão não respirar os fumos.
FI: Vältettävä palamisessa tai räjähdyksessä muodostuvan savun hengittämistä.
SV: Undvik inandning av rök vid brand eller explosion.
S42
ES: Durante las fumigaciones/pulverizaciones, úsese equipo respiratorio adecuado [denominación(es) adecuada(s) a especificar por el fabricante].
DA: Brug egnet åndedrætsværn ved rygning/sprøjtning (den eller de pågældende betegnelser angives af fabrikanten).
DE: Beim Räuchern/Versprühen geeignetes Atemschutzgerät anlegen (geeignete Bezeichnung(en) vom Hersteller anzugeben).
EL: Κατά τη διάρκεια υποκαπνισμού/ψεκάσματος χρησιμοποιείτε κατάλληλη αναπνευστική συσκευή (η κατάλληλη διατύπωση καθορίζεται από τον παραγωγό).
EN: During fumigation/spraying wear suitable respiratory equipment (appropriate wording to be specified by the manufacturer).
FR: Pendant les fumigations/pulvérisations, porter un appareil respiratoire approprié [terme(s) approprié(s) à indiquer par le fabricant].
IT: Durante le fumigazioni/polimerizzazioni usare un apparecchio respiratorio adatto [termine(i) appropriato(i) da precisare da parte del produttore].
NL: Tijdens de ontsmetting/bespuiting een geschikte adembescherming dragen (geschikte term(en) door de fabrikant aan te geven).
PT: Durante as fumigações/pulverizações usar equipamento respiratório adequado [termo(s) adequado(s) a indicar pelo produtor].
FI: Kaasutuksen/ruiskutuksen aikana käytettävä sopivaa hengityksensuojainta (oikean sanamuodon valitsee valmistaja/maahantuoja).
SV: Använd lämpligt andningsskydd vid gasning/sprutning (specificeras av tillverkaren).
S43
ES: En caso de incendio, utilizar ... (los medios de extinción los debe especificar el fabricante). (Si el agua aumenta el riesgo, se deberá añadir: "No usar nunca agua").
DA: Brug ... ved brandslukning (den nøjagtige type brandslukningsudstyr angives af fabrikanten. Såfremt vand ikke må bruges tilføjes: "Brug ikke vand").
DE: Zum Löschen ... (vom Hersteller anzugeben) verwenden. (Wenn Wasser die Gefahr erhöht, anfügen: "Kein Wasser verwenden".).
EL: Σε περίπτωση πυρκαγιάς χρησιμοποιείτε ... (Αναφέρεται το ακριβές είδος μέσων πυρόσβεσης. Εάν το νερό αυξάνει τον κίνδυνο, προστίθεται: "Μη χρησιμοποιείτε ποτέ νερό").
EN: In case of fire, use ... (indicate in the space the precise type of fire-fighting equipment. If water increases risk, add "Never use water").
FR: En cas d'incendie, utiliser ... (moyens d'extinction à préciser par le fabricant. Si l'eau augmente les risques, ajouter: "Ne jamais utiliser d'eau").
IT: In caso di incendio usare ... (mezzi estinguenti idonei da indicarsi da parte del fabbricante. Se l'acqua aumenta il rischio precisare "Non usare acqua").
NL: In geval van brand ... gebruiken (blusmiddelen aan te duiden door de fabrikant. Indien water het risico vergroot toevoegen: "Nooit water gebruiken.").
PT: Em caso de incêndio, utilizar ... (meios de extinção a especificar pelo produtor. Se a água aumentar os riscos, acrescentar "Nunca utilizar água").
FI: Sammutukseen käytettävä ... (ilmoitettava sopiva sammutusmenetelmä. Jos vesi lisää vaaraa, lisättävä sanat: Sammutukseen ei saa käyttää vettä).
SV: Vid brandsläckning använd ... (ange lämplig metod. Om vatten ökar riskerna, lägg till: "Använd aldrig vatten").
S45
ES: En caso de accidente o malestar, acúdase inmediatamente al médico (si es posible, muéstresele la etiqueta).
DA: Ved ulykkestilfælde eller ved ildebefindende er omgående lægebehandling nødvendig; vis etiketten, hvis det er muligt.
DE: Bei Unfall oder Unwohlsein sofort Arzt zuziehen (wenn möglich, dieses Etikett vorzeigen).
EL: Σε περίπτωση ατυχήματος ή αν αισθανθείτε αδιαθεσία ζητήστε αμέσως ιατρική συμβουλή (δείξτε την ετικέττα αν είναι δυνατό).
EN: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
FR: En cas d'accident ou de malaise, consulter immédiatement un médecin (si possible, lui montrer l'étiquette).
IT: In caso di incidente o di malessere consultare immediatamente il medico (se possibile, mostrargli l'etichetta).
NL: Bij een ongeval of indien men zich onwel voelt, onmiddellijk een arts raadplegen (indien mogelijk hem dit etiket tonen).
PT: Em caso de acidente ou de indisposição, consultar imediatamente o médico (se possível mostrar-lhe o rótulo).
FI: Onnettomuuden sattuessa tai tunnettaessa pahoinvointia hakeuduttava heti lääkärin hoitoon (näytettävä tätä etikettiä, mikäli mahdollista).
SV: Vid olycksfall, illamående eller annan påverkan, kontakta omedelbart läkare. Visa om möjligt etiketten.
S46
ES: En caso de ingestión, acúdase inmediatamente al médico y muéstresele la etiqueta o el envase.
DA: Ved indtagelse, kontakt omgående læge og vis denne beholder eller etiket.
DE: Bei Verschlucken sofort ärztlichen Rat einholen und Verpackung oder Etikett vorzeigen.
EL: Σε περίπτωση κατάποσης ζητήστε αμέσως ιατρική συμβουλή και δείξτε αυτό το δοχείο ή την ετικέττα.
EN: If swallowed, seek medical advice immediately and show this container or label.
FR: En cas d'ingestion, consulter immédiatement un médecin et lui montrer l'emballage ou l'étiquette.
IT: In caso d'ingestione consultare immediatamente il medico e mostrargli il contenitore o l'etichetta.
NL: In geval van inslikken onmiddellijk een arts raadplegen en verpakking of etiket tonen.
PT: Em caso de ingestão, consultar imediatamente o médico e mostrar-lhe a embalagem ou o rótulo.
FI: Jos ainetta on nielty, hakeuduttava heti lääkärin hoitoon ja näytettävä tämä pakkaus tai etiketti.
SV: Vid förtäring kontakta genast läkare och visa denna förpackning eller etiketten.
S47
ES: Consérvese a una temperatura no superior a ... °C (a especificar por el fabricante).
DA: Må ikke opbevares ved temperaturer på over ... °C (angives af fabrikanten).
DE: Nicht bei Temperaturen über ... °C aufbewahren (vom Hersteller anzugeben).
EL: Να διατηρείται σε θερμοκρασία που δεν υπερβαίνει τους ... °C (καθορίζεται από τον παραγωγό).
EN: Keep at temperature not exceeding ... °C (to be specified by the manufacturer).
FR: Conserver à une température ne dépassant pas ... °C (à préciser par le fabricant).
IT: Conservare a temperatura non superiore a ... °C (da precisare da parte del fabbricante).
NL: Bewaren bij een temperatuur beneden ... °C (aan te geven door de fabrikant).
PT: Conservar a uma temperatura que não exceda ... °C (a especificar pelo produtor).
FI: Säilytettävä alle ... °C:n lämpötilassa (valmistaja/maahantuoja ilmoittaa lämpötilan).
SV: Förvaras vid en temperatur som inte överstiger ... °C (anges av tillverkaren).
S48
ES: Consérvese húmedo con ... (medio apropiado a especificar por el fabricante).
DA: Holdes befugtet med ... (passende middel angives af fabrikanten).
DE: Feucht halten mit ... (geeignetes Mittel vom Hersteller anzugeben).
EL: Να διατηρείται υγρό με ... (το κατάλληλο υλικό καθορίζεται από τον παραγωγό).
EN: Keep wet with ... (appropriate material to be specified by the manufacturer).
FR: Maintenir humide avec ... (moyen approprié à préciser par le fabricant).
IT: Mantenere umido con ... (mezzo appropriato da precisare da parte del fabbricante).
NL: Inhoud vochtig houden met ... (middel aan te geven door de fabrikant).
PT: Manter húmido com ... (material adequado a especificar pelo produtor).
FI: Säilytettävä kosteana ... (valmistaja/maahantuoja ilmoittaa sopivan aineen).
SV: Innehållet skall hållas fuktigt med ... (lämpligt material anges av tillverkaren).
S49
ES: Consérvese únicamente en el recipiente de origen.
DA: Må kun opbevares i den originale emballage.
DE: Nur im Originalbehälter aufbewahren.
EL: Διατηρείται μόνο μέσα στο αρχικό δοχείο.
EN: Keep only in the original container.
FR: Conserver uniquement dans le récipient d'origine.
IT: Conservare soltanto nel recipiente originale.
NL: Uitsluitend in de oorspronkelijke verpakking bewaren.
PT: Conservar unicamente no recipiente de origem.
FI: Säilytettävä vain alkuperäispakkauksessa.
SV: Förvaras endast i originalförpackningen.
S50
ES: No mezclar con ... (a especificar por el fabricante).
DA: Må ikke blandes med ... (angives af fabrikanten).
DE: Nicht mischen mit ... (vom Hersteller anzugeben).
EL: Να μην αναμειχθεί με ... (καθορίζεται από τον παραγωγό).
EN: Do not mix with ... (to be specified by the manufacturer).
FR: Ne pas mélanger avec ... (à spécifier par le fabricant).
IT: Non mescolare con ... (da specificare da parte del fabbricante).
NL: Niet vermengen met ... (aan te geven door de fabrikant).
PT: Não misturar com ... (a especificar pelo produtor).
FI: Ei saa sekoittaa ... (valmistaja/maahantuoja ilmoittaa aineen) kanssa.
SV: Blanda inte med ... (anges av tillverkaren).
S51
ES: Úsese únicamente en lugares bien ventilados.
DA: Må kun bruges på steder med god ventilation.
DE: Nur in gut gelüfteten Bereichen verwenden.
EL: Να χρησιμοποιείται μόνο σε καλά αεριζόμενο χώρο.
EN: Use only in well-ventilated areas.
FR: Utiliser seulement dans des zones bien ventilées.
IT: Usare soltanto in luogo ben ventilato.
NL: Uitsluitend op goed geventileerde plaatsen gebruiken.
PT: Utilizar somente em locais bem ventilados.
FI: Huolehdittava hyvästä ilmanvaihdosta.
SV: Sörj för god ventilation.
S52
ES: No usar sobre grandes superficies en locales habitados.
DA: Bør ikke anvendes til større flader i beboelses- eller opholdsrum.
DE: Nicht großflächig für Wohn- und Aufenthaltsräume zu verwenden.
EL: Δεν συνιστάται η χρήση σε ευρείες επιφάνειες σε εσωτερικούς χώρους.
EN: Not recommended for interior use on large surface areas.
FR: Ne pas utiliser sur de grandes surfaces dans les locaux habités.
IT: Non utilizzare su grandi superfici in locali abitati.
NL: Niet voor gebruik op grote oppervlakken in woon- en verblijfruimten.
PT: Não utilizar em grandes superfícies nos locais habitados.
FI: Ei suositella sisäkäyttöön laajoilla pinnoilla.
SV: Olämpligt för användning inomhus vid behandling av stora ytor.
S53
ES: Evítese la exposición - recábense instrucciones especiales antes del uso.
DA: Undgå enhver kontakt - indhent særlige anvisninger før brug.
DE: Exposition vermeiden - vor Gebrauch besondere Anweisungen einholen.
EL: Αποφεύγετε την έκθεση - εφοδιαστείτε με τις ειδικές οδηγίες πριν από τη χρήση.
EN: Avoid exposure - obtain special instructions before use.
FR: Éviter l'exposition - se procurer des instructions spéciales avant l'utilisation.
IT: Evitare l'esposizione - procurarsi speciali istruzioni prima dell'uso.
NL: Blootstelling vermijden - vóór gebruik speciale aanwijzingen raadplegen.
PT: Evitar a exposição - obter instruções específicas antes da utilização.
FI: Vältettävä altistumista - ohjeet luettava ennen käyttöä.
SV: Undvik exponering - Begär specialinstruktioner före användning.
S56
ES: Elimínense esta sustancia y su recipiente en un punto de recogida pública de residuos especiales o peligrosos.
DA: Aflever dette materiale og dets beholder til et indsamlingssted for farligt affald og problemaffald.
DE: Dieses Produkt und seinen Behälter der Problemabfallentsorgung zuführen.
EL: Το υλικό αυτό και ο περιέκτης του να εναποτεθούν σε χώρο συλλογής επικινδύνων ή ειδικών αποβλήτων.
EN: Dispose of this material and its container to hazardous or special waste collection point.
FR: Éliminer ce produit et son récipient dans un centre de collecte des déchets dangereux ou spéciaux.
IT: Smaltire questo materiale e i relativi contenitori in un punto di raccolta rifiuti pericolosi o speciali.
NL: Deze stof en de verpakking naar inzamelpunt voor gevaarlijk of bijzonder afval brengen.
PT: Eliminar este produto e o seu recipiente, enviando-os para local autorizado para a recolha de resíduos perigosos ou especiais.
FI: Tämä aine ja sen pakkaus on toimitettava ongelmajätteen vastaanottopaikkaan.
SV: Lämna detta material och dess behållare till insamlingsställe för farligt avfall.
S57
ES: Utilícese un envase de seguridad adecuado para evitar la contaminación del medio ambiente.
DA: Skal indesluttes forsvarligt for at undgå miljøforurening.
DE: Zur Vermeidung einer Kontamination der Umwelt geeigneten Behälter verwenden.
EL: Να χρησιμοποιηθεί ο κατάλληλος περιέκτης για να αποφευχθεί μόλυνση του περιβάλλοντος.
EN: Use appropriate container to avoid environmental contamination.
FR: Utiliser un récipient approprié pour éviter toute contamination du milieu ambiant.
IT: Usare contenitori adeguati per evitare l'inquinamento ambientale.
NL: Neem passende maatregelen om verspreiding in het milieu te voorkomen.
PT: Utilizar um recipiente adequado para evitar a contaminação do ambiente.
FI: Käytettävä sopivaa säilytystapaa ympäristön likaantumisen ehkäisemiseksi.
SV: Förvaras på lämpligt sätt för att undvika miljöförorening.
S59
ES: Remitirse al fabricante o proveedor para obtener información sobre su recuperación/reciclado.
DA: Indhent oplysninger om genvinding/genanvendelse hos producenten/leverandøren.
DE: Informationen zur Wiederverwendung/Wiederverwertung beim Hersteller/Lieferanten erfragen.
EL: Ζητήστε πληροφορίες από τον παραγωγό/προμηθευτή για ανάκτηση/ανακύκλωση.
EN: Refer to manufacturer/supplier for information on recovery/recycling.
FR: Consulter le fabricant/fournisseur pour des informations relatives à la récupération/au recyclage.
IT: Richiedere informazioni al produttore/fornitore per il recupero/riciclaggio.
NL: Raadpleeg fabrikant/leverancier voor informatie over terugwinning/recycling.
PT: Solicitar ao produtor/fornecedor informações relativas à sua recuperação/reciclagem.
FI: Hanki valmistajalta/luovuttajalta tietoja uudelleenkäytöstä/kierrätyksestä.
SV: Rådfråga tillverkare/leverantör om återvinning/återanvändning.
S60
ES: Elimínense el producto y su recipiente como residuos peligrosos.
DA: Dette materiale og dets beholder skal bortskaffes som farligt affald.
DE: Dieses Produkt und sein Behälter sind als gefährlicher Abfall zu entsorgen.
EL: Το υλικό και ο περιέκτης του να θεωρηθούν κατά τη διάθεση τους επικίνδυνα απόβλητα.
EN: This material and its container must be disposed of as hazardous waste.
FR: Éliminer le produit et son récipient comme un déchet dangereux.
IT: Questo materiale e il suo contenitore devono essere smaltiti come rifiuti pericolosi.
NL: Deze stof en de verpakking als gevaarlijk afval afvoeren.
PT: Este produto e o seu recipiente devem ser eliminados como resíduos perigosos.
FI: Tämä aine ja sen pakkaus on käsiteltävä ongelmajätteenä.
SV: Detta material och dess behållare skall tas om hand som farligt avfall.
S61
ES: Evítese su liberación al medio ambiente. Recábense instrucciones específicas de la ficha de datos de seguridad.
DA: Undgå udledning til miljøet. Se særlig vejledning/leverandørbrugsanvisning.
DE: Freisetzung in die Umwelt vermeiden. Besondere Anweisungen einholen/Sicherheitsdatenblatt zu Rate ziehen.
EL: Αποφύγετε την ελευθέρωσή του στο περιβάλλον. Αναφερθείτε σε ειδικές οδηγίες/δελτίο δεδομένων ασφαλείας.
EN: Avoid release to the environment. Refer to special instructions/safety data sheets.
FR: Éviter le rejet dans l'environnement. Consulter les instructions spéciales/la fiche de données de sécurité.
IT: Non disperdere nell'ambiente. Riferirsi alle istruzioni speciali/ schede informative in materia di sicurezza.
NL: Voorkom lozing in het milieu. Vraag om speciale instructies/veiligheidskaart.
PT: Evitar a libertação para o ambiente. Obter instruções específicas/fichas de segurança.
FI: Vältettävä päästämistä ympäristöön. Lue erityisohjeet/käyttöturvallisuustiedote.
SV: Undvik utsläpp till miljön. Läs särskilda instruktioner/varuinformationsblad.
S62
ES: En caso de ingestión no provocar el vómito: acúdase inmediatamente al médico y muéstresele la etiqueta o el envase.
DA: Ved indtagelse, undgå at fremprovokere opkastning: kontakt omgående læge og vis denne beholder eller etiket.
DE: Bei Verschlucken kein Erbrechen herbeiführen. Sofort ärztlichen Rat einholen und Verpackung oder dieses Etikett vorzeigen.
EL: Σε περίπτωση κατάποσης να μην προκληθεί εμετός: ζητήστε αμέσως ιατρική συμβουλή και δείξτε αυτό το δοχείο ή την ετικέττα του.
EN: If swallowed, do not induce vomiting: seek medical advice immediately and show this container or label.
FR: En cas d'ingestion, ne pas faire vomir. Consulter immédiatement un médecin et lui montrer l'emballage ou l'étiquette.
IT: In caso di ingestione non provocare il vomito: consultare immediatamente il medico e mostrargli il contenitore o l'etichetta.
NL: Bij inslikken niet het braken opwekken; direct een arts raadplegen en de verpakking of het etiket tonen.
PT: Em caso de ingestão, não provocar o vómito. Consultar imediatamente um médico e mostrar-lhe a embalagem ou o rótulo.
FI: Jos kemikaalia on nielty, ei saa oksennuttaa: hakeuduttava välittömästi lääkärin hoitoon ja näytettävä tämä pakkaus tai etiketti.
SV: Vid förtäring, framkalla ej kräkning. Kontakta genast läkare och visa denna förpackning eller etiketten.
S63
ES: En caso de accidente por inhalación, alejar a la víctima de la zona contaminada y mantenerla en reposo.
DA: Ved ulykkestilfælde ved indånding bringes tilskadekomne ud i frisk luft og holdes i ro.
DE: Bei Unfall durch Einatmen: Verunfallten an die frische Luft bringen und ruhigstellen.
EL: Σε περίπτωση ατυχήματος λόγω εισπονής: απομακρύνετε το θύμα από το μολυσμένο χώρο και αφήστε το να ηρεμήσει.
EN: In case of accident by inhalation: remove casualty to fresh air and keep at rest.
FR: En cas d'accident par inhalation, transporter la victime hors de la zone contaminée et la garder au repos.
IT: In caso di incidente per inalazione, allontanare l'infortunato dalla zona contaminata e mantenerlo a riposo.
NL: Bij een ongeval door inademing: slachtoffer in de frisse lucht brengen en laten rusten.
PT: Em caso de inalação acidental, remover a vítima da zona contaminada e mantê-la em repouso.
FI: Jos ainetta on onnettomuuden sattuessa hengitetty: siirrä henkilö raittiiseen ilmaan ja pidä hänet levossa.
SV: Vid olycksfall via inandning, flytta den drabbade till frisk luft och låt vila.
S64
ES: En caso de ingestión, enjuáguese la boca con agua (solamente si la persona está consciente).
DA: Ved indtagelse, skyl munden med vand (kun hvis personen er ved bevidsthed).
DE: Bei Verschlucken Mund mit Wasser ausspülen (nur wenn Verunfallter bei Bewusstsein ist).
EL: Σε περίπτωση κατάποσης, ξεπλύνετε το στόμα με νερό (μόνο εφόσον το θύμα διατηρεί τις αισθήσεις του).
EN: If swallowed, rinse mouth with water (only if the person is conscious).
FR: En cas d'ingestion, rincer la bouche avec de l'eau (seulement si la personne est consciente).
IT: In caso di ingestione, sciacquare la bocca con acqua (solamente se l'infortunato è cosciente).
NL: Bij inslikken, mond met water spoelen (alleen als de persoon bij bewustzijn is).
PT: Em caso de ingestão, lavar repetidamente a boca com água (apenas se a vítima estiver consciente).
FI: Jos ainetta on nielty, huuhtele suu vedellä (vain jos henkilö on tajuissaan).
SV: Vid förtäring, skölj munnen med vatten (endast om personen är vid medvetande)
Combinación de frases-S
Kombination af S-sætninger
Kombination der S-Sätze
Συνδυασμός των S-φράσεων
Combination of S-phrases
Combinaison des phrases S
Combinazioni delle frasi S
Combinatie van S-zinnen
Combinação das frases S
Yhdistetyt S-lausekkeet
Sammansatta S-fraser
S1/2
ES: Consérvese bajo llave y manténgase fuera del alcance de los niños.
DA: Opbevares under lås og utilgængeligt for børn.
DE: Unter Verschluss und für Kinder unzugänglich aufbewahren.
EL: Φυλάξτε το κλειδωμένο και μακρυά από παιδιά.
EN: Keep locked up and out of the reach of children.
FR: Conserver sous clef et hors de portée des enfants.
IT: Conservare sotto chiave e fuori della portata dei bambini.
NL: Achter slot en buiten bereik van kinderen bewaren.
PT: Guardar fechado à chave e fora do alcance das crianças.
FI: Säilytettävä lukitussa tilassa ja lasten ulottumattomissa.
SV: Förvaras i låst utrymme och oåtkomligt för barn.
S3/7
ES: Consérvese el recipiente bien cerrado y en lugar fresco.
DA: Emballagen opbevares tæt lukket på et køligt sted.
DE: Behälter dicht geschlossen halten und an einem kühlen Ort aufbewahren.
EL: Διατηρείστε το δοχείο ερμητικά κλεισμένο σε δροσερό μέρος.
EN: Keep container tightly closed in a cool place.
FR: Conserver le récipient bien fermé dans un endroit frais.
IT: Tenere il recipiente ben chiuso in luogo fresco.
NL: Gesloten verpakking op een koele plaats bewaren.
PT: Conservar em recipiente bem fechado em lugar fresco.
FI: Säilytettävä tiiviisti suljettuna viileässä paikassa.
SV: Förpackningen förvaras väl tillsluten och svalt.
S3/9/14
ES: Consérvese en lugar fresco y bien ventilado y lejos de ... (materiales incompatibles, a especificar por el fabricante).
DA: Opbevares køligt, godt ventileret og adskilt fra ... (uforligelige stoffer angives af fabrikanten).
DE: An einem kühlen, gut gelüfteten Ort, entfernt von ... aufbewahren (die Stoffe, mit denen Kontakt vermieden werden muss, sind vom Hersteller anzugeben).
EL: Διατηρείται σε δροσερό και καλά αεριζόμενο μέρος μακρυά από ... (ασύμβατα υλικά που υποδεικνύονται από τον παραγωγό).
EN: Keep in a cool, well-ventilated place away from ... (incompatible materials to be indicated by the manufacturer).
FR: Conserver dans un endroit frais et bien ventilé à l'écart des ... (matières incompatibles à indiquer par le fabricant).
IT: Conservare in luogo fresco e ben ventilato lontano da ... (materiali incompatibili da precisare da parte del fabbricante).
NL: Bewaren op een koele, goed geventileerde plaats verwijderd van ... (stoffen waarmee contact vermeden dient te worden, aan te geven door de fabrikant).
PT: Conservar em lugar fresco e bem ventilado ao abrigo de ... (matérias incompatíveis a indicar pelo produtor).
FI: Säilytettävä erillään ... (yhteensopimattomat aineet ilmoittaa valmistaja/maahantuoja) viileässä paikassa, jossa on hyvä ilmanvaihto.
SV: Förvaras svalt, på väl ventilerad plats åtskilt från ... (oförenliga ämnen anges av tillverkaren).
S3/9/14/49
ES: Consérvese únicamente en el recipiente de origen, en lugar fresco y bien ventilado y lejos de ... (materiales incompatibles, a especificar por el fabricante).
DA: Må kun opbevares i originalemballagen på et køligt, godt ventileret sted og adskilt fra ... (uforligelige stoffer angives af fabrikanten).
DE: Nur im Originalbehälter an einem kühlen, gut gelüfteten Ort, entfernt von ... aufbewahren (die Stoffe, mit denen Kontakt vermieden werden muss, sind vom Hersteller anzugeben).
EL: Διατηρείται μόνο μέσα στο αρχικό δοχείο σε δροσερό και καλά αεριζόμενο μέρος μακρυά από ... (ασύμβατα υλικά που υποδεικνύονται από τον παραγωγό).
EN: Keep only in the original container in a cool, well-ventilated place away from ... (incompatible materials to be indicated by the manufacturer).
FR: Conserver uniquement dans le récipient d'origine dans un endroit frais et bien ventilé à l'écart de ... (matières incompatibles à indiquer par le fabricant).
IT: Conservare soltanto nel contenitore originale in luogo fresco e ben ventilato lontano da ... (materiali incompatibili da precisare da parte del fabbricante).
NL: Uitsluitend in de oorspronkelijke verpakking bewaren op een koele, goed geventileerde plaats verwijderd van ... (stoffen waarmee contact vermeden dient te worden, aan te geven door de fabrikant).
PT: Conservar unicamente no recipiente de origem, em lugar fresco e bem ventilado ao abrigo de ... (matérias incompatíveis a indicar pelo produtor).
FI: Säilytettävä alkuperäispakkauksessa viileässä paikassa, jossa on hyvä ilmanvaihto erillään ... (yhteensopimattomat aineet ilmoittaa valmistaja/maahantuoja).
SV: Förvaras endast i originalförpackningen på sval, väl ventilerad plats åtskilt från ... (oförenliga ämnen anges av tillverkaren).
S3/9/49
ES: Consérvese únicamente en el recipiente de origen, en lugar fresco y bien ventilado.
DA: Må kun opbevares i originalemballagen på et køligt, godt ventileret sted.
DE: Nur im Originalbehälter an einem kühlen, gut gelüfteten Ort aufbewahren.
EL: Διατηρείται μόνο μέσα στο αρχικό δοχείο σε δροσερό και καλά αεριζόμενο μέρος.
EN: Keep only in the original container in a cool, well-ventilated place.
FR: Conserver uniquement dans le récipient d'origine dans un endroit frais et bien ventilé.
IT: Conservare soltanto nel contenitore originale in luogo fresco e ben ventilato.
NL: Uitsluitend in de oorspronkelijke verpakking bewaren op een koele, goed geventileerde plaats.
PT: Conservar unicamente no recipiente de origem, em lugar fresco e bem ventilado.
FI: Säilytettävä alkuperäispakkauksessa viileässä paikassa, jossa on hyvä ilmanvaihto.
SV: Förvaras endast i originalförpackningen på sval, väl ventilerad plats.
S3/14
ES: Consérvese en lugar fresco y lejos de ... (materiales incompatibles, a especificar por el fabricante).
DA: Opbevares køligt og adskilt fra ... (uforligelige stoffer angives af fabrikanten).
DE: An einem kühlen, von ... entfernten Ort aufbewahren (die Stoffe, mit denen Kontakt vermieden werden muss, sind vom Hersteller anzugeben).
EL: Διατηρείται σε δροσερό μέρος μακρυά από ... (ασύμβατα υλικά που υποδεικνύονται από τον παραγωγό).
EN: Keep in a cool place away from ... (incompatible materials to be indicated by the manufacturer).
FR: Conserver dans un endroit frais à l'écart des ... (matières incompatibles à indiquer par le fabricant).
IT: Conservare in luogo fresco lontano da ... (materiali incompatibili da precisare da parte del fabbricante).
NL: Bewaren op een koele plaats verwijderd van ... (stoffen waarmee contact vermeden dient te worden, aan te geven door de fabrikant).
PT: Conservar em lugar fresco ao abrigo de ... (matérias incompatíveis a indicar pelo produtor).
FI: Säilytettävä viileässä erillään ... (yhteensopimattomat aineet ilmoittaa valmistaja/maahantuoja).
SV: Förvaras svalt och åtskilt från ... (oförenliga ämnen anges av tillverkaren).
S7/8
ES: Manténgase el recipiente bien cerrado y en lugar seco.
DA: Emballagen skal holdes tæt lukket og opbevares tørt.
DE: Behälter trocken und dicht geschlossen halten.
EL: Το δοχείο να διατηρείται ερμητικά κλεισμένο και να προστατεύεται από την υγρασία.
EN: Keep container tightly closed and dry.
FR: Conserver le récipient bien fermé et à l'abri de l'humidité.
IT: Conservare il recipiente ben chiuso e al riparo dall'umidità.
NL: Droog houden en in een goed gesloten verpakking bewaren.
PT: Conservar o recipiente bem fechado e ao abrigo da humidade.
FI: Säilytettävä kuivana ja tiiviisti suljettuna.
SV: Förpackningen förvaras väl tillsluten och torrt.
S7/9
ES: Manténgase el recipiente bien cerrado y en lugar bien ventilado.
DA: Emballagen skal holdes tæt lukket og opbevares på et godt ventileret sted.
DE: Behälter dicht geschlossen an einem gut gelüfteten Ort aufbewahren.
EL: Το δοχείο να διατηρείται ερμητικά κλεισμένο και σε καλά αεριζόμενο μέρος.
EN: Keep container tightly closed and in a well-ventilated place.
FR: Conserver le récipient bien fermé et dans un endroit bien ventilé.
IT: Tenere il recipiente ben chiuso e in luogo ben ventilato.
NL: Gesloten verpakking op een goed geventileerde plaats bewaren.
PT: Manter o recipiente bem fechado em local bem ventilado.
FI: Säilytettävä tiiviisti suljettuna paikassa, jossa on hyvä ilmanvaihto.
SV: Förpackningen förvaras väl tillsluten på väl ventilerad plats.
S7/47
ES: Manténgase el recipiente bien cerrado y consérvese a una temperatura no superior a ... °C (a especificar por el fabricante).
DA: Emballagen skal holdes tæt lukket og opbevares ved temperaturer på ikke over ... °C (angives af fabrikanten).
DE: Behälter dicht geschlossen und nicht bei Temperaturen über ... °C aufbewahren (vom Hersteller anzugeben).
EL: Διατηρείστε το δοχείο καλά κλεισμένο σε θερμοκρασία που δεν υπερβαίνει τους ... °C (να καθοριστεί από τον παραγωγό).
EN: Keep container tightly closed and at a temperature not exceeding ... °C (to be specified by the manufacturer).
FR: Conserver le récipient bien fermé et à une température ne dépassant pas ... °C (à préciser par le fabricant).
IT: Tenere il recipiente ben chiuso e a temperatura non superiore a ... °C (da precisare da parte del fabbricante).
NL: Gesloten verpakking bewaren bij een temperatuur beneden ... °C (aan te geven door de fabrikant).
PT: Manter o recipiente bem fechado e conservar a uma temperatura que não exceda ... °C (a especificar pelo produtor).
FI: Säilytettävä tiiviisti suljettuna ja alle ... °C:n lämpötilassa (valmistaja/maahantuoja ilmoittaa lämpötilan).
SV: Förpackningen förvaras väl tillsluten vid en temperatur som inte överstiger ... °C (anges av tillverkaren).
S20/21
ES: No comer, ni beber, ni fumar durante su utilización.
DA: Der må ikke spises, drikkes eller ryges under brugen.
DE: Bei der Arbeit nicht essen, trinken oder rauchen.
EL: Όταν το χρησιμοποιείτε μην τρώτε, μην πίνετε, μην καπνίζετε.
EN: When using do not eat, drink or smoke.
FR: Ne pas manger, ne pas boire et ne pas fumer pendant l'utilisation.
IT: Non mangiare, né bere, né fumare durante l'impiego.
NL: Niet eten, drinken of roken tijdens gebruik.
PT: Não comer, beber ou fumar durante a utilização.
FI: Syöminen, juominen ja tupakointi kielletty kemikaalia käytettäessä.
SV: Ät inte, drick inte eller rök inte under hanteringen.
S24/25
ES: Evítese el contacto con los ojos y la piel.
DA: Undgå kontakt med huden og øjnene.
DE: Berührung mit den Augen und der Haut vermeiden.
EL: Αποφεύγετε επαφή με το δέρμα και τα μάτια.
EN: Avoid contact with skin and eyes.
FR: Éviter le contact avec la peau et les yeux.
IT: Evitare il contatto con gli occhi e con la pelle.
NL: Aanraking met de ogen en de huid vermijden.
PT: Evitar o contacto com a pele e os olhos.
FI: Varottava kemikaalin joutumista iholle ja silmiin.
SV: Undvik kontakt med huden och ögonen.
S27/28
ES: Después del contacto con la piel, quítese inmediatamente toda la ropa manchada o salpicada y lávese inmediata y abundantemente con ... (productos a especificar por el fabricante).
DA: Kommer stof på huden, tages tilsmudset tøj straks af og der vaskes med store mængder ... (angives af fabrikanten).
DE: Bei Berührung mit der Haut beschmutzte, getränkte Kleidung sofort ausziehen und Haut sofort abwaschen mit viel ... (vom Hersteller anzugeben).
EL: Σε περίπτωση επαφής με το δέρμα, αφαιρέστε αμέσως όλα τα μολυσμένα ρούχα και πλύνετε αμέσως με άφθονο ... (το είδος του υγρού καθορίζεται από τον παραγωγό).
EN: After contact with skin, take off immediately all contaminated clothing, and wash immediately with plenty of ... (to be specified by the manufacturer).
FR: Après contact avec la peau, enlever immédiatement tout vêtement souillé ou éclaboussé et se laver immédiatement et abondamment avec ... (produits appropriés à indiquer par le fabricant).
IT: In caso di contatto con la pelle, togliersi di dosso immediatamente gli indumenti contaminati e lavarsi immediatamente e abbondantemente con ... (prodotti idonei da indicarsi da parte del fabbricante).
NL: Na contact met de huid, alle besmette kleding onmiddellijk uittrekken en de huid onmiddellijk wassen met veel ... (aan te geven door de fabrikant).
PT: Em caso de contacto com a pele, retirar imediatamente toda a roupa contaminada e lavar imediata e abundantemente com ... (produto adequado a indicar pelo produtor).
FI: Ihokosketuksen jälkeen, saastunut vaatetus on riisuttava välittömästi ja roiskeet huuhdeltava välittömästi runsaalla määrällä ... (aineen ilmoittaa valmistaja/maahantuoja).
SV: Vid kontakt med huden, tag genast av alla nedstänkta kläder och tvätta genast med mycket ... (anges av tillverkaren).
S29/35
ES: No tirar los residuos por el desagüe; elimínense los residuos del producto y sus recipientes con todas las precauciones posibles.
DA: Må ikke tømmes i kloakafløb; materialet og dets beholder skal bortskaffes på en sikker måde.
DE: Nicht in die Kanalisation gelangen lassen; Abfälle und Behälter müssen in gesicherter Weise beseitigt werden.
EL: Μην αδειάζετε το υπόλοιπο τουπεριεχομένου στην αποχέτευση, διαθέστε αυτό το υλικό και τον περιέκτη του κατά ασφαλή τρόπο.
EN: Do not empty into drains; dispose of this material and its container in a safe way.
FR: Ne pas jeter les résidus à l'égout; ne se débarrasser de ce produit et de son récipient qu'en prenant toutes les précautions d'usage.
IT: Non gettare i residui nelle fognature; non disfarsi del prodotto e del recipiente se non con le dovute precauzioni.
NL: Afval niet in de gootsteen werpen; stof en verpakking op veilige wijze afvoeren.
PT: Não deitar os resíduos no esgoto; não eliminar o produto e o seu recipiente sem tomar as precauções de segurança devidas.
FI: Ei saa tyhjentää viemäriin; tämä aine ja sen pakkaus on hävitettävä turvallisesti.
SV: Töm ej i avloppet, oskadliggör produkt och förpackning på säkert sätt.
S29/56
ES: No tirar los residuos por el desagüe; elimínese esta sustancia y su recipiente en un punto de recogida pública de residuos especiales o peligrosos.
DA: Må ikke tømmes i kloakafløb, aflever dette materiale og dets beholder til et indsamlingssted for farligt affald og problemaffald.
DE: Nicht in die Kanalisation gelangen lassen; dieses Produkt und seinen Behälter der Problemabfallentsorgung zuführen.
EL: Μην αδειάζετε το υπόλοιπο του περιεχομένου στην αποχέτευση. Το υλικό αυτό και ο περιέκτης του να εναποτεθούν σε δημόσιο χώρο συλλογής επικινδύνων ή ειδικών αποβλήτων.
EN: Do not empty into drains, dispose of this material and its container at hazardous or special waste collection point.
FR: Ne pas jeter les résidus à l'égout, éliminer ce produit et son récipient dans un centre de collecte des déchets dangereux ou spéciaux.
IT: Non gettare i residui nelle fognature; smaltire questo materiale e i relativi contenitori in un punto di raccolta rifiuti pericolosi o speciali.
NL: Afval niet in de gootsteen werpen; deze stof en de verpakking naar een inzamelpunt voor gevaarlijk of bijzonder afval brengen.
PT: Não deitar os resíduos no esgoto; eliminar este produto e o seu recipiente enviando-os para local autorizado para a recolha de resíduos perigosos ou especiais.
FI: Ei saa tyhjentää viemäriin; tämä aine ja sen pakkaus on toimitettava ongelmajätteen vastaanottopaikkaan.
SV: Töm ej i avloppet, lämna detta material och dess behållare till insamlingsställe för farligt avfall.
S36/37
ES: Úsense indumentaria y guantes de protección adecuados.
DA: Brug særligt arbejdstøj og egnede beskyttelseshandsker.
DE: Bei der Arbeit geeignete Schutzhandschuhe und Schutzkleidung tragen.
EL: Φοράτε κατάλληλη προστατευτική ενδυμασία και γάντια.
EN: Wear suitable protective clothing and gloves.
FR: Porter un vêtement de protection et des gants appropriés.
IT: Usare indumenti protettivi e guanti adatti.
NL: Draag geschikte handschoenen en beschermende kleding.
PT: Usar vestuário de protecção e luvas adequadas.
FI: Käytettävä sopivaa suojavaatetusta ja suojakäsineitä.
SV: Använd lämpliga skyddskläder och skyddshandskar.
S36/37/39
ES: Úsense indumentaria y guantes adecuados y protección para los ojos/la cara.
DA: Brug særligt arbejdstøj, egnede beskyttelseshandsker og -briller/ansigtsskærm.
DE: Bei der Arbeit geeignete Schutzkleidung, Schutzhandschuhe und Schutzbrille/Gesichtsschutz tragen.
EL: Φοράτε κατάλληλη προστατευτική ενδυμασία, γάντια και συσκευή προστασίας ματιών/προσώπου.
EN: Wear suitable protective clothing, gloves and eye/face protection.
FR: Porter un vêtement de protection approprié, des gants et un appareil de protection des yeux/du visage.
IT: Usare indumenti protettivi e guanti adatti e proteggersi gli occhi/la faccia.
NL: Draag geschikte beschermende kleding, handschoenen en een beschermingsmiddel voor de ogen/het gezicht.
PT: Usar vestuário de protecção, luvas e equipamento protector para os olhos/face adequados.
FI: Käytettävä sopivaa suojavaatetusta, suojakäsineitä ja silmien- tai kasvonsuojainta.
SV: Använd lämpliga skyddskläder, skyddshandskar samt skyddsglasögon eller ansiktsskydd.
S36/39
ES: Úsense indumentaria adecuada y protección para los ojos/la cara.
DA: Brug særligt arbejdstøj og egnede beskyttelsesbriller/ansigtsskærm.
DE: Bei der Arbeit geeignete Schutzkleidung und Schutzbrille/Gesichtsschutz tragen.
EL: Φοράτε κατάλληλη προστατευτική ενδυμασία και συσκευή προστασίας ματιών/προσώπου.
EN: Wear suitable protective clothing and eye/face protection.
FR: Porter un vêtement de protection approprié et un appareil de protection des yeux/du visage.
IT: Usare indumenti protettivi adatti e proteggersi gli occhi/la faccia.
NL: Draag geschikte beschermende kleding en een beschermingsmiddel voor de ogen/het gezicht.
PT: Usar vestuário de protecção e equipamento protector para os olhos/face adequados.
FI: Käytettävä sopivaa suojavaatetusta ja silmien- tai kasvonsuojainta.
SV: Använd lämpliga skyddskläder samt skyddsglasögon eller ansiktsskydd.
S37/39
ES: Úsense guantes adecuados y protección para los ojos/la cara.
DA: Brug egnede beskyttelseshandsker og -briller/ansigtsskærm under arbejdet.
DE: Bei der Arbeit geeignete Schutzhandschuhe und Schutzbrille/Gesichtsschutz tragen.
EL: Φοράτε κατάλληλα γάντια και συσκευή προστασίας ματιών/προσώπου.
EN: Wear suitable gloves and eye/face protection.
FR: Porter des gants appropriés et un appareil de protection des yeux/du visage.
IT: Usare guanti adatti e proteggersi gli occhi/la faccia.
NL: Draag geschikte handschoenen en een beschermingsmiddel voor de ogen/het gezicht.
PT: Usar luvas e equipamento protector para os olhos/face adequados.
FI: Käytettävä sopivia suojakäsineitä ja silmien- tai kasvonsuojainta.
SV: Använd lämpliga skyddshandskar samt skyddsglasögon eller ansiktsskydd.
S47/49
ES: Consérvese únicamente en el recipiente de origen y a temperatura no superior a ... °C (a especificar por el fabricante).
DA: Må kun opbevares i originalemballagen ved en temperatur på ikke over ... °C (angives af fabrikanten).
DE: Nur im Originalbehälter bei einer Temperatur von nicht über ... °C (vom Hersteller anzugeben) aufbewahren.
EL: Διατηρείται μόνο μέσα στο αρχικό δοχείο σε θερμοκρασία που δέν υπερβαίνει τους ... °C (καθορίζεται από τον παραγωγό).
EN: Keep only in the original container at a temperature not exceeding ... °C (to be specified by the manufacturer).
FR: Conserver uniquement dans le récipient d'origine à une température ne dépassant pas ... °C (à préciser par le fabricant).
IT: Conservare soltanto nel contenitore originale a temperatura non superiore a ... °C (da precisare da parte del fabbricante).
NL: Uitsluitend in de oorspronkelijke verpakking bewaren bij een temperatuur beneden ... °C (aan te geven door de fabrikant).
PT: Conservar unicamente no recipiente de origem a temperatura que não exceda ... °C (a especificar pelo produtor).
FI: Säilytettävä alkuperäispakkauksessa alle ... °C:n lämpötilassa (valmistaja/maahantuoja ilmoittaa lämpötilan).
SV: Förvaras endast i originalförpackningen vid en temperatur som inte överstiger ... °C (anges av tillverkaren).
ANNEX 5A
EN: B.13/14. Mutagenicity - reverse mutation test using bacteria.
(Does not concern the ES version)
(Does not concern the DA version)
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ANNEX 5B
FR: L'administration du témoin positif par une voie différente de celle utilisée pour la substance d'essai est acceptable.
(Does not concern the ES version)
(Does not concern the DA version)
(Does not concern the DE version)
(Does not concern the EL version)
(Does not concern the EN version)
(Does not concern the IT version)
(Does not concern the NL version)
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ANNEX 5C
EN:
>REFERENCE TO A GRAPHIC>
(Does not concern the ES version)
(Does not concern the DA version)
(Does not concern the DE version)
(Does not concern the EL version)
(Does not concern the FR version)
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ANNEX 5D
B.26. SUBCHRONIC ORAL TOXICITY TEST
REPEATED DOSE 90-DAY ORAL TOXICITY STUDY IN RODENTS
1. METHOD
This subchronic oral toxicity test method is a replicate of the OECD TG 408 (1998).
1.1. INTRODUCTION
In the assessment and evaluation of the toxic characteristics of a chemical, the determination of subchronic oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained from acute or repeated dose 28-day toxicity tests. The 90-day study provides information on the possible health hazards likely to arise from repeated exposure over a prolonged period of time covering post-weaning maturation and growth well into adulthood. The study will provide information on the major toxic effects, indicate target organs and the possibility of accumulation, and can provide an estimate of a no observed adverse effect level of exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.
The method places additional emphasis on neurological endpoints and gives an indication of immunological and reproductive effects. The need for careful clinical observations of the animals, so as to obtain as much information as possible, is also stressed. This study should allow for the identification of chemicals with the potential to cause neurotoxic, immunological or reproductive organ effects, which may warrant further in-depth investigation.
See also General Introduction Part B.
1.2. DEFINITIONS
Dose: is the amount of test substance administered. Dose is expressed as weight (g, mg) or as weight of test substance per unit weight of test animal (e.g. mg/kg), or as constant dietary concentrations (ppm).
Dosage: is a general term comprising of dose, its frequency and the duration of dosing.
NOAEL: is the abbreviation for no observed adverse effect level and is the highest dose level where no adverse treatment-related findings are observed.
1.3. PRINCIPLE OF THE TEST METHOD
The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 90 days. During the period of administration the animals are observed closely for signs of toxicity. Animals which die or are killed during the test are necropsied and, at the conclusion of the test, surviving animals are also killed and necropsied.
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations of animals
Healthy animals, which have been acclimated to laboratory conditions for at least five days and have not been subjected to previous experimental procedures, should be used. The test animals should be characterised as to species, strain, source, sex, weight and/or age. Animals should be randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. Each animal should be assigned a unique identification number.
1.4.2. Preparations of doses
The test substance is administered by gavage or via the diet or drinking water. The method of oral administration is dependent on the purpose of the study, and the physical/chemical properties of the test material.
Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance under the conditions of administration should be determined.
1.4.3. Test conditions
1.4.3.1. Experimental animals
The preferred species is the rat, although other rodent species, e.g. the mouse, may be used. Commonly used laboratory strains of young healthy adult animals should be employed. The females should be nulliparous and non-pregnant. Dosing should begin as soon as possible after weaning and, in any case, before the animals are nine weeks old. At the commencement of the study the weight variation of animals used should be minimal and not exceed ± 20 % of the mean weight of each sex. Where the study is conducted as a preliminary to a long-term chronic toxicity study, animals from the same strain and source should be used in both studies.
1.4.3.2. Number and sex
At least 20 animals (10 female and 10 male) should be used at each dose level. If interim kills are planned, the number should be increased by the number of animals scheduled to be killed before the completion of the study. Based on previous knowledge of the chemical or a close analogue, consideration should be given to including an additional satellite group of 10 animals (five per sex) in the control and in the top dose group for observation, after the treatment period, of reversibility or persistence of any toxic effects. The duration of this post-treatment period should be fixed appropriately with regard to the effects observed.
1.4.3.3. Dose levels
At least three dose levels and a concurrent control shall be used, except where a limit test is conducted (see 1.4.3.4). Dose levels may be based on the results of repeated dose or range finding studies and should take into account any existing toxicological and toxicokinetic data available for the test substance or related materials. Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no observed adverse effect level (NOAEL) at the lowest dose level. Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6-10) between dosages.
The control group shall be an untreated group or a vehicle-control group if a vehicle is used in administering the test substance. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to those in the test groups. If a vehicle is used, the control group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet, and causes reduced dietary intake, then a pair-fed control group may be useful in distinguishing between reductions due to palatability or toxicological alterations in the test model.
Consideration should be given to the following characteristics of the vehicle and other additives, as appropriate: effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.
1.4.3.4. Limit test
If a test at one dose level, equivalent to at least 1000 mg/kg body weight/day, using the procedures described for this study, produces no observed adverse effects and if toxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.
1.5. PROCEDURE
1.5.1. Administration of doses
The animals are dosed with the test substance daily seven days each week for a period of 90 days. Any other dosing regime, e.g. five days per week, needs to be justified. When the test substance is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100 g body weight, except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.
For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animal's body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a 90-day study is used as a preliminary to a long-term chronic toxicity study, a similar diet should be used in both studies.
1.5.2. Observations
The observation period should be at least 90 days. Animals in a satellite group scheduled for follow-up observations should be kept for an appropriate period without treatment to detect persistence of, or recovery from toxic effects.
General clinical observations should be made at least once a day, preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animals should be recorded. At least twice daily, usually at the beginning and end of each day, all animals are inspected for signs of morbidity and mortality.
At least once prior to the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage, preferably in a standard arena and at similar times on each occasion. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the observation conditions are minimal. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) should also be recorded (1).
Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study, preferably in all animals but at least in the high dose and control groups. If changes in the eyes are detected all animals should be examined.
Towards the end of the exposure period and in any case not earlier than in week 11, sensory reactivity to stimuli of different types (1) (e.g. auditory, visual and proprioceptive stimuli) (2), (3), (4), assessment of grip strength (5) and motor activity assessment (6) should be conducted. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used.
Functional observations conducted towards the end of the study may be omitted when data on functional observations are available from other studies and the daily clinical observations did not reveal any functional deficits.
Exceptionally, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.
1.5.2.1. Body weight and food/water consumption
All animals should be weighed at least once a week. Measurements of food consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly. Water consumption may also be considered for dietary or gavage studies during which drinking activity may be altered.
1.5.2.2. Haematology and clinical biochemistry
Blood samples should be taken from a named site and stored, if applicable, under appropriate conditions. At the end of the test period, samples are collected just prior to or as part of the procedure for killing the animals.
The following haematological examinations should be made at the end of the test period and when any interim blood samples may have been collected: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count and a measure of blood clotting time/potential.
Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from each animal just prior to or as part of the procedure for killing the animals (apart from those found moribund and/or intercurrently killed). In a similar manner to haematological investigations, interim sampling for clinical biochemical tests may be performed. Overnight fasting of the animals prior to blood sampling is recommended(1). Determinations in plasma or serum should include sodium, potassium, glucose, total cholesterol, urea, blood urea nitrogen, creatinine, total protein and albumin, and more than two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, and sorbitol dehydrogenase). Measurements of additional enzymes (of hepatic or other origin) and bile acids, which may provide useful information under certain circumstances, may also be included.
Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.
In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphorus, fasting triglycerides, specific hormones, methaemoglobin and cholinesterase. These need to be identified for chemicals in certain classes or on a case-by-case basis.
Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect from a given substance.
If historical baseline data are inadequate, consideration should be given as to whether haematological and clinical biochemistry variables need to be determined before dosing commences; it is generally not recommended that this data be generated before treatment (7).
1.5.2.3. Gross necropsy
All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen, brain and heart of all animals (apart from those found moribund and/or intercurrently killed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.
The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and medulla/pons), spinal cord (at three levels: cervical, mid-thoracic and lumbar), pituitary, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines (including Peyer's patches), liver, pancreas, kidneys, adrenals, spleen, heart, trachea and lungs (preserved by inflation with fixative and then immersion), aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary bladder, gall bladder (mouse), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, a section of bone marrow (and/or a fresh bone marrow aspirate), skin and eyes (if changes were observed during ophthalmological examinations). The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.
1.5.2.4. Histopathology
Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups. These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.
All gross lesions should be examined.
When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.
2. DATA AND REPORTING
2.1. DATA
Individual data should be provided. Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the time of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion.
When applicable, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods and the data to be analysed should be selected during the design of the study.
2.2. TEST REPORT
The test report must include the following information:
2.2.1. Test substance:
- physical nature, purity and physico-chemical properties,
- identification data,
- vehicle (if appropriate): justification for choice of vehicle, if other than water.
2.2.2. Test species:
- species and strain used,
- number, age and sex of animals,
- source, housing conditions, diet etc.,
- individual weights of animals at the start of the test.
2.2.3. Test conditions:
- rationale for dose level selection,
- details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation,
- details of the administration of the test substance,
- actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water test substance concentration (ppm) to the actual dose, if applicable,
- details of food and water quality.
2.2.4. Results:
- body weight and body weight changes,
- food consumption, and water consumption, if applicable,
- toxic response data by sex and dose level, including signs of toxicity,
- nature, severity and duration of clinical observations (whether reversible or not),
- results of ophthalmological examination,
- sensory activity, grip strength and motor activity assessments (when available),
- haematological tests with relevant base-line values,
- clinical biochemistry tests with relevant base-line values,
- terminal body weight, organ weights and organ/body weight ratios,
- necropsy findings,
- a detailed description of all histopathological findings,
- absorption data if available,
- statistical treatment of results, where appropriate;
Discussion of results.
Conclusions.
3. REFERENCES
(1) IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60.
(2) Tupper, D. E., Wallace, R.B. (1980). Utility of the Neurologic Examination in Rats. Acta Neurobiol. Exp., 40, pp. 999-1003.
(3) Gad, S. C. (1982). A Neuromuscular Screen for Use in Industrial Toxicology. J. Toxicol. Environ. Health, 9, pp. 691-704.
(4) Moser, V. C., Mc Daniel, K. M., Phillips, P. M. (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol., 108, pp. 267-283.
(5) Meyer O. A., Tilson H. A., Byrd W. C., Riley M. T. (1979). A Method for the Routine Assessment of Fore- and Hind- limb grip Strength of Rats and Mice. Neurobehav. Toxivol., 1, pp. 233-236.
(6) Crofton K. M., Howard J. L., Moser V. C., Gill M. W., Reiter L. W., Tilson H. A., MacPhail R. C. (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol., 13, pp. 599-609.
(7) Weingand K., Brown G., Hall R. et al. (1996). "Harmonisation of Animal Clinic Pathology Testing in Toxicity and Safety Studies", Fundam. & Appl. Toxicol., 29, pp. 198-201.
(1) For a number of measurements in serum and plasma, most notably for glucose, overnight fasting would be preferable. The major reason for this preference is that the increased variability which would inevitably result from non-fasting, would tend to mask more subtle effects and make interpretation difficult. On the other hand, however, overnight fasting may interfere with the general metabolism of the animals and, particularly in feeding studies, may disturb the daily exposure to the test substance. If overnight fasting is adopted, clinical biochemical determinations should be performed after the conduct of functional observations of the study.
ANNEX 5E
B.27. SUBCHRONIC ORAL TOXICITY TEST
REPEATED DOSE 90-DAY ORAL TOXICITY STUDY IN NON-RODENTS
1. METHOD
This subchronic oral toxicity test method is a replicate of the OECD TG 409 (1998).
1.1. INTRODUCTION
In the assessment and evaluation of the toxic characteristics of a chemical, the determination of subchronic oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained from acute or repeated dose 28-day toxicity tests. The 90-day study provides information on the possible health hazards likely to arise from repeated exposure over a period of rapid growth and into young adulthood. The study will provide information on the major toxic effects, indicate target organs and the possibility of accumulation, and can provide an estimate of a no observed adverse effect level of exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.
The test method allows for the identification in non-rodent species of adverse effects of chemical exposure and should only be used:
- where effects observed in other studies indicate a need for clarification/characterisation in a second, non-rodent species, or
- where toxicokinetic studies indicate that the use of a specific non-rodent species is the most relevant choice of laboratory animal, or
- where other specific reasons justify the use of a non-rodent species.
See also General Introduction Part B.
1.2. DEFINITIONS
Dose: is the amount of test substance administered. Dose is expressed as weight (g, mg) or as weight of test substance per unit weight of test animal (e.g., mg/kg), or as constant dietary concentrations (ppm).
Dosage: is a general term comprising of dose, its frequency and the duration of dosing.
NOAEL: is the abbreviation for no observed adverse effect level and is the highest dose level where no adverse treatment-related findings are observed.
1.3. PRINCIPLE OF THE TEST METHOD
The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 90 days. During the period of administration the animals are observed closely for signs of toxicity. Animals which die or are killed during the test are necropsied and at the conclusion of the test surviving animals are also killed and necropsied.
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Selection of animal species
The commonly used non-rodent species is the dog, which should be of a defined breed; the beagle is frequently used. Other species, e.g. swine, mini-pigs, may also be used. Primates are not recommended and their use should be justified. Young, healthy animals should be employed, and in the case of the dog, dosing should begin preferably at 4-6 months and not later than nine months of age. Where the study is conducted as a preliminary to a long-term chronic toxicity study, the same species/breed should be used in both studies.
1.4.2. Preparation of animals
Healthy young animals, which have been acclimated to laboratory conditions and have not been subjected to previous experimental procedures, should be used. The duration of acclimatisation will depend upon the selected test species and their source. At least five days for dogs or purpose bred swine from a resident colony and at least two weeks for these animals if from external sources are recommended. The test animals should be characterised as to species, strain, source, sex, weight and/or age. Animals should be randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. Each animal should be assigned a unique identification number.
1.4.3. Preparations of doses
The test substance may be administered in the diet or in the drinking water, by gavage or in capsules. The method of oral administration is dependent on the purpose of the study, and the physical-chemical properties of the test material.
Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance under the conditions of administration should be determined.
1.5. PROCEDURE
1.5.1. Number and sex of animals
At least eight animals (four female and four male) should be used at each dose level. If interim kills are planned, the number should be increased by the number of animals scheduled to be killed before the completion of the study. The number of animals at the termination of the study must be adequate for a meaningful evaluation of toxic effects. Based on previous knowledge of the substance or a close analogue, consideration should be given to including an additional satellite group of eight animals (four per sex) in control and in top dose group for observation after the treatment period of reversibility or persistence of any toxic effects. The duration of this post-treatment period should be fixed appropriately with regard to the effects observed.
1.5.2. Dosage
At least three dose levels and a concurrent control shall be used, except where a limit test is conducted (see 1.5.3). Dose levels may be based on the results of repeated dose or range finding studies and should take into account any existing toxicological and toxicokinetic data available for the test compound or related materials. Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no observed adverse effect level (NOAEL) at the lowest dose level. Two- to fourfold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of about 6-10) between dosages.
The control group shall be an untreated group or a vehicle-control group if a vehicle is used in administering the test substance. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to those in the test groups. If a vehicle is used, the control group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet, and causes reduced dietary intake, then a pair-fed control group may be useful in distinguishing between reductions due to palatability or toxicological alterations in the test model.
Consideration should be given to the following characteristics of the vehicle and other additives, as appropriate: effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.
1.5.3. Limit test
If a test at one dose level, equivalent to at least 1000 mg/kg body weight/day, using the procedures described for this study, produces no observed adverse effects and if toxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.
1.5.4. Administration of doses
The animals are dosed with the test substance daily seven days each week for a period of 90 days. Any other dosing regime, e.g. five days per week, needs to be justified. When the test substance is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. Normally the volume should be kept as low as possible. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.
For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animal's body weight may be used; any alternative used must be specified. For a substance administered by gavage or by capsule, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where the 90-day study is used as a preliminary to a long term chronic toxicity study, a similar diet should be used in both studies.
1.5.5. Observations
The observation period should be at least 90 days. Animals in a satellite group scheduled for follow-up observations should be kept for an appropriate period without treatment to detect persistence of, or recovery from toxic effects.
General clinical observations should be made at least once a day, preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animals should be recorded. At least twice daily, usually at the beginning and end of each day, all animals should be inspected for signs of morbidity and mortality.
At least once prior to the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made, where practical outside the home cage in a standard arena and preferably at similar times on each occasion. Effort should be made to ensure that variations in the observation conditions are minimal. Signs of toxicity should be carefully recorded, including time of onset, degree and duration. Observations should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or any bizarre behaviour should also be recorded.
Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study, preferably in all animals but at least in the high dose and control groups. If treatment related changes in the eyes are detected all animals should be examined.
1.5.5.1. Body weight and food/water consumption
All animals should be weighed at least once a week. Measurements of food consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly. Water consumption may also be considered for dietary or gavage studies during which drinking activity may be altered.
1.5.5.2. Haematology and clinical biochemistry
Blood samples should be taken from a named site and stored, if applicable, under appropriate conditions. At the end of the test period, samples are collected just prior to or as part of the procedure for killing the animals.
Haematology, including haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count and a measure of clotting potential such as clotting time, prothrombin time, or thromboplastin time should be investigated at the start of the study, then either at monthly intervals or midway through the test period and finally at the end of the test period.
Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from all animals at the start, then either at monthly intervals or midway through the test and finally at the end of the test period. Test areas which should be considered are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the test substance. Animals should be fasted for a period appropriate to the species prior to blood sampling. Suggested determinations include calcium, phosphorus, chloride, sodium, potassium, fasting glucose, alanine aminotransferase, aspartate aminotransferase, ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements.
Urinalysis determinations should be performed at least at the start, then midway and finally at the end of the study using timed urine volume collection. Urinalysis determinations include appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells. Additional parameters may be employed where necessary to extend the investigation of observed effect(s).
In addition, studies to investigate markers of general tissue damage should be considered. Other determinations which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methaemoglobin, and cholinesterase inhibition. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects. These need to be identified for chemicals in certain classes or on a case-by-case basis.
Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect from a given substance.
1.5.5.3. Gross necropsy
All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver with gall bladder, kidneys, adrenals, testes, epididymides, ovaries, uterus, thyroid (with parathyroids), thymus, spleen, brain and heart of all animals (apart from those found moribund and/or inter-currently killed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.
The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and medulla/pons), spinal cord (at three levels: cervical, mid-thoracic and lumbar), pituitary, eyes, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines (including Peyer's patches), liver, gall bladder, pancreas, kidneys, adrenals, spleen, heart, trachea and lungs, aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary bladder, lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, a section of bone marrow (and/or a fresh bone marrow aspirate) and skin. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.
1.5.5.4. Histopathology
Full histopathology should be carried out on the preserved organs and tissues in at least all animals in control and high dose group. The examination should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.
All gross lesions should be examined.
When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.
2. DATA AND REPORTING
2.1. DATA
Individual data should be provided. Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the time of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion.
When applicable, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods and the data to be analysed should be selected during the design of the study.
2.2. TEST REPORT
The test report must include the following information:
2.2.1. Test substance:
- physical nature, purity and physico-chemical properties,
- identification data.
- vehicle (if appropriate): justification for choice of vehicle, if other than water.
2.2.2. Test species:
- species and strain used,
- number, age and sex of animals,
- source, housing conditions, diet etc.,
- individual weights of animals at the start of the test.
2.2.3. Test conditions:
- rationale for dose level selection,
- details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation,
- details of the administration of the test substance,
- actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water test substance concentration (ppm) to the actual dose, if applicable,
- details of food and water quality.
2.2.4. Results
- body weight/body weight changes,
- food consumption, and water consumption, if applicable,
- toxic response data by sex and dose level, including signs of toxicity,
- nature, severity and duration of clinical observations (whether reversible or not),
- ophthalmological examination,
- haematological tests with relevant base-line values,
- clinical biochemistry tests with relevant base-line values,
- terminal body weight, organ weights and organ/body weight ratios,
- necropsy findings,
- a detailed description of all histopathological findings,
- statistical treatment of results, where appropriate.
Discussion of results.
Conclusions.
ANNEX 5F
C.14. FISH JUVENILE GROWTH TEST
1. METHOD
This growth toxicity test method is a replicate of the OECD TG 215 (2000).
1.1. INTRODUCTION
This test is designed to assess the effects of prolonged exposure to chemicals on the growth of juvenile fish. It is based on a method, developed and ring-tested (1) (3) within the European Union, for assessing the effects of chemicals on the growth of juvenile rainbow trout (Oncorynchus mykiss) under flow-through conditions. Other well documented species may be used. For example, experience has been gained from growth tests with zebrafish (Danio rerio) (2) (4) (5) and ricefish (medaka, Oryzias latipes) (6) (7) (8).
See also General Introduction Part C.
1.2. DEFINITIONS
Lowest observed effect concentration (LOEC): is the lowest tested concentration of a test substance at which the substance is observed to have a significant effect (at p < 0,05) when compared with the control. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC.
No observed effect concentration (NOEC): is the test concentration immediately below the LOEC.
ECx: in this test method is the concentration of the test substance which causes a x % variation in growth rate of the fish when compared with controls.
Loading rate: is the wet weight of fish per volume of water.
Stocking density: is the number of fish per volume of water.
Individual fish specific growth rate: expresses the growth rate of one individual based on its initial weight.
Tank-average specific growth rate: expresses the mean growth rate of a tank population at one concentration.
Pseudo specific growth rate: expresses the individual growth rate compared to the mean initial weight of the tank population.
1.3. PRINCIPLE OF THE TEST METHOD
Juvenile fish in exponential growth phase are placed, after being weighted, in test chambers and are exposed to a range of sublethal concentrations of the test substance dissolved in water preferably under flow-through, or, if not possible, under appropriate semi-static (static-renewal) conditions. The test duration is 28 days. Fish are fed daily. The food ration is based on initial fish weights and may be recalculated after 14 days. At the end of the test, the fish are weighed again. Effects on growth rates are analysed using a regression model in order to estimate the concentration that would cause a x % variation in growth rate, i.e. ECx (e.g. EC10, EC20, or EC30). Alternatively, the data may be compared with control values in order to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC).
1.4. INFORMATION ON THE TEST SUBSTANCE
Results of an acute toxicity test (see test method C.1) preferably performed with the species chosen for this test, should be available. This implies that the water solubility and the vapour pressure of the test substance are known and a reliable analytical method is available for the quantification of the substance in the test solutions with known and reported accuracy and limit of detection is available.
Useful information includes the structural formula, purity of the substance, stability in water and light, pKa, Pow and results of a test for ready biodegradability (see test method C.4).
1.5. VALIDITY OF THE TEST
For the test to be valid the following conditions apply:
- the mortality in the control(s) must not exceed 10 % at the end of the test,
- the mean weight of fish in the control(s) must have increased enough to permit the detection of the minimum variation of growth rate considered as significant. A ring-test (3) has shown that for rainbow trout the mean weight of fish in the controls must have increased by at least the half (i.e. 50 %) of their mean initial weight over 28 days; e.g. initial weight: 1 g/fish (= 100 %), final weight after 28 days: >= 1,5 g/fish (>= 150 %),
- the dissolved oxygen concentration must have been at least 60 % of the air saturation value (ASV) throughout the test,
- the water temperature must not differ by more than ± 1 °C between test chambers at any one time during the test and should be maintained within a range of 2 °C within the temperature ranges specified for the test species (Appendix 1).
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
Normal laboratory equipment and especially the following:
(a) oxygen and pH meters;
(b) equipment for determination of water hardness and alkalinity;
(c) adequate apparatus for temperature control and preferably continuous monitoring;
(d) tanks made of chemically inert material and of suitable capacity in relation to the recommended loading and stocking density (see section 1.8.5 and Appendix 1);
(e) suitably accurate balance (i.e. accurate to ± 0,5 %).
1.6.2. Water
Any water in which the test species shows suitable long-term survival and growth may be used as a test water. It should be of constant quality during the period of the test. The pH of the water should be within the range 6,5 to 8,5, but during a given test it should be within a range of ± 0,5 pH units. Hardness above 140 mg/l (as CaCO3) is recommended. In order to ensure that the dilution water will not unduly influence the test result (for example by complexion of test substance), samples should be taken at intervals for analysis. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd and Ni), major anions and cations (e.g. Ca, Mg, Na, K, Cl and SO4), pesticides (e.g. total organophosphorus and total organochlorine pesticides), total organic carbon and suspended solids should be made, for example, every three months where a dilution water is known to be relatively constant in quality. If water quality has been demonstrated to be constant over at least one year, determinations can be less frequent and intervals extended (e.g. every six months). Some chemical characteristics of an acceptable dilution water are listed in Appendix 2.
1.6.3. Test solutions
Test solutions of the chosen concentrations are prepared by dilution of a stock solution.
The stock solution should preferably be prepared by simply mixing or agitating the test substance in the diluition water by using mechanical means (e.g. stirring or ultrasonication). Saturation columns (solubility columns) can be used for achieving a suitable concentrated stock solution.
The use of solvents or dispersants (solubilising agents) may be required in some cases in order to produce a suitably concentrated stock solution. Examples of suitable solvents are acetone, ethanol, methanol, dimethylsulfoxide, dimethylformamide and triethyleneglycol. Examples of suitable dispersants are Cremophor RH40, Tween 80, Methylcellulose 0,01 % and HCO-40. Care should be taken when using readily biodegradable agents (e.g. acetone) and/or highly volatile compounds as these can cause problems with bacterial built-up in flow-through tests. When a solubilising agent is used it must have no significant effects on the fish growth nor visible adverse effects on the juvenile as revealed by a solvent-only control.
For flow-through tests, a system which continually dispenses and dilutes a stock solution of the test substance (e.g. metering pump, proportional diluter, saturator system) is required to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals, preferably daily, during the test and should not vary by more than 10 % throughout the test. A ring-test (3) has shown that, for rainbow trout, a frequency of water removal during the test of 6 litres/g of fish/day is acceptable (see section 1.8.2.2).
For semi-static (renewal) tests, the frequency of medium renewal will depend on the stability of the test substance, but a daily water renewal is recommended. If, from preliminary stability tests (see section 1.4), the test substance concentration is not stable (i.e. outside the range 80-120 % of nominal or falling below 80 % of the measured initial concentration) over the renewal period, consideration should be given to the use of a flow-through test.
1.6.4. Selection of species
Rainbow trout (Oncorhynchus mykiss) is the recommended species for this test since most experience has been gained from ring-test with this species (1) (3). However, other well documented species can be used but the test procedure may have to be adapted to provide suitable test conditions. For example, experience is also available with zebrafish (Danio rerio) (4) (5) and ricefish (medaka, Oryzias latipes) (6) (7) (8). The rationale for the selection of the species and the experimental method should be reported in this case.
1.6.5. Holding of fish
The test fish shall be selected from a population of a single stock, preferably from the same spawning, which has been held for at least two weeks prior to the test under conditions of water quality and illumination similar to those used in the test. They should be fed a minimum ration of 2 % body weight per day and preferably 4 % body weight per day throughout the holding period and during the test.
Following a 48-hour setting-in period, mortalities are recorded and the following criteria applied:
- mortalities of greater than 10 % of the population in seven days: reject the entire batch,
- mortalities of between 5 % and 10 % of the population: acclimation for seven additional days; if more than 5 % mortality during the second seven days, reject the entire batch,
- mortalities of less than 5 % of the population in seven days: accept the batch.
Fish should not receive treatment for disease in the two weeks preceding the test, or during the test.
1.7. TEST DESIGN
The "test design" relates to the selection of the number and spacing of the test concentrations, the number of tanks at each concentration level and the number of fish per tank. Ideally, the test design should be chosen with regard to:
(a) the objective of the study;
(b) the method of statistical analysis that will be used;
(c) the availability and cost of experimental resources.
The statement of the objective should, if possible, specify the statistical power at which a given size of difference (e.g. in growth rate) is required to be detected or, alternatively, the precision with which the ECx (e.g. with x = 10, 20, or 30, and preferably not less than 10) is required to be estimated. Without this, a firm prescription of the size of the study cannot be given.
It is important to recognise that a design which is optimal (makes best use of resources) for use with one method of statistical analysis is not necessarily optimal for another. The recommended design for the estimation of a LOEC/NOEC would not therefore be the same as that recommended for analysis by regression.
In most of cases, regression analysis is preferable to the analysis of variance, for reasons discussed by Stephan and Rogers (9). However, when no suitable regression model is found (r2 < 0,9) NOEC/LOEC should be used.
1.7.1. Design for analysis by regression
The important considerations in the design of a test to be analysed by regression are:
(a) the effect concentration (e.g. EC10,20,30) and the concentration range over which the effect of the test substance is of interest, should necessarily be spanned by the concentrations included in the test. The precision with which estimates of effect concentrations can be made, will be best when the effect concentration is in the middle of the range of concentrations tested. A preliminary range-finding test may be helpful in selecting appropriate test concentrations;
(b) to enable satisfactory statistical modelling, the test should include at least one control tank and five additional tanks at different concentrations. Where appropriate, when a solubilising agent is used, one control containing the solubilising agent at the highest tested concentration should be run in addition to the test series (see sections 1.8.3 and 1.8.4);
(c) an appropriate geometric series or logarithmic series (10) (see Appendix 3) may be used. Logarithmic spacing of test concentration is to be preferred;
(d) if more than six tanks are available, the additional tanks should either be used to provide replication or distributed across the range of concentrations in order to enable closer spacing of the levels. Either of these measures are equally desirable.
1.7.2. Design for estimation of a NOEC/LOEC using analysis of variance (ANOVA)
There should preferably be replicate tanks at each concentration, and statistical analysis should be at the tank level (11). Without replicate tanks, no allowance can be made for variability between tanks beyond that due to individual fish. However, experience has shown (12) that between-tank variability was very small compared with within-tank (i.e. between-fish) variability in the case examined. Therefore a relatively acceptable alternative is to perform statistical analysis at the level of individual fish.
Conventionally, at least five test concentrations in a geometric series with a factor preferably not exceeding 3,2 are used.
Generally, when tests are performed with replicate tanks, the number of replicate control tanks and therefore the number of fish should be the double of the number in each of the test concentrations, which should be of equal size (13) (14) (15). On the opposite, in absence of replicate tanks, the number of fish in the control group should be the same as the number in each test concentration.
If the ANOVA is to be based on tanks rather than individual fish (which would entail either individual marking of the fish or the use of "pseudo" specific growth rates (see section 2.1.2)), there is a need for enough replication of tanks to enable the standard deviation of "tanks within concentrations" to be determined. This means that the degrees of freedom for error in the analysis of variance should be at least 5 (11). If only the controls are replicated, there is a danger that the error variability will be biased because it may increase with the mean value of the growth rate in question. Since growth rate is likely to decrease with increasing concentration, this will tend to lead to an overestimate of the variability.
1.8. PROCEDURE
1.8.1. Selection and weighing of test fish
It is important to minimise variation in weight of the fish at the beginning of the test. Suitable size ranges for the different species recommended for use in this test are given in Appendix 1. For the whole batch of fish used in the test, the range in individual weights at the start of the test should ideally be kept to within ± 10 % of the arithmetic mean weight and, in any case, should not exceed 25 %. It is recommended to weight a subsample of fish before the test in order to estimate the mean weigh.
Food should be withheld from the stock population for 24 hours prior to the start of the test. Fish should then be chosen at random. Using a general anaesthetic (e.g. an aqueous solution of 100 mg/l tricaine methane sulphonate (MS 222) neutralised by the addition of two parts of sodium bicarbonate per part of MS 222), fish should be weighted individually as wet weights (blotted dry) to the precision given in Appendix 1. Those fish with weights within the intended range should be retained and then should be randomly distributed between the test vessels. The total wet weight of fish in each test vessel should be recorded. The use of anaesthetics likewise handling of fish (including blotting and weighing) may cause stress and injuries to the juvenile fish, in particular for those species of small size. Therefore handling of juvenile fish must be done with the utmost care to avoid stressing and injuring test animals.
The fish are weighed again on day 28 of the test (see section 1.8.6). However, if it is deemed necessary to recalculate the food ration, fish can be weighed again on day 14 of the test (see section 1.8.2.3). Another method such as the photographic method could be used to determine changes in fish size on the basis of which food rations could be adjusted.
1.8.2. Conditions of exposure
1.8.2.1. Duration
The test duration is >= 28 days.
1.8.2.2. Loading rates and stocking densities
It is important that the loading rate and stocking density is appropriate for the test species used (see Appendix 1). If the stocking density is too high, then overcrowding stress will occur leading to reduced growth rates and possibly to disease. If it is too low, territorial behaviour may be induced which could also affect growth. In any case, the loading rate should be low enough in order that a dissolved oxygen concentration of at least 60 % ASV can be maintained without aeration. A ring-test (3) has shown that, for rainbow trout, a loading rate of 16 trouts of 3-5 g in a 40-litre volume is acceptable. Recommended frequency of water removal during the test is 6 litres/g of fish/day.
1.8.2.3. Feeding
The fish should be fed with an appropriate food (Appendix 1) at a sufficient rate to induce acceptable growth rate. Care should be taken to avoid microbial growth and water turbidity. For rainbow trout, a rate of 4 % of their body weight per day is likely to satisfy these conditions (3) (16) (17) (18). The daily ration may be divided into two equal portions and given to the fish in two feeds per day, separated by at least five hours. The ration is based on the initial total fish weight for each test vessel. If the fish are weighted again on day 14, the ration is then recalculated. Food should be withheld from the fish 24 hours prior to weighing.
Uneaten food and fecal material should be removed from the test vessels each day by carefully cleaning the bottom of each tank using a suction.
1.8.2.4. Light and temperature
The photoperiod and water temperature should be appropriate for the test species (Appendix 1).
1.8.3. Test concentrations
Normally five concentrations of the test substance are required, regardless of the test design (see section 1.7.2). Prior knowledge of the toxicity of the test substance (e.g. from an acute test and/or from range-finding studies) should help in selecting appropriate test concentrations. Justification should be given if fewer than five concentrations are used. The highest tested concentration should not exceed the substance solubility limit in water.
Where a solubilising agent is used to assist in stock solution preparation, its final concentration should not be greater than 0,1 ml/l and should preferably be the same in all test vessels (see section 1.6.3). However, every effort should be made to avoid use of such materials.
1.8.4. Controls
The number of dilution-water controls depends on the test design (see sections 1.7-1.7.2). If a solubilising agent is used, then the same number of solubilising-agent controls as dilution-water controls should also be included.
1.8.5. Frequency of analytical determinations and measurements
During the test, the concentrations of test substance are determined at regular intervals (see below).
In flow-through tests, the flow rates of diluent and toxicant stock solution should be checked at intervals, preferably daily, and should not vary by more than 10 % throughout the test. Where the test substance concentrations are expected to be within ± 20 % of the nominal values (i.e. within the range 80-120 %; see sections 1.6.2 and 1.6.3), it is recommended that, as a minimum, the highest and lowest test concentrations be analysed at the start of the test and at weekly intervals thereafter. For the test where the concentration of the test substance is not expected to remain within ± 20 % of nominal (on the basis of stability data of the test substance), it is necessary to analyse all test concentrations, but following the same regime.
In semi-static (renewal) tests where the concentration of the test substance is expected to remain within ± 20 % of the nominal values, it is recommended that, as a minimum, the highest and lowest test concentrations be analysed when freshly prepared and immediately prior to renewal at the start of the study and weekly thereafter. For tests where the concentration of the test substance is not expected to remain within ± 20 % of nominal, all test concentrations must be analysed following the same regime as for more stable substances.
It is recommended that results be based on measured concentrations. However, if evidence is available to demonstrate that the concentration of the test substance in solution has been satisfactorily maintained within ± 20 % of the nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured values.
Samples may need to be filtered (e.g. using a 0,45 μm pore size) or centrifuged. Centrifugation is the recommended procedure. However, if the test material does not adsorb to filters, filtration may also be acceptable.
During the test, dissolved oxygen, pH and temperature should be measured in all test vessels. Total hardness, alkalinity and salinity (if relevant) should be measured in the controls and one vessel at the highest concentration. As a minimum, dissolved oxygen and salinity (if relevant) should be measured three times (at the beginning, middle and end of the test). In semi-static tests, it is recommended that dissolved oxygen be measured more frequently, preferably before and after each water renewal or at least once a week. The pH should be measured at the beginning and end of each water renewal in static renewal test and at least weekly in flow-through tests. Hardness and alkalinity should be measured once each test. Temperature should preferably be monitored continuously in at least one test vessel.
1.8.6. Observations
Weight: at the end of the test all surviving fish must be weighed as wet weights (blotted dry) either in groups by test vessel or individually. Weighing of animals by test vessel is preferred to individual weights which require that fish be individually marked. In the case of the measurement of individual weights for determination of individual fish specific growth rate, the marking technique selected should avoid stressing the animals (alternatives to freeze marking may be appropriate, e.g. the use of coloured fine fishing line).
The fish should be examined daily during the test period and any external abnormalities (such as haemorrhage, discoloration) and abnormal behaviour noted. Any mortalities should be recorded and the dead fish removed as soon as possible. Dead fish are not replaced, the loading rate and stocking density being sufficient to avoid effects on growth through changes in number of fish per tank. However, the feeding rate will need to be adjusted.
2. DATA AND REPORTING
2.1. TREATMENT OF RESULTS
It is recommended that a statistician be involved in both the design and analysis of the test since this test method allows for considerable variation in experimental design as for example, in the number of test chambers, number of test concentrations, number of fish, etc. In view of the options available in test design, specific guidance on statistical procedure is not given here.
Growth rates should not be calculated for test vessels where the mortality exceeds 10 %. However, mortality rate should be indicated for all test concentrations.
Whichever method is used to analyse the data, the central concept is the specific growth rate r between time t1 and time t2. This can be defined in several ways depending on whether fish are individually marked or not or whether a tank average is required.
>REFERENCE TO A GRAPHIC>
>REFERENCE TO A GRAPHIC>
>REFERENCE TO A GRAPHIC>
where,
r1= individual fish specific growth rate
r2= tank-average specific growth rate
r3= "pseudo" specific growth rate
w1, w2= weights of a particular fish at times t1 and t2, respectively
loge w1= logarithm of the weight of an individual fish at the start of the study period
loge w2= logarithm of the weight of an individual fish at the end of the study period
>REFERENCE TO A GRAPHIC>
= average of the logarithms of the values w1 for the fish in the tank at the start of the study period
>REFERENCE TO A GRAPHIC>
= average of the logarithms of the values w2 for the fish in the tank at the end of the study period
t1, t2= time (days) at start and end of study period
r1, r2, r3 can be calculated for the 0-28 days period and, where appropriate (i.e. when measurement at day 14 has been done) for the 0-14 and 14-28 days periods.
2.1.1. Analysis of results by regression (concentration-response modelling)
This method of analysis fits a suitable mathematical relationship between the specific growth rate and concentration, and hence enables the estimation of the "ECx" i.e. any required EC value. Using this method the calculation of r for individual fish (r1) is not necessary and instead, the analysis can be based on the tank-average value of r (r2). This last method is preferred. It is also more appropriate in case of the use of smallest species.
The tank-average specific growth rates (r2) should be plotted graphically against concentration, in order to inspect the concentration response relationship.
For expressing the relationship between r2 and concentration, an appropriate model should be chosen and its choice must be supported by appropriate reasoning.
If the numbers of fish surviving in each tank are unequal, then the process of model fitting, whether simple or non-linear, should be weighted to allow for unequal sizes of groups.
The method of fitting the model must enable an estimate of, for example, the EC20 and of its dispersion (either standard error or confidence interval) to be derived. The graph of the fitted model should be shown in relation to the data so that the adequacy of the fit of the model can be seen (9) (19) (20) (21).
2.1.2. Analysis of results for the estimation of the LOEC
If the test has included replication of tanks at all concentration levels, the estimation of the LOEC could be based on an analysis of variance (ANOVA) of the tank-average specific growth rate (see section 2.1), followed by a suitable method (e.g. Dunnett's or Williams' test (13) (14) (15) (22)) of comparing the average r for each concentration with the average r for the controls to identify the lowest concentration for which this difference is significant at a 0,05 probability level. If the required assumptions for parametric methods are not met - non-normal distribution (e.g. Shapiro-Wilk's test) or heterogeneous variance (Bartlett's test), consideration should be given to transforming the data to homogenise variances prior to performing the ANOVA, or to carrying out a weighted ANOVA.
If the test has not included replication of tanks at each concentration, an ANOVA based on tanks will be insensitive or impossible. In this situation, an acceptable compromise is to base the ANOVA on the "pseudo" specific growth rate r3 for individual fish.
The average r3 for each test concentration may then be compared with the average r3 for the controls. The LOEC can then be identified as before. It must be recognised that this method provides no allowance for, nor protection against, variability between tanks, beyond that which is accounted for by the variability between individual fish. However, experience has shown (9) that between-tank variability was very small compared with within-tank (i.e. between-fish) variability. If individual fish are not included in the analysis, the method of outlier identification and justification for its use must be provided.
2.2. INTERPRETATION OF RESULTS
The results should be interpreted with caution where measured toxicant concentrations in test solutions occur at levels near the detection limit of the analytical method or, in semi static tests, when the concentration of the test substance decreases between freshly prepared solution and before renewal.
2.3. TEST REPORT
The test report must include the following information:
2.3.1. Test substance:
- physical nature and relevant physical-chemical properties,
- chemical identification data including purity and analytical method for quantification of the test substance where appropriate.
2.3.2. Test species
- scientific name, possibly,
- strain, size, supplier, any pre-treatment, etc.
2.3.3. Test conditions:
- test procedure used (e.g. semi-static/renewal, flow-through, loading, stocking density, etc.),
- test design (e.g. number of test vessels, test concentrations and replicates, number of fish per vessel),
- method of preparation of stock solutions and frequency of renewal (the solubilising agent and its concentration must be given, when used),
- the nominal test concentrations, the means of the measured values and their standard deviations in the test vessels and the method by which these were attained and evidence that the measurements refer to the concentrations of the test substance in true solution,
- dilution water characteristics: pH, hardness, alkalinity, temperature, dissolved oxygen concentration, residual chlorine levels (if measured), total organic carbon, suspended solids, salinity of the test medium (if measured) and any other measurements made,
- water quality within test vessels: pH, hardness, temperature and dissolved oxygen concentration,
- detailed information on feeding, (e.g. type of food(s), source, amount given and frequency).
2.3.4. Results:
- evidence that controls met the validity criterion for survival, and data on mortalities occurring in any of the test concentrations,
- statistical analytical techniques used, statistics based on replicates or fish, treatment of data and justification of techniques used,
- tabulated data on individual and mean fish weights on days 0, 14 (if measured) and 28 values of tank-average or pseudo specific growth rates (as appropriate) for the periods 0-28 days or possibly 0-14 and 14-28,
- results of the statistical analysis (i.e. regression analysis or ANOVA) preferably in tabular and graphical form and the LOEC (p = 0,05) and the NOEC or ECx with, when possible, standard errors, as appropriate,
- incidence of any unusual reactions by the fish and any visible effects produced by the test substance.
3. REFERENCES
(1) Solbe J. F. de LG (1987). Environmental Effects of Chemicals (CFM 9350 SLD). Report on a UK Ring Test of a Method for Studying the Effects of Chemicals on the Growth Rate of Fish. WRc Report No PRD 1388-M/2.
(2) Meyer, A., Bierman, C. H. and Orti, G. (1993). The phylogenetic position of the zebrafish (Danio rerio), a model system in developmental biology: an invitation to the comparative method. Proc. R. Soc. Lond. B. 252, pp. 231-236.
(3) Ashley S., Mallett M. J. and Grandy N. J. (1990). EEC Ring Test of a Method for Determining the Effects of Chemicals on the Growth Rate of Fish. Final Report to the Commission of the European Communities. WRc Report No EEC 2600-M.
(4) Crossland N. O. (1985). A method to evaluate effects of toxic chemicals on fish growth. Chemosphere, 14, pp. 1855-1870.
(5) Nagel R., Bresh H., Caspers N., Hansen P. D., Market M., Munk R., Scholz N. and Höfte B. B. (1991). Effect of 3,4-dichloroaniline on the early life stages of the Zebrafish (Brachydanio rerio): results of a comparative laboratory study. Ecotox. Environ. Safety, 21, pp. 157-164.
(6) Yamamoto, Tokio. (1975). Series of stock cultures in biological field. Medaka (killifish) biology and strains. Keigaku Publish. Tokio, Japan.
(7) Holcombe, G. W., Benoit D. A., Hammermeister, D. E., Leonard, E. N. and Johnson, R. D. (1995). Acute and long-term effects of nine chemicals on the Japanese medaka (Oryzias latipes). Arch. Environ. Conta. Toxicol. 28, pp. 287-297.
(8) Benoit, D. A., Holcombe, G. W. and Spehar, R. L. (1991). Guidelines for conducting early life toxicity tests with Japanese medaka (Oryzias latipes). Ecological Research Series EPA-600/3-91-063. US Environmental Protection Agency, Duluth, Minnesota.
(9) Stephan C. E. and Rogers J. W. (1985). Advantages of using regression analysis to calculate results of chronic toxicity tests. Aquatic Toxicology and Hazard Assessment: Eighth Symposium, ASTM STP 891, R. C. Bahner and D. J. Hansen, eds., American Society for Testing and Materials, Philadelphia, pp. 328-338.
(10) Environment Canada (1992). Biological test method: toxicity tests using early life stages of salmonid fish (rainbow trout, coho salmon, or atlantic salmon). Conservation and Protection, Ontario, Report EPS 1/RM/28, 81 pp.
(11) Cox D. R. (1958). Planning of experiments. Wiley Edt.
(12) Pack S. (1991). Statistical issues concerning the design of tests for determining the effects of chemicals on the growth rate of fish. Room Document 4, OECD Ad Hoc Meeting of Experts on Aquatic Toxicology, WRc Medmenham, UK, 10-12 December 1991.
(13) Dunnett C. W. (1955). A Multiple Comparisons Procedure for Comparing Several Treatments with a Control, J. Amer. Statist. Assoc., 50, pp. 1096-1121.
(14) Dunnett C. W. (1964). New tables for multiple comparisons with a control. Biometrics, 20, pp. 482-491.
(15) Williams D. A. (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27, pp. 103-117.
(16) Johnston, W. L., Atkinson, J. L., Glanville N. T. (1994). A technique using sequential feedings of different coloured food to determine food intake by individual rainbow trout, Oncorhynchus mykiss: effect of feeding level. Aquaculture 120, pp. 123-133.
(17) Quinton, J. C. and Blake, R. W. (1990). The effect of feed cycling and ration level on the compensatory growth response in rainbow trout, Oncorhynchus mykiss. Journal of Fish Biology, 37, pp. 33-41.
(18) Post, G. (1987). Nutrition and Nutritional Diseases of Fish. Chapter IX in Testbook of Fish Health. T.F.H. Publications, Inc. Neptune City, New Jersey, USA. 288 pp.
(19) Bruce, R. D. and Versteeg D. J. (1992). A statistical procedure for modelling continuous toxicity data. Environ. Toxicol. Chem. 11, pp. 1485-1494.
(20) DeGraeve, G. M., Cooney, J. M., Pollock, T. L., Reichenbach, J. H., Dean, Marcus, M. D. and McIntyre, D. O. (1989). Precision of EPA seven-day fathead minnow larval survival and growth test; intra and interlaboratory study. Report EA-6189 (American Petroleum Institute Publication, No 4468). Electric Power Research Institute, Palo Alto, CA.
(21) Norbert-King T. J. (1988). An interpolation estimate for chronic toxicity: the ICp approach. US Environmental Protection Agency. Environmental Research Lab., Duluth, Minnesota. Tech. Rep. No 05-88 of National Effluent Toxicity Assesment Center. Sept. 1988. 12 pp.
(22) Williams D. A. (1972). The comparison of several dose levels with a zero dose control. Biometrics 28, pp. 510-531.
APPENDIX 1
FISH SPECIES RECOMMENDED FOR TESTING AND SUITABLE TEST CONDITIONS
>TABLE>
APPENDIX 2
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER
>TABLE>
APPENDIX 3
LOGARITHMIC SERIES OF CONCENTRATIONS SUITABLE FOR TOXICITY TEST (9)
>TABLE>
C.15. FISH, SHORT-TERM TOXICITY TEST ON EMBRYO AND SAC-FRY STAGES
1. METHOD
This short-term toxicity test method is a replicate of the OECD TG 212 (1998).
1.1. INTRODUCTION
This short-term toxicity test on fish embryo and sac-fry stages is a short-term test in which the life stages from the newly fertilised egg to the end of the sac-fry stage are exposed. No feeding is provided in the embryo and sac-fry test, and the test should thus be terminated while the sac-fry are still nourished from the yolk sac.
The test is intended to define lethal, and to a limited extent, sublethal effects of chemicals on the specific stages and species tested. This test would provide useful information in that it could (a) form a bridge between lethal and sublethal tests, (b) be used as a screening test for either a full early life stage test or for chronic toxicity tests and (c) be used for testing species where husbandry techniques are not sufficiently advanced to cover the period of change from endogenous to exogenous feeding.
It should be borne in mind that only tests incorporating all stages of the life-cycle of fish are generally liable to give an accurate estimate of the chronic toxicity of chemicals to fish, and that any reduced exposure with respect to life stages may reduce the sensitivity and thus underestimate the chronic toxicity. It is therefore expected that the embryo and sac-fry test would be less sensitive than a full early life stage test, particularly with respect to chemicals with high lipophilicity (log Pow > 4) and chemicals with a specific mode of toxic action. However smaller differences in sensitivity between the two tests would be expected for chemicals with a non-specific, narcotic mode of action (1).
Prior to the publication of this test, most experience with this embryo and sac-fry test has been with the freshwater fish Danio rerio Hamilton-Buchanan (Teleostei, Cyprinidae - common name zebrafish). More detailed guidance on test performance for this species is therefore given in Appendix 1. This does not preclude the use of other species for which experience is also available (Tables IA and IB).
1.2. DEFINITIONS
Lowest observed effect concentration (LOEC): is the lowest tested concentration of a test substance at which the substance is observed to have a significant effect (at p < 0,05) when compared with the control. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC.
No observed effect concentration (NOEC): is the test concentration immediately below the LOEC.
1.3. PRINCIPLE OF THE TEST
The embryo and sac-fry stages of fish are exposed to a range of concentrations of the test substance dissolved in water. Within the protocol a choice is possible between a semi-static and a flow-through procedure. The choice depends on the nature of the test substance. The test is begun by placing fertilised eggs in the test chambers and is terminated just before the yolk sac of any larvae in any of the test chambers has been completely absorbed or before mortalities by starvation start in controls. Lethal and sub-lethal effects are assessed and compared with control values to determine the lowest observed effect concentration and hence the no observed effect concentration. Alternatively, they may be analysed using a regression model in order to estimate the concentration that would cause a given percentage effect (i.e. LC/ECx, where x is a defined % effect).
1.4. INFORMATION ON THE TEST SUBSTANCE
Results of an acute toxicity test (see method C.1) preferably performed with the species chosen for this test, should be available. The results may be useful in selecting an appropiate range of test concentrations in the early life stages test. Water solubility (including solubility in the test water) and the vapour pressure of the test substance should be known. A reliable analytical method for the quantification of the substance in the test solutions with known and reported accuracy and limit of detection should be available.
Information on the test substance which is useful in establishing the test conditions includes the structural formula, purity of the substance, stability in light, stability under the conditions of the test, pKa, Pow and results of a test for ready biodegradability (see method C.4).
1.5. VALIDITY OF THE TEST
For a test to be valid, the following conditions apply:
- overall survival of fertilised eggs in the controls and where relevant, in the solvent-only vessels must be greater than or equal to the limits defined in Appendices 2 and 3,
- the dissolved oxygen concentration must be between 60 and 100 % of the air saturation value (ASV) throughout the test,
- the water temperature must not differ by more than ± 1,5 °C between test chambers or between successive days at any time during the test and should be within the temperature ranges specified for the test species (Appendices 2 and 3).
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Test chambers
Any glass or other chemically inert vessels can be used. The dimensions of the vessels should be large enough to allow compliance with the loading rate (see section 1.7.1.2). It is recommended that test chambers be randomly positioned in the test area. A randomised block design with each treatment being present in each block is preferable to a completely randomised design when there are systematic effects in the laboratory that can be controlled using blocking. Blocking, if used, should be taken account of in the subsequent data analysis. The test chambers should be shielded from unwanted disturbance.
1.6.2. Selection of fish species
Recommended fish species are given in Table 1A. This does not preclude the use of other species (examples are given in Table 1B), but the test procedure may have to be adapted to provide suitable test conditions. The rationale for the selection of the species and the experimental method should be reported in this case.
1.6.3. Holding of the brood fish
Details on holding the brood stock under satisfactory conditions may be found in OECD TG 210(1) and in references (2) (3) (4) (5) (6).
1.6.4. Handling of embryos and larvae
Embryos and larvae may be exposed, within the main vessel, in smaller vessels fitted with mesh sides or ends to permit a flow of test solution through the vessel. Non-turbulent flow through these small vessels may be induced by suspending them from an arm arranged to move the vessel up and down but always keeping the organisms submerged; a siphon-flush system can also be used. Fertilised eggs of salmonid fishes can be supported on racks or meshes with apertures sufficiently large to allow larvae to drop through after hatching. The use of pasteur pipettes is appropriate to remove the embryos and larvaes in the semi-static tests with complete daily renewal (see paragraph 1.6.6).
Where egg containers, grids or meshes have been used to hold eggs within the main test vessel, these restraints should be removed after the larvae hatch(2), except that meshes should be retained to prevent the escape of the fish. If there is a need to transfer the larvae, they should not be exposed to the air and nets should not be used to release fish from egg containers (such a caution may not be necessary for some less fragile species, e.g. the carp). The timing of this transfer varies with the species and transfer may not always be necessary. For the semi-static technique, beakers or shallow containers may be used, and, if necessary, equipped with a mesh screen slightly elevated above the bottom of the beaker. If the volume of these containers is sufficient to comply with loading requirements, (see 1.7.1.2) no transfer of embryo or larvae may be necessary.
1.6.5. Water
Any water which conforms to the chemical characteristics of an acceptable dilution water as listed in Appendix 4 and in which the test species shows control survival at least as good as that described in Appendices 2 and 3 is suitable as a test water. It should be of constant quality during the period of the test. The pH should remain within a range of ± 0,5 pH units. In order to ensure that the dilution water will not unduly influence the test result (for example by complexation of test substance), or adversely affect the performance of the brood stock, samples should be taken at intervals for analysis. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd and Ni), major anions and cations (e.g. Ca, Mg, Na, K, Cl and SO4), pesticides (e.g. total organophosphorus and total organochlorine pesticides), total organic carbon and suspended solids should be made, for example, every three months, where a dilution water is known to be relatively constant in quality. If water quality has been demonstrated to be constant over at least one year, determinations can be less frequent and intervals extended (e.g. every six months).
1.6.6. Test solutions
Test solutions of the chosen concentrations are prepared by dilution of a stock solution.
The stock solution should preferably be prepared by simply mixing or agitating the test substance in the dilution water by using mechanical means (e.g. stirring and ultrasonication). Saturation colums (solubility columns) can be used for achieving a suitable concentrated stock solution. As far as possible, the use of solvents or dispersants (solubilising agents) should be avoided; however, such coupounds may be required in some cases in order to produced a suitably concentrated stock solution. Examples of suitable solvents are acetone, ethanol, methanol, dimethylformamide and triethyleneglycol. Examples of suitable dispersants are Cremophor RH40, Tween 80, methylcellulose 0,01 % and HCO-40. Care should be taken when using readily biodegradable agents (e.g. acetone) and/or highly volatile as these can cause problems with bacterial built-up in flow-through tests. When a solubilising agent is used it must have no significant effect on survival nor visible adverse effect on the early-life stages as revealed by a solvent-only control. However, every effort should be made to avoid the use of such materials.
For the semi-static technique, two different renewal procedures may be followed; either (i) new test solutions are prepared in clean vessels and surviving eggs and larvae gently transferred into the new vessels in a small volume of old solution, avoiding exposure to air, or (ii) the test organisms are retained in the vessels whilst a proportion (at least three quarters) of the test water is changed. The frequency of medium renewal will depend on the stability of the test substance, but a daily water renewal is recommended. If, from preliminary stability tests (see section 1.4), the test substance concentration is not stable (i.e.outside the range 80-120 % of nominal or falling below 80 % of the measured initial concentration) over the renewal period, consideration should be given to the use of a flow-through test. In any case, care should be taken to avoid stressing the larvae during the water renewal operation.
For flow-through tests, a system which continually dispenses and dilutes a stock solution of the test substance (e.g. metering pump, proportional diluter, saturator system) is required to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals, preferably daily, and should not vary by more than 10 % throughout the test. A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (2).
1.7. PROCEDURE
Useful information on the performance of fish embryo and sac-fry toxicity tests is available in the literature, some examples of which are included in the literature section of this text (7) (8) (9).
1.7.1. Conditions of exposure
1.7.1.1. Duration
The test should start preferably within 30 minutes after the eggs have been fertilised. The embryos are immersed in the test solution before, or as soon as possible after, commencement of the blastodisc cleavage stage and in any case before the onset of the gastrula stage. For eggs obtained from commercial supplier, it may not be possible to start the test immediately after fertilisation. As the sensitivity of the test may be seriously influenced by delaying the start of the test, the test should be initiated within eight hours after fertilisation. As larvae are not fed during the exposure period, the test should be terminated just before the yolk sac of any larvae in any of the test chambers has been completely absorbed or before mortalities by starvation start in controls. The duration will depend upon the species used. Some recommended durations are given in Appendices 2 and 3.
1.7.1.2. Loading
The number of fertilised eggs at the start of the test should be sufficient to meet statistical requirements. They should be randomly distributed among treatments, and at least 30 fertilised eggs, divided equally (or as equally as possible since it can be difficult to obtain equal batches when using some species) between at least three replicate test chambers, should be used per concentration. The loading rate (biomass per volume of test solution) should be low enough in order that a dissolved oxygen concentration of at least 60 % ASV can be maintained without aeration. For flow-through tests, a loading rate not exceeding 0,5 g/l per 24 hours and not exceeding 5 g/l of solution at any time has bean recommended (2).
1.7.1.3. Light and temperature
The photoperiod and test water temperature should be appropriate for the test species (Appendices 2 and 3). For the purpose of temperature monitoring, it may be appropriate to use an additional test vessel.
1.7.2. Test concentrations
Normally, five concentrations of the test substance spaced by a constant factor not exceeding 3,2 are required. The curve relating LC50 to period of exposure in the acute study should be considered when selecting the range of test concentrations. The use of fewer than five concentrations, for example in limit tests, and a narrower concentration interval may be appropriate in some circumstances. Justification should be provided if fewer than five concentrations are used. Concentrations of the substance higher than the 96 hour LC50 or 100 mg/l, whichever is the lower, need not be tested. Substances should not be tested above their solubility limit in in the test water.
When a solubilising agent is used to aid preparation of test solutions (see section 1.6.6), its final concentration in the test vessels should not be greater than 0,1 ml/l and should be the same in all test vessels.
1.7.3. Controls
One dilution-water control (replicated as appropriate) and also, if relevant, one control containing the solubilising-agent (replicated as appropriate) should be run in addition to the test series.
1.7.4. Frequency of analytical determinations and measurements
During the test, the concentrations of the test substance are determined at regular intervals.
In semi-static tests where the concentration of the test substance is expected to remain within ± 20 % of the nominal (i.e. within the range 80-120 %; see section 1.4 and 1.6.6), it is recommended that, as a minimum, the highest and lowest test concentrations be analysed when freshly prepared and immediately prior to renewal on at least three occasions spaced evenly over the test (i.e. analyses should be made on a sample from the same solution - when freshly prepared and at renewal).
For tests where the concentration of the test substance is not expected to remain within ± 20 % of nominal (on the basis of stability data of the substance), it is necessary to analyse all test concentrations, when freshly prepared and at renewal, but following the same regime (i.e. on at least three occasions spaced evenly over the test). Determination of test substance concentrations prior to renewal need only be performed on one replicate vessel at each test concentration. Determinations should be made no more than seven days apart. It is recommended that results be based on measured concentrations. However, if evidence is available to demonstrate that the concentration of the test substance in solution has been satisfactorily maintained within ± 20 % of the nominal or measured initial concentration throughout the test, then results can be based on nominal or measured initial values.
For flow-through tests, a similar sampling regime to that described for semi-static tests is appropriate (but measurement of "old" solutions is not applicable in this case). However, if the test duration is more than seven days, it may be advisable to increase the number of sampling occasions during the first week (e.g. three sets of measurements) to ensure that the test concentrations are remaining stable.
Samples may need to be centrifuged or filtered (e.g. using a 0,45 ìm pore size). However, since neither centrifuging nor filtration appears always to separate the non-bioavailable fraction of the test substance from that which is bioavailable, samples may not be subjected to those treatments.
During the test, dissolved oxygen, pH and temperature should be measured in all test vessels. Total hardness and salinity (if relevant) should be measured in the controls and one vessel at the highest concentration. As a minimum, dissolved oxygen and salinity (if relevant) should be measured three times (at the beginning, middle and end of the test). In semi-static tests, it is recommended that dissolved oxygen be measured more frequently, preferably before and after each water renewal or at least once at week. Ôhe pH should be measured at the beginning and end of each water renewal in semi-static test and at least weekly in flow-through tests. Hardness should be measured once each test. Temperature should be measured daily and it should preferably be monitored continuously in at least one test vessel.
1.7.5. Observations
1.7.5.1. Stage of embryonic development
The embryonic stage (i.e. gastrula stage) at the beginning of exposure to the test substance should be verified as precisely as possible. This can be done using a representative sample of eggs suitably preserved and cleared. The literature may also be consulted for the description and illustration of embryonic stages (2) (5) (10) (11).
1.7.5.2. Hatching and survival
Observations on hatching and survival should be made at least once daily and numbers recorded. It may be desirable to make more frequent observations at the beginning of the test (e.g. each 30 minutes during the first three hours), since in some cases, survival times can be more relevant than only the number of deaths (e.g. when there are acute toxic effects). Dead embryos and larvae should be removed as soon as observed since they can decompose rapidly. Extreme care should be taken when removing dead individuals not to knock or physically damage adjacent eggs/larvae, these being extremely delicate and sensitive. Criteria for death vary according to life stage:
- for eggs: particularly in the early stages, a marked loss of translucency and change in colouration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance,
- for embryos: absence of body movement and/or absence of heartbeat and/or opaque discoloration in species whose embryos are normally translucent,
- for larvae: immobility and/or absence of respiratory movement and/or absence of heartbeat and/or white opaque colouration of central nervous system and/or lack of reaction mechanical stimulus.
1.7.5.3. Abnormal appearance
The number of larvae showing abnormality of body form and/or pigmentation, and the stage of yolk-sac absorption, should be recorded at adequate intervals depending on the duration of the test and the nature of the abnormality described. It should be noted that abnormal embryos and larvae occur naturally and can be of the order of several per cent in the control(s) in some species. Abnormal animals should only be removed from the test vessels on death.
1.7.5.4. Abnormal behaviour
Abnormalities, e.g. hyperventilation, uncoordinated swimming, and atypical quiescence should be recorded at adequate intervals depending on the duration of the test. These effects, although difficult to quantify, can, when observed, aid in the interpretation of mortality data i.e. provide information on the mode of toxic action of the substance.
1.7.5.5. Length
At the end of the test, measurement of individual lengths is recommended; standard, fork or total length may be used. If however, caudal fin rot or fin erosion occurs, standard lengths should be used. Generally, in a well-run test, the coefficient of variation for length among replicates in the controls should be <= 20 %.
1.7.5.6. Weight
At the end of the test, individual weights can be measured; dry weights (24 hours at 60 °C) are preferable to wet weights (blotted dry). Generally, in a well-run test, the coefficient of variation for weight among replicates in the controls should be <= 20 %.
These observations will result in some or all of the following data being available for statistical analysis:
- cumulative mortality,
- numbers of healthy larvae at end of test,
- time to start of hatching and end of hatching (i.e. 90 % hatching in each replicate),
- numbers of larvae hatching each day,
- length (and weight) of surviving animals at end of the test,
- numbers of larvae that are deformed or of abnormal appearance,
- numbers of larvae exhibiting abnormal behaviour.
2. DATA AND REPORTING
2.1. TREATMENT OF RESULTS
It is recommended that a statistician be involved in both the design and analysis of the test since the method allows for considerable variation in experimental design as, for example, in the number of test chambers, number of test concentrations, starting number of fertilised eggs and in the parameters measured. In view of the options available in test design, specific guidance on statistical procedures is not given here.
If LOEC/NOECs are to be estimated, it will be necessary for variations to be analysed within each set of replicates using analysis of variance (ANOVA) or contingency table procedures. In order to make a multiple comparison between the results at the individual concentrations and those for the controls, Dunnett's method may be found useful (12) (13). Other useful examples are also available (14) (15). The size of the effect detectable using ANOVA or other procedures (i.e. the power of the test) should be calculated and reported. It should be noted that not all the observations listed in section 1.7.5.6 are suitable for statistical analysis using ANOVA. For example, cumulative mortality and numbers of healthy larvae at the end of the test could be analysed using probit methods.
If LC/ECxs are to be estimated, (a) suitable curve(s), such as the logistic curve, should be fitted to the data of interest using a statistical method such as least squares or non-linear least squares. The curve(s) should be parameterised so that the LC/ECx of interest and its standard error can be estimated directly. This will greatly ease the calculation of the confidence limits around the LC/ECx. Unless there are good reasons to prefer different confidence levels, two-sided 95 % confidence should be quoted. The fitting procedure should preferably provide a means for assessing the significance of the lack of fit. Graphical methods for fitting curves can be used. Regression analysis is suitable for all observations listed in section 1.7.5.6.
2.2. INTERPRETATION OF RESULTS
The results should be interpreted with caution where measured toxicant concentrations in test solutions occur at levels near the detection limit of the analytical method. The interpretation of results for concentrations above the water solubility of the substance should also be made with care.
2.3. TEST REPORT
The test report must include the following information:
2.3.1. Test substance:
- physical nature and relevant physical-chemical properties,
- chemical identification data, including purity and analytical method for quantification of the tests substance where appropriate.
2.3.2. Test species:
- scientific name, strain, numbers of parental fish (i.e. how many females were used for providing the required numbers of eggs in the test), source and method of collection of the fertilised eggs and subsequent handling.
2.3.3. Test conditions:
- test procedure used (e.g. semi-static or flow-through, time period from fertilisation to start the test, loading, etc),
- photoperiod(s),
- test design (e.g. number of test chambers and replicates, number of embryos per replicate),
- method of preparation of stock solutions and frequency of renewal (the solubilising agent and its concentration must be given, when used),
- the nominal test concentrations, the measured values, their means and their standard deviations in the test vessels and the method by which these were attained and, if the test substance is soluble in water at concentrations below those tested, evidence should be provided that the measurements refer to the concentrations of the test substance in solution,
- dilution water characteristics: pH, hardness, temperature, dissolved oxygen concentration, residual chlorine levels (if measured), total organic carbon, suspended solids, salinity of the test medium (if measured) and any other measurements made,
- water quality within test vessels: pH, hardness, temperature and dissolved oxygen concentration.
2.3.4. Results:
- results from any preliminary studies on the stability of the test substance,
- evidence that controls met the overall survival acceptability standard of the test species (Appendices 2 and 3),
- data on mortality/survival at embryo and larval stages and overall mortality/survival,
- days to hatch and numbers hatched,
- data for length (and weight),
- incidence and description of morphological abnormalities, if any,
- incidence and description of behavioural effects, if any,
- statistical analysis and treatment of data,
- for tests analysed using ANOVA, the lowest observed effect concentration (LOEC) at p = 0,05 and the no observed effect concentration (NOEC) for each response assessed, including a description of the statistical procedures used and an indication of what size of effect could be detected,
- for tests analysed using regression techniques, the LC/ECx and confidence intervals and a graph of the fitted model used for its calculation,
- explanation for any deviation from this testing method.
3. REFERENCES
(1) Kristensen P. (1990). Evaluation of the Sensitivity of Short Term Fish Early Life Stage Tests in Relation to other FELS Test Methods. Final report to the Commission of the European Communities, 60 pp. June 1990.
(2) ASTM (1988). Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. American Society for Testing and Materials. E 1241-88. 26 pp.
(3) Brauhn J. L. and Schoettger R. A. (1975). Acquisition and Culture of Research Fish: Rainbow trout, Fathead minnows, Channel Catfish and Bluegills. p. 54, Ecological Research Series, EPA-660/3-75-011, Duluth, Minnesota.
(4) Brungs W. A. and Jones B. R. (1977). Temperature Criteria for Freshwater Fish: Protocol and Procedures. p. 128, Ecological Research Series EPA-600/3-77-061, Duluth, Minnesota.
(5) Laale H. W. (1977). The Biology and Use of the Zebrafish (Brachydanio rerio) in Fisheries Research. A Literature Review. J. Biol. 10, pp. 121-173.
(6) Legault R. (1958). A Technique for Controlling the Time of Daily Spawning and Collecting Eggs of the Zebrafish, Brachydanio rerio (Hamilton-Buchanan) Copeia, 4, pp. 328-330.
(7) Dave G., Damgaard B., Grande M., Martelin J. E., Rosander B. and Viktor T. (1987). Ring Test of an Embryo-larval Toxicity Test with Zebrafish (Brachydanio rerio) Using Chromium and Zinc as Toxicants. Environmental Toxicology and Chemistry, 6, pp. 61-71.
(8) Birge J. W., Black J. A. and Westerman A. G. (1985). Short-term Fish and Amphibian Embryo-larval Tests for Determining the Effects of Toxicant Stress on Early Life Stages and Estimating Chronic Values for Single Compounds and Complex Effluents. Environmental Toxicology and Chemistry 4, pp. 807-821.
(9) Van Leeuwen C. J., Espeldoorn A. and Mol F. (1986). Aquatic Toxicological Aspects of Dithiocarbamates and Related Compounds. III. Embryolarval Studies with Rainbow Trout (Salmo gairdneri). J. Aquatic Toxicology, 9, pp. 129-145.
(10) Kirchen R. V. and W. R. West (1969). Teleostean Development. Carolina Tips 32(4): 1-4. Carolina Biological Supply Company.
(11) Kirchen R. V. and W. R. West (1976). The Japanese Medaka. Its care and Development. Carolina Biological Supply Company, North Carolina. 36 pp.
(12) Dunnett C. W. (1955). A Multiple Comparisons Procedure for Comparing Several Treatments with Control. J. Amer. Statist. Assoc., 50, pp. 1096-1121.
(13) Dunnett C. W. (1964). New Tables for Multiple Comparisons with a Control. Biometrics, 20, pp. 482-491.
(14) Mc Clave J. T., Sullivan J. H. and Pearson J.G. (1980). Statistical Analysis of Fish Chronic Toxicity Test Data. Proceedings of 4th Aquatic Toxicology Symposium, ASTM, Philadelphia.
(15) Van Leeuwen C. J., Adema D. M. M. and Hermes J. (1990). Quantitative Structure-Activity Relationships for Fish Early Life Stage Toxicity. Aquatic Toxicology, 16, pp. 321-334.
(16) Environment Canada. (1992). Toxicity Tests Using Early Life Stages of Salmonid Fish (Rainbow Trout, Coho Salmon or Atlantic Salmon). Biological Test Method Series. Report EPS 1/RM/28, December 1992, 81 pp.
(17) Dave G. and Xiu R. (1991). Toxicity of Mercury, Nickel, Lead and Cobalt to Embryos and Larvae of Zebrafish, Brachydanio rerio. Arch. of Environmental Contamination and Toxicology, 21, pp. 126-134.
(18) Meyer A., Bierman C. H. and Orti G. (1993). The phylogenetic position of the Zebrafish (Danio rerio), a model system in developmental biology - an invitation to the comperative methods. Proc. Royal Society of London, Series B, 252: pp. 231-236.
(19) Ghillebaert F., Chaillou C., Deschamps F. and Roubaud P. (1995). Toxic Effects, at Three pH Levels, of Two Reference Molecules on Common Carp Embryo. Ecotoxicology and Environmental Safety 32, pp. 19-28.
(20) US EPA, (1991). Guidelines for Culturing the Japanese Medaka, Oryzias latipes. EPA report EPA/600/3-91/064, Dec. 1991, EPA, Duluth.
(21) US EPA, (1991). Guidelines for Conducting Early Life Stage Toxicity Tests with Japanese Medaka, (Oryzias latipes). EPA report EPA/600/3-91/063, Dec. 1991, EPA, Duluth.
(22) De Graeve G. M., Cooney J. D., McIntyre D. O., Poccocic T. L., Reichenbach N. G., Dean J. H. and Marcus M. D. (1991). Validity in the performance of the seven-day Fathead minnow (Pimephales promelas) larval survival and growth test: an intra- and interlaboratory study. Environ. Tox. Chem. 10, pp. 1189-1203.
(23) Calow P. (1993). Handbook of Ecotoxicology, Blackwells, Oxford. Vol. 1, Chapter 10: Methods for spawning, culturing and conducting toxicity tests with Early Life stages of Estuarine and Marine fish.
(24) Balon E. K. (1985). Early life history of fishes: New developmental, ecological and evolutionary perspectives, Junk Publ., Dordrecht, 280 pp.
(25) Blaxter J. H. S. (1988). Pattern and variety in development, in: W. S. Hoar and D. J. Randall eds., Fish Physiology, Vol. XIA, Academic press, pp. 1-58.
TABLE 1A: Fish species recommended for testing
>TABLE>
TABLE 1B: Examples of other well-documented species which have also been used
>TABLE>
APPENDIX 1
GUIDANCE ON PERFORMANCE OF A TOXICITY TEST ON EMBRYOS AND SAC-FRY OF ZEBRAFISH (BRACHYDANIO RERIO)
INTRODUCTION
The zebrafish originates from the Coromandel coast of India where it inhabits fast-flowing streams. It is a common aquarium fish of the carp family, and information about procedures for its care and culture can be found in standard reference books on tropical fish. Its biology and use in fishery research have been reviewed by Laale (1).
The fish rarely exceeds 45 mm in length. The body is cylindrical with 7-9 dark-blue horizontal silvery stripes. These stripes run into the caudal and anal fins. The back is olive-green. Males are slimmer than females. Females are more silvery and the abdomen is distended, particularly prior to spawning.
Adult fishes are able to tolerate large fluctuations in temperature, pH and hardness. However, in order to get healthy fish which produce eggs of good quality, optimal conditions should be provided.
During spawning the male pursues and butts the female, and as the eggs are expelled they are fertilised. The eggs, which are transparent and non-adhesive, fall to the bottom where they may be eaten by the parents. Spawning is influenced by light. If the morning light is adequate, the fish usually spawns in the early hours following daybreak.
A female can produce batches of several hundreds of eggs at weekly intervals.
CONDITIONS OF PARENTAL FISH, REPRODUCTION AND EARLY-LIFE STAGES
Select a suitable number of healthy fish and keep these in a suitable water (e.g. Appendix 4) for at least two weeks prior to the intended spawning. The group of fish should be allowed to breed at least once before producing the batch of eggs used in the test. The density of fish during this period should not exceed 1 gram of fish per litre. Regular changes of water or the use of purification systems will enable the density to be higher. The temperature in the holding tanks should be maintained at 25 ± 2 °C. The fish should be provided with a varied diet, which may consist of, for example, appropriate commercial dry food, live newly hatched Arthemia, chironomids, Daphnia, white worms (Enchytraeids).
Two procedures are outlined below, which in practice have led to a sufficient batch of healthy, fertilised eggs for a test to be run:
(i) Eight females and 16 males are placed in a tank containing 50 litres of dilution water, shielded from direct light and left as undisturbed as possible for at least 48 hours. A spawning tray is placed at the bottom of the aquarium in the afternoon the day before start of the test. The spawning tray consists of a frame (plexi-glass or other suitable material), 5-7 cm high with a 2-5 mm coarse net attached at the top and a 10-30 μm fine net at the bottom. A number of "spawning-trees", consisting of untwisted nylon rope, are attached to the coarse net of the frame. After the fish have been left in dark for 12 hours, a faint light is turned on which will initiate the spawning. Two to four hours after spawning, the spawning tray is removed and the eggs collected. The spawning tray will prevent the fish from eating the eggs and at the same time permit an easy collection of the eggs. The group of fish should have spawned at least once before the spawning from which eggs are used for testing.
(ii) Five to 10 male and female fish are housed individually at least two weeks prior to the intended spawning. After 5-10 days, the abdomens of the females will be distended and their genital papillae visible. Male fish lack papillae. Spawning is performed in spawning tanks equipped with a false mesh bottom (as above). The tank is filled with dilution water, so that the depth of water above the mesh is 5-10 cm. One female and two males are placed in the tank the day before the intended spawning. The water temperature is gradually increased one degree higher than the acclimatisation temperature. The light is turned off and the tank is left as undisturbed as possible. In the morning a faint light is turned on which will initiate spawning. After 2-4 hours, the fish are removed and the eggs collected. If larger batches of eggs are needed than can be obtained from one female, a sufficient number of spawning tanks may be set-up in parallel. By recording the reproduction success of the individual females prior to the test (size of batch and quality), those females with highest reproduction success may be selected for breeding.
The eggs should be transferred to the test vessels by means of glass tubes (inner diameter not less than 4 mm) provided with a flexible suction bulb. The amount of water accompanying the eggs on their transfer should be as small as possible. The eggs are heavier than water and sink out of the tube. Care should be taken to prevent eggs (and larvae) coming into contact with the air. Microscopic examination of sample(s) of the batch(es) should be carried out to ensure that there are no irregularities in the first developmental stages. Disinfection of the eggs is not allowed.
The mortality rate of the eggs is highest within the first 24 hours after fertilisation. A mortality of 5-40 % is often seen during this period. Eggs degenerate as a result of unsuccessful fertilisation or development failures. The quality of the batch of eggs seems to depend on the female fish, as some females consistently produce good quality eggs, others never will. Also the development rate and the rate of hatching vary from one batch to another. The successfully fertilised eggs and the yolk sac larvae survive well, normally above 90 %. At 25 °C the eggs will hatch 3-5 days after fertilization and the yolk sac will be absorbed approximately 13 days after fertilisation.
The embryonic development has been well defined by Hisaoka and Battle (2). Due to the transparency of the eggs and post-hatch larvae, the development of the fish may be followed and the presence of malformations may be observed. Approximately four hours after spawning, the non-fertilised eggs may be distinguished from the fertilised (3). For this examination, eggs and larvae are placed in test vessels of small volume and studied under a microscope.
The test conditions, which apply to the early life stages, are listed in Appendix 2. Optimal values for pH values and hardness of the dilution water are 7,8 and 250 mg CaCO3/l respectively.
CALCULATIONS AND STATISTICS
A two-stage approach is proposed. First, the data on mortality, abnormal development and hatching-time are analysed statistically. Then, for those concentrations at which no adverse effects on any of these parameters have been detected, the body length is statistically evaluated. This approach is advisable since the toxicant may selectively kill smaller fish, delay hatching-time and induce gross malformations, thus leading to biased length measurements. Furthermore, there will be roughly the same number of fish to be measured per treatment, ensuring the validity of the test statistics.
LC50 AND EC50 DETERMINATIONS
The percentage of surviving eggs and larvae is calculated and corrected for mortality in the controls in accordance with Abbott's formula (4):
>REFERENCE TO A GRAPHIC>
where,
P= corrected % survival
P'= % survival observed in the test concentration
C= survival in the control
If possible, the LC50 is determined by a suitable method at the end of the test.
If the inclusion of morphological abnormalities in the EC50 statistic is desired, guidance can be found in Stephan (5).
ESTIMATION OF LOEC AND NOEC
An objective of the egg and sac-fry test is to compare the non-zero concentrations with the control, i.e. to determine the LOEC. Therefore multiple comparison procedures should be utilised (6) (7) (8) (9) (10).
REFERENCES
(1) Laale H. W. (1977). The Biology and Use of the Zebrafish (Brachydanio rerio) in Fisheries Research. A Literature Review. J. Fish Biol. 10, pp. 121-173.
(2) Hisaoka K. K. and Battle H. I. (1958). The Normal Development Stages of the Zebrafish Brachydanio rerio (Hamilton-Buchanan) J. Morph., 102, 311 pp.
(3) Nagel R. (1986). Untersuchungen zur Eiproduktion beim Zebrabärbling (Brachydanio rerio Hamilton-Buchanan). Journal of Applied Ichthyology, 2, pp. 173-181.
(4) Finney D. J. (1971). Probit Analysis, 3rd ed., Cambridge University Press, Great Britain, pp. 1-333.
(5) Stephan C. E. (1982). Increasing the Usefulness of Acute Toxicity Tests. Aquatic Toxicology and Hazard Assessment: Fifth Conference, ASTM STP 766, J. G. Pearson, R. B. Foster and W. E. Bishop, Eds., American Society for Testing and Materials, pp. 69-81.
(6) Dunnett C. W. (1955). A Multiple Comparisons Procedure for Comparing Several Treatments with a Control. J. Amer. Statist. Assoc., 50, pp. 1096-1121.
(7) Dunnett C. W. (1964). New Tables for Multiple Comparisons with a Control. Biometrics, 20, pp. 482-491.
(8) Williams D. A. (1971). A Test for Differences Between Treatment Means when Several Dose Levels are Compared with a Zero Dose Control. Biometrics, 27, pp. 103-117.
(9) Williams D. A. (1972). The Comparison of Several Dose Levels with a Zero Dose Control. Biometrics 28, pp. 519-531.
(10) Sokal R. R. and Rohlf F. J. (1981). Biometry, the Principles and Practice of Statistics in Biological Research, W. H. Freeman and Co., San Francisco.
APPENDIX 2
TEST CONDITIONS, DURATION AND SURVIVAL CRITERIA FOR RECOMMENDED SPECIES
>TABLE>
APPENDIX 3
TEST CONDITIONS, DURATION AND SURVIVAL CRITERIA FOR OTHER WELL-DOCUMENTED SPECIES
>TABLE>
APPENDIX 4
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER
>TABLE>
C.16. HONEYBEES - ACUTE ORAL TOXICITY TEST
1. METHOD
This acute toxicity test method is a replicate of the OECD TG 213 (1998).
1.1. INTRODUCTION
This toxicity test is a laboratory method, designed to assess the oral acute toxicity of plant protection products and other chemicals, to adult worker honeybees.
In the assessment and evaluation of toxic characteristics of substances, determination of acute oral toxicity in honeybees may be required, e.g. when exposure of bees to a given chemical is likely. The acute oral toxicity test is carried out to determine the inherent toxicity of pesticides and other chemicals to bees. The results of this test should be used to define the need for further evaluation. In particular, this method can be used in step-wise programmes for evaluating the hazards of pesticides to bees, based on sequential progression from laboratory toxicity tests to semi-field and field experiments (1). Pesticides can be tested as active substances (a.s.) or as formulated products.
A toxic standard should be used to verify the sensitivity of the bees and the precision of the test procedure.
1.2. DEFINITIONS
Acute oral toxicity: is the adverse effects occurring within a maximum period of 96 hours (h) of an oral administration of a single dose of test substance.
Dose: is the amount of test substance consumed. Dose is expressed as mass (μg) of test substance per test animal (μg/bee). The real dose for each bee can not be calculated as the bees are fed collectively, but an average dose can be estimated (totally consumed test substance/number of test bees in one cage).
LD50 (median lethal dose) oral: is a statistically derived single dose of a substance that can cause death in 50 % of animals when administered by the oral route. The LD50 value is expressed in (μg) of test substance per bee. For pesticides, the test substance may be either an active substance (a.s.) or a formulated product containing one or more than one active substance.
Mortality: an animal is recorded as dead when it is completely immobile.
1.3. PRINCIPLE OF THE TEST METHOD
Adult worker honeybees (Apis mellifera) are exposed to a range of doses of the test substance dispersed in sucrose solution. The bees are then fed the same diet, free of the test substance. Mortality is recorded daily during at least 48 h and compared with control values. If the mortality rate is increasing between 24 h and 48 h whilst control mortality remains at an accepted level, i.e. <= 10 %, it is appropriate to extend the duration of the test to a maximum of 96 h. The results are analysed in order to calculate the LD50 at 24 h and 48 h and, in case the study is prolonged, at 72 h and 96 h.
1.4. VALIDITY OF THE TEST
For a test to be valid, the following conditions apply:
- the average mortality for the total number of controls must not exceed 10 % at the end of the test,
- the LD50 of the toxic standard meets the specified range.
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Collection of bees
Young adult worker bees of the same race should be used, i.e. bees of the same age, feeding status, etc. Bees should be obtained from adequately fed, healthy, as far as possible disease-free and queen-right colonies with known history and physiological status. They could be collected in the morning of use or in the evening before test and kept under test conditions to the next day. Bees collected from frames without brood are suitable. Collection in early spring or late autumn should be avoided as the bees have a changed physiology during this time. If tests must be conduced in early spring or late autumn, bees can be emerged in an incubator and reared for one week with "bee bread" (pollen collected from the comb) and sucrose solution. Bees treated with chemical substances, such as antibiotics, anti-varroa products, etc., should not be used for toxicity test for four weeks from the time of the end of the last treatment.
1.5.2. Housing and feeding conditions
Easy to clean and well-ventilated cages are used. Any appropriate material can be used, e.g. stainless steel, wire mesh, plastic or disposable wooden cages, etc. Groups of 10 bees per cage are preferred. The size of test cages should be appropriate to the number of bees, i.e. providing adequate space.
The bees should be held in the dark in an experimental room at a temperature of 25 ± 2 °C. The relative humidity, normally around 50-70 %, should be recorded throughout the test. Handling procedures, including treatment and observations may be conducted under (day) light. Sucrose solution in water with a final concentration of 500 g/l (50 % w/v) is used as food. After given test doses, food should be provided ad libitum. The feeding system should allow recording food intake for each cage (see section 1.6.3.1). A glass tube (approximately 50 mm long and 10 mm wide with the open end narrowed to about 2 mm diameter) can be used.
1.5.3. Preparation of bees
The collected bees are randomly allocated to test cages, which are randomly placed in the experimental room.
The bees may be starved for up to 2 h before the initiation of the test. It is recommended that the bees are deprived of food prior to treatment so that all bees are equal in terms of their gut contents at the start of the test. Moribund bees should be rejected and replaced by healthy bees before starting the test.
1.5.4. Preparation of doses
Where the test substance is a water miscible compound this may be dispersed directly in 50 % sucrose solution. For technical products and substances of low water solubility, vehicles such as organic solvent, emulsifiers or dispersants of low toxicity to bees may be used (e.g. acetone, dimethylformamide, dimethylsulfoxide). The concentration of the vehicle depends on the solubility of the test substance and it should be the same for all concentrations tested. However, a concentration of the vehicle of 1 % is generally appropriate and should not be exceeded.
Appropriate control solutions should be prepared, i.e. where a solvent or a dispersant is used to solubilise the test substance, two separate control groups should be used: a solution in water, and a sucrose solution with the solvent/carrier at the concentration used in dosing solutions.
1.6. PROCEDURE
1.6.1. Test and control groups
The number of doses and replicates tested should meet the statistical requirements for determination of LD50 with 95 % confidence limits. Normally, five doses in a geometric series, with a factor not exceeding 2,2, and covering the range for LD50, are required for the test. However, the dilution factor and the number of concentrations for dosage have to be determined in relation to the slope of the toxicity curve (dose versus mortality) and with consideration taken to the statistical method which is chosen for analysis of the results. A range-finding test enables the choice of the appropriate concentrations for dosage.
A minimum of three replicate test groups, each of 10 bees, should be dosed with each test concentration. A minimum of three control batches, each of 10 bees, should be run in addition to the test series. Control batches should also be included for the solvents/carriers used (see section 1.5.4).
1.6.2. Toxic standard
A toxic standard should be included in the test series. At least three doses should be selected to cover the expected LD50 value. A minimum of three replicate cages, each containing ten bees, should be used with each test dose. The preferred toxic standard is dimethoate, for which the reported oral LD50-24 h is in the range 0,10-0,35 ìg a.s./bee (2). However, other toxic standards would be acceptable where sufficient data can be provided to verify the expected dose response (e.g. parathion).
1.6.3. Exposure
1.6.3.1. Administration of doses
Each test group of bees must be provided with 100-200 ìl of 50 % sucrose solution in water, containing the test substance at the appropriate concentration. A larger volume is required for products of low solubility, low toxicity or low concentration in the formulation, as higher proportions in the sucrose solution have to be used. The amount of treated diet consumed per group should be monitored. Once consumed (usually within 3-4 h), the feeder should be removed from the cage and replaced with one containing sucrose solution alone. The sucrose solutions are then provided ad libitum. For some compounds, at higher concentrations rejection of test dose may result in little or no food being consumed. After a maximum of 6 h, unconsumed treated diet should be replaced with the sucrose solution alone. The amount of treated diet consumed should be assessed (e.g. measurement of volume/weight of treated diet remaining).
1.6.3.2. Duration
The duration of the test is preferably 48 h after the test solution has been replaced with sucrose solution alone. If mortality continues to rise by more than 10 % after the first 24 h, the test duration should be extended to a maximum of 96 h provided that control mortality does not exceed 10 %.
1.6.4. Observations
Mortality is recorded at 4 h after starting the test and thereafter at 24 h and 48 h (i.e. after giving dose). If a prolonged observation period is required, further assessments should be made at 24-h intervals, up to a maximum of 96 h, provided that the control mortality does not exceed 10 %.
The amount of diet consumed per group should be estimated. Comparison of the rates of consumption of treated and untreated diet within the given 6 h can provide information about palatability of the treated diet.
All abnormal behavioural effects observed during the testing period should be recorded.
1.6.5. Limit test
In some cases (e.g. when a test substance is expected to be of low toxicity) a limit test may be performed, using 100 ìg a.s./bee in order to demonstrate that the LD50 is greater than this value. The same procedure should be used, including three replicate test groups for the test dose, the relevant controls, the assessment of the amount of treated diet comsumed, and the use of the toxic standard. If mortalities occur, a full study should be conducted. If sublethal effects are observed (see section 1.6.4), these should be recorded.
2. DATA AND REPORTING
2.1. DATA
Data should be summarised in tabular form, showing for each treatment group, as well as control and toxic standard groups, the number of bees used, mortality at each observation time and number of bees with adverse behaviour. Analyse the mortality data by appropriate statistical methods (e.g. probit analysis, moving average, binomial probability) (3) (4). Plot dose-response curves at each recommended observation time and calculate the slopes of the curves and the median lethal doses (LD50) with 95 % confidence limits. Corrections for control mortality could be made using Abbott's correction (4) (5). Where treated diet is not completely consumed, the dose of test substance consumed per group should be determined. LD50 should be expressed in ìg of test substance per bee.
2.2. TEST REPORT
The test report must include the following information:
2.2.1. Test substance:
- physical nature and relevant physical-chemical properties (e.g. stability in water, vapour pressure),
- chemical identification data, including structural formula, purity (i.e. for pesticides, the identity and concentration of active substance(s)).
2.2.2. Test species:
- scientific name, race, approximate age (in weeks), collection method, date of collection,
- information on colonies used for collection of test bees including health, any adult disease, any pre-treatment, etc.
2.2.3. Test conditions:
- temperature and relative humidity of experimental room,
- housing conditions including type, size and material of cages,
- methods of preparation of stock and test solutions (the solvent and its concentration must be given, when used),
- method of preparation of stock solutions and frequency of renewal (the solubilising agent and its concentration must be given, when used),
- test design, e.g. number and test concentrations used, number of controls; for each test concentration and control, number of replicate cages and number of bees per cage,
- date of test.
2.2.4. Results:
- results of preliminary range-finding study if performed,
- raw data: mortality at each dose tested at each observation time,
- graph of the dose-response curves at the end of the test,
- LD50 values with 95 % confidence limits, at each of the recommended observation times, for test substance and toxic standard,
- statistical procedures used for determining the LD50,
- mortality in controls,
- other biological effects observed or measured e.g. abnormal behaviour of the bees (including rejection of the test dose), rate of consumption of diet in treated and untreated groups,
- any deviation from the test procedures described here and any other relevant information.
3. REFERENCES
(1) EPPO/Council of Europe (1993). Decision-Making Scheme for the Environmental Risk Assessment of Plant Protection Products - Honeybees. EPPO Bulletin, Vol. 23, N.1, pp. 151-165. March 1993.
(2) Gough, H. J., McIndoe, E.C., Lewis, G.B. (1994). The use of dimethoate as a reference compound in laboratory acute toxicity tests on honeybees (Apis mellifera L.) 1981-1992. Journal of Apicultural Research, 22, pp. 119-125.
(3) Litchfield, J.T. and Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments. Jour. Pharmacol. and Exper. Ther., 96, pp. 99-113.
(4) Finney, D. J. (1971). Probit Analysis. 3rd ed., Cambridge, London and New York.
(5) Abbott, W. S. (1925). A method for computing the effectiveness of an insecticide. Jour. Econ. Entomol., 18, pp. 265-267.
C.17. HONEYBEES - ACUTE CONTACT TOXICITY TEST
1. METHOD
This acute toxicity test method is a replicate of the OECD TG 214 (1998).
1.1. INTRODUCTION
This toxicity test is a laboratory method, designed to assess the acute contact toxicity of plant protection products and other chemicals to adult worker honeybees.
In the assessment and evaluation of toxic characteristics of substances, determination of acute contact toxicity in honeybees may be required, e.g. when exposure of bees to a given chemical is likely. The acute contact toxicity test is carried out to determine the inherent toxicity of pesticides and other chemicals to bees. The results of this test should be used to define the need for further evaluation. In particular, this method can be used in step-wise programmes for evaluating the hazards of pesticides to bees, based on sequential progression from laboratory toxicity tests to semi-field and field experiments (1). Pesticides can be tested as active substances (a.s.) or as formulated products.
A toxic standard should be used to verify the sensitivity of the bees and the precision of the test procedure.
1.2. DEFINITIONS
Acute oral toxicity: is the adverse effects occurring within a maximum period of 96 hours of a topical application of a single dose of a substance.
Dose: is the amount of test substance applied. Dose is expressed as mass (ìg) of test substance per test animal (ìg/bee).
LD50 (median lethal dose) contact: is a statistically derived single dose of a substance that can cause death in 50 % of animals when administered by the contact. The LD50 value is given in ìg of test substance per bee. For pesticides, the test substance may be either an active substance (a.s.) or a formulated product containing one or more than one active substance.
Mortality: an animal is recorded as dead when it is completely immobile.
1.3. PRINCIPLE OF THE TEST METHOD
Adult worker honeybees (Apis mellifera) are exposed to a range of doses of the test substance dissolved in appropriate carrier, by direct application to the thorax (droplets). The test duration is 48 h. If the mortality rate is increasing between 24 h and 48 h whilst control mortality remains at an accepted level, i.e. <= 10 %, it is appropriate to extend the duration of the test to a maximum of 96 h. Mortality is recorded daily and compared with control values. The results are analysed in order to calculate the LD50 at 24 h and 48 h, and in case the study is prolonged at 72 h and 96 h.
1.4. VALIDITY OF THE TEST
For a test to be valid, the following conditions apply:
- the average mortality for the total numbers of controls must not exceed 10 % at the end of the test,
- the LD50 of the toxic standard meets the specified range.
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Collection of bees
Young adult worker bees should be used, i.e. bees of the same age, feeding status, race etc. Bees should be obtained from adequately fed, healthy, as far as possible disease-free and queen-right colonies with known history and physiological status. They could be collected in the morning of use or in the evening before test and kept under test conditions to the next day. Bees collected from frames without brood are suitable. Collection in early spring or late autumn should be avoided, as the bees have a changed physiology during the time. If tests have to be conduced in early spring or late autumn, bees can be emerged in an incubator and reared for one week with "bee bread" (pollen collected from the comb) and sucrose solution. Bees treated with chemical substances, such as antibiotics, anti-varroa products, etc., should not be used for toxicity test for four weeks from the time of the end of the last treatment.
1.5.2. Housing and feeding conditions
Easy to clean and well-ventilated cages are used. Any appropriate material can be used, e.g. stainless steel, wire mesh, plastic, disposable wooden cages, etc. The size of test cages should be appropriate to the number of bees, i.e. providing adequate space. Groups of ten bees per cage are preferred.
The bees should be held in the dark in an experimental room at a temperature of 25 ± 2 °C. The relative humidity, normally around 50-70 %, should be recorded throughout the test. Handling procedures, including treatment and observations may be conducted under (day) light. Sucrose solution in water with a final concentration of 500 g/l (50 % w/v) should be used as food and provided ad libitum during the test time, using a bee feeder. This can be a glass tube (approximately 50 mm long and 10 mm wide with the open end narrowed to about 2 mm diameter).
1.5.3. Preparation of bees
The collected bees may be anaesthetised with carbon dioxide or nitrogen for application of the test substance. The amount of anaesthetic used and time of exposure should be minimised. Moribund bees should be rejected and replaced by healthy bees before starting the test.
1.5.4. Preparation of doses
The test substance is to be applied as solution in a carrier, i.e. an organic solvent or a water solution with a wetting agent. As organic solvent, acetone is preferred but other organic solvents of low toxicity to bees may be used (e.g. dimethylformamide, dimethylsulfoxide). For water dispersed formulated products and highly polar organic substances not soluble in organic carrier solvents, solutions may be easier to apply if prepared in a weak solution of a commercial wetting agent (e.g. Agral, Cittowett, Lubrol, Triton, Tween).
Appropriate control solutions should be prepared, i.e. where a solvent or a dispersant is used to solubilise the test substance, two separate control groups should be used, one treated with water, and one treated with the solvent/dispersant.
1.6. PROCEDURE
1.6.1. Test and control groups
The number of doses and replicates tested should meet the statistical requirements for determination LD50 with 95 % confidence limits. Normally five doses in a geometric series, with a factor not exceeding 2,2, and covering the range for LD50, are required for the test. However, the number of doses have to be determined in relation to the slope of the toxicity curve (dose versus mortality) and with consideration taken to the statistical method which is chosen for analysis of the results. A range-finding test enables the choice of the appropriate doses.
A minimum of three replicate test groups, each of 10 bees, should be dosed with each test concentration.
A minimum of three control batches, each of 10 bees, should be run in addition to the test series. If an organic solvent or a wetting agent is used three additional control batches of each 10 bees for the solvent or the wetting agent have to be included.
1.6.2. Toxic standard
A toxic standard must be included in the test series. At least three doses should be selected to cover the expected LD50 value. A minimum of three replicate cages, each containing 10 bees, should be used with each test dose. The preferred toxic standard is dimethoate, for which the reported contact LD50-24 h is in the range 0,10-0,30 ìg a.s./bee (2). However, other toxic standards would be acceptable where sufficient data can be provided to verify the expected dose response (e.g. parathion).
1.6.3. Exposure
1.6.3.1. Administration of doses
Anaesthetised bees are individually treated by topical application.The bees are randomly assigned to the different test doses and controls. A volume of 1 ìl of solution containing the test substance at the suitable concentration should be applied with a microapplicator to the dorsal side of the thorax of each bee. Other volumes may be used, if justified. After application, the bees are allocated to test cages and supplied with sucrose solutions.
1.6.3.2. Duration
The duration of the test is preferably 48 h. If mortality increases by more than 10 % between 24 h and 48 h, the test duration should be extended up to a maximum of 96 h provided that control mortality does not exceed 10 %.
1.6.4. Observations
Mortality is recorded at 4 h after dosing and thereafter at 24-h and 48 h. If a prolonged observation period is required, further assessments should be made, at 24-h intervals, to a maximum of 96 h, provided that the control mortality does not exceeding 10 %.
All abnormal behavioural effects observed during the testing period should be recorded.
1.6.5. Limit test
In some cases (e.g. when a test substance is expected to be of low toxicity) limit test may be performed, using 100 ìg a.s./bee in order to demonstrate that the LD50 is greater than this value. The same procedure should be used, including three replicate test groups for the test dose, the relevant controls, and the use of the toxic standard. If mortalities occur, a full study should be conducted. If sublethal effects are observed (see section 1.6.4) these should be recorded.
2. DATA AND REPORTING
2.1. DATA
Data should be summarised in tabular form, showing for each treatment group, as well as, control and toxic standard groups, the number of bees used, mortality at each observation time and number of bees with adverse behaviour. Analyse the mortality data by appropriate statistical methods (e.g. probit analysis, moving average, binomial probability) (3) (4). Plot dose-response curves at each recommended observation time (i.e. 24 h, 48 h and, if relevant, 72 h, 96 h) and calculate the slopes of the curves and the median lethal doses (LD50) with 95 % confidence limits. Corrections for control mortality could be made using Abbott's correction (4) (5). LD50 should be expressed in ìg of test substance per bee.
2.2. TEST REPORT
The test report must include the following information:
2.2.1. Test substance:
- physical nature and physical-chemical properties (e.g. stability in water, vapour pressure),
- chemical identification data, including structural formula, purity (i.e. for pesticides, the identity and concentration of active substance(s)).
2.2.2. Test species:
- scientific name, race, approximate age (in weeks), collection method, date of collection,
- information on colonies used for collection of test bees including health, any adult disease, any pre-treatment, etc.
2.2.3. Test conditions:
- temperature and relative humidity of experimental room,
- housing conditions including type, size and material of cages,
- methods of administration of test substance, e.g. carrier solvent used, volume of test solution applied anaesthetics used,
- test design, e.g. number and test doses used, number of controls; for each test dose and control, number of replicate cages and number of bees per cage,
- date of test.
2.2.4. Results:
- results of preliminary range-finding study if performed,
- raw data: mortality at each concentration tested at each observation time,
- graph of the dose-response curves at the end of the test,
- LD50 values, with 95 % confidence limits, at each of the recommended observation times, for test substance and toxic standard,
- statistical procedures used for determining the LD50,
- mortality in controls,
- other biological effects observed or measured and any abnormal responses of the bees,
- any deviation from the test method procedures described here and any other relevant information.
3. REFERENCES
(1) EPPO/Council of Europe (1993). Decision-Making Scheme for the Environmental Risk Assessment of Plant Protection Products - Honeybees. EPPO bulletin, Vol. 23, N.1, pp. 151-165. March 1993.
(2) Gough, H. J., McIndoe, E. C., Lewis, G. B. (1994). The use of dimethoate as a reference compound in laboratory acute toxicity tests on honeybees (Apis mellifera L.), 1981-1992. Journal of Apicultural Research 22, pp. 119-125.
(3) Litchfield, J. T. and Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments. Jour. Pharmacol. and Exper. Ther., 96, pp. 99-113.
(4) Finney, D. J. (1971). Probit Analysis. 3rd ed., Cambridge, London and New York.
(5) Abbott, W. S. (1925). A method for computing the effectiveness of an insecticide. Jour. Econ. Entomol. 18, pp. 265-267.
C.18. ADSORPTION/DESORPTION USING A BATCH EQUILIBRIUM METHOD
1. METHOD
This method is a replicate of the OECD TG 106, for the Determination of Soil Adsorption/Desorption, using a Batch Equilibrium Method (2000).
1.1. INTRODUCTION
The method takes into account a ring test and a workshop for soil selection for the development of an adsorption test (1) (2) (3) (4) and also existing guidelines at national level (5) (6) (7) (8) (9) (10) (11).
Adsorption/desorption studies are useful for generating essential information on the mobility of chemicals and their distribution in the soil, water and air compartments of the biosphere (12) (13) (14) (15) (16) (17) (18) (19) (20) (21). The information can be used in the prediction or estimation, for example, of the availability of a chemical for degradation (22) (23), transformation and uptake by organisms (24); leaching through the soil profile (16) (18) (19) (21) (25) (26) (27) (28); volatility from soil (21) (29) (30); run-off from land surfaces into natural waters (18) (31) (32). Adsorption data can be used for comparative and modelling purposes (19) (33) (34) (35).
The distribution of a chemical between soil and aqueous phases is a complex process depending on a number of different factors: the chemical nature of the substance (12) (36) (37) (38) (39) (40), the characteristics of the soil (4) (12) (13) (14) (41) (42) (43) (44) (45) (46) (47) (48) (49), and climatic factors such as rainfall, temperature, sunlight and wind. Thus, the numerous phenomena and mechanisms involved in the process of adsorption of a chemical by soil cannot be completely defined by a simplified laboratory model such as the present method. However, even if this attempt cannot cover all the environmentally possible cases, it provides valuable information on the environmental relevance of the adsorption of a chemical.
See also General Introduction.
1.2. SCOPE
The method is aimed at estimating the adsorption/desorption behaviour of a substance on soils. The goal is to obtain a sorption value which can be used to predict partitioning under a variety of environmental conditions; to this end, equilibrium adsorption coefficients for a chemical on various soils are determined as a function of soil characteristics (e.g. organic carbon content, clay content and soil texture and pH). Different soil types have to be used in order to cover as widely as possible the interactions of a given substance with naturally occurring soils.
In this method, adsorption represents the process of the binding of a chemical to surfaces of soils; it does not distinguish between different adsorption processes (physical and chemical adsorption) and such processes as surface catalysed degradation, bulk adsorption or chemical reaction. Adsorption that will occur on colloids particles (diameter < 0,2 ìm) generated by the soils is not taken into account.
The soil parameters that are believed most important for adsorption are: organic carbon content (3) (4) (12) (13) (14) (41) (43) (44) (45) (46) (47) (48); clay content and soil texture (3) (4) (41) (42) (43) (44) (45) (46) (47) (48) and pH for ionisable compounds (3) (4) (42). Other soil parameters which may have an impact on the adsorption/desorption of a particular substance are the effective cation exchange capacity (ECEC), the content of amorphous iron and aluminium oxides, particularly for volcanic and tropical soils (4), as well as the specific surface (49).
The test is designed to evaluate the adsorption of a chemical on different soil types with a varying range of organic carbon content, clay content and soil texture, and pH. It comprises three tiers:
Tier 1: Preliminary study in order to determine:
- the soil/solution ratio,
- the equilibrium time for adsorption and the amount of test substance adsorbed at equilibrium,
- the adsorption of the test substance on the surfaces of the test vessels and the stability of the test substance during the test period.
Tier 2: Screening test: the adsorption is studied in five different soil types by means of adsorption kinetics at a single concentration and determination of distribution coefficient Kd and Koc.
Tier 3: Determination of Freundlich adsorption isotherms to determine the influence of concentration on the extent of adsorption on soils.
Study of desorption by means of desorption kinetics/Freundlich desorption isotherms (Appendix 1).
1.3. DEFINITIONS AND UNITS
>TABLE>
1.4. PRINCIPLE OF THE TEST METHOD
Known volumes of solutions of the test substance, non-labelled or radiolabelled, at known concentrations in 0,01 M CaCl2 are added to soil samples of known dry weight which have been pre-equilibrated in 0,01 M CaCl2. The mixture is agitated for an appropriate time. The soil suspensions are then separated by centrifugation and, if so wished, filtration and the aqueous phase is analysed. The amount of test substance adsorbed on the soil sample is calculated as the difference between the amount of test substance initially present in solution and the amount remaining at the end of the experiment (indirect method).
As an option, the amount of the test substance adsorbed can also be directly determined by analysis of soil (direct method). This procedure which involves step-wise soil extraction with appropriate solvent, is recommended in cases where the difference in the solution concentration of the substance cannot be accurately determined. Examples of such cases are: adsorption of the test substance on surface of the test vessels, instability of the test substance in the time scale of the experiment, weak adsorption giving only small concentration change in the solution; and strong adsorption yielding low concentration which cannot be accurately determined. If radiolabelled substance is used, the soil extraction may be avoided by analysis of the soil phase by combustion and liquid scintillation counting. However, liquid scintillation counting is an unspecific technique which cannot differentiate between parental and transformation products; therefore it should be used only if the test chemical is stable for the duration of the study.
1.5. INFORMATION ON THE TEST SUBSTANCE
Chemical reagents should be of analytical grade. The use of non-labelled test substances with known composition and preferably at least 95 % purity or of radiolabelled test substances with known composition and radio-purity, is recommended. In the case of short half-life tracers, decay corrections should be applied.
Before carrying out a test for adsorption-desorption, the following information about the test substance should be available:
(a) water solubility (A.6);
(b) vapour pressure (A.4) and/or Henry's Law Constant;
(c) abiotic degradation: hydrolysis as a function of pH (C.7);
(d) partition coefficient (A.8);
(e) ready biodegradability (C.4) or aerobic and anaerobic transformation in soil;
(f) pKa of ionisable substances;
(g) direct photolysis in water (i.e. UV-Vis absorption spectrum in water, quantum yield) and photodegradation on soil.
1.6. APPLICABILITY OF THE TEST
The test is applicable to chemical substances for which an analytical method with sufficient accuracy is available. An important parameter that can influence the reliability of the results, especially when the indirect method is followed, is the stability of the test substance in the time scale of the test. Thus, it is a prerequisite to check the stability in a preliminary study; if a transformation in the time scale of the test is observed, it is recommended that the main study be performed by analysing both soil and aqueous phases.
Difficulties may arise in conducting this test for test substances with low water solubility (Sw < 10-4 g l-1), as well as for highly charged substances, due to the fact that the concentration in the aqueous phase cannot be measured analytically with sufficient accuracy. In these cases, additional steps have to be taken. Guidance on how to deal with these problems is given in the relevant sections of this method.
When testing volatile substances, care should be taken to avoid losses during the study.
1.7. DESCRIPTION OF THE METHOD
1.7.1. Apparatus and chemical reagents
Standard laboratory equipment, especially the following:
(a) Tubes or vessels to conduct the experiments. It is important that these tubes or vessels:
- fit directly in the centrifuge apparatus in order to minimise handling and transfer errors,
- be made of an inert material, which minimises adsorption of the test substance on its surface.
(b) Agitation device: overhead shaker or equivalent equipment; the agitation device should keep the soil in suspension during shaking.
(c) Centrifuge: preferably high-speed, e.g. centrifugation forces > 3000 g, temperature controlled, capable of removing particles with a diameter greater than 0,2 μm from aqueous solution. The containers should be capped during agitation and centrifugation to avoid volatility and water losses; to minimise adsorption on them, deactivated caps such as teflon lined screw caps should be used.
(d) Optional: filtration device; filters of 0,2 μm porosity, sterile, single use. Special care should be taken in the choice of the filter material, to avoid any losses of the test substance on it; for poorly soluble test substances, organic filter material is not recommended.
(e) Analytical instrumentation, suitable for measuring the concentration of the test chemical.
(f) Laboratory oven, capable of maintaining a temperature of 103 °C to 110 °C.
1.7.2. Characterisation and selection of soils
The soils should be characterised by three parameters considered to be largely responsible for the adsorptive capacity: organic carbon, clay content and soil texture, and pH. As already mentioned (see Scope) other physico-chemical properties of the soil may have an impact on the adsorption/desorption of a particular substance and should be considered in such cases.
The methods used for soil characterisation are very important and can have a significant influence on the results. Therefore, it is recommended that soil pH should be measured in a solution of 0,01 M CaCl2 (that is the solution used in adsorption/desorption testing) according to the corresponding ISO method (ISO-10390-1). It is also recommended that the other relevant soil properties be determined according to standard methods (for example ISO "Handbook of Soil Analysis"); this permits the analysis of sorption data to be based on globally standardised soil parameters. Some guidance for existing standard methods of soil analysis and characterisation is given in references (50-52). For calibration of soil test methods, the use of reference soils is recommended.
Guidance for selection of soils for adsorption/desorption experiments is given in Table 1. The seven selected soils cover soil types encountered in temperate geographical zones. For ionisable test substances, the selected soils should cover a wide range of pH, in order to be able to evaluate the adsorption of the substance in its ionised and unionised forms. Guidance on how many different soils to use at the various stages of the test is given under 1.9. Performance of the test.
If other soil types are preferred, they should be characterised by the same parameters and should have similar variation in properties to those described in Table 1, even if they do not match the criteria exactly.
Table 1: Guidance for selection of soil samples for adsorption-desorption
>TABLE>
1.7.3. Collection and storage of soil samples
1.7.3.1. Collection
No specific sampling techniques or tools are recommended; the sampling technique depends on the purpose of the study (53) (54) (55) (56) (57) (58).
The following should be considered:
(a) detailed information on the history of the field site is necessary; this includes location, vegetation cover, treatments with pesticides and/or fertilisers, biological additions or accidental contamination. Recommendations of the ISO standard on soil sampling (ISO 10381-6) should be followed with respect to the description of the sampling site;
(b) the sampling site has to be defined by UTM (Universal Transversal Mercator-Projection/European Horizontal Datum) or geographical coordinates; this could allow recollection of a particular soil in the future or could help in defining soil under various classification systems used in different countries. Also, only A horizon up to a maximum depth of 20 cm should be collected. Especially for the soil type n. 7 if a Oh horizon is present as part of the soil, it should be included in the sampling.
The soil samples should be transported using containers and under temperature conditions which guarantee that the initial soil properties are not significantly altered.
1.7.3.2. Storage
The use of soils freshly taken from the field is preferred. Only if this is not possible soil can be stored at ambient temperature and should be kept air-dried. No limit on the storage time is recommended, but soils stored for more than three years should be re-analysed prior to the use with respect to their organic carbon content, pH and CEC.
1.7.3.3. Handling and preparation of soil samples for the test
The soils are air-dried at ambient temperature (preferably between 20-25 °C). Disaggregation should be performed with minimal force, so that the original texture of the soil will be changed as little as possible. The soils are sieved to a particle size <= 2 mm; recommendations of the ISO standard on soil sampling (ISO 10381-6) should be followed with respect to the sieving process. Careful homogenisation is recommended, as this enhances the reproducibility of the results. The moisture content of each soil is determined on three aliquots with heating at 105 °C until there is no significant change in weight (approximately 12 h). For all calculations the mass of soil refers to oven dry mass, i.e. the weight of soil corrected for moisture content.
1.7.4. Preparation of the test substance for application to soil
The test substance is dissolved in a solution of 0,01 M CaCl2 in distilled or deionised water; the CaCl2 solution is used as the aqueous solvent phase to improve centrifugation and minimise cation exchange. The concentration of the stock solution should preferably be three orders of magnitude higher than the detection limit of the analytical method used. This threshold safeguards accurate measurements with respect to the methodology followed in this method; additionally, the stock solution concentration should be below water solubility of the test substance.
The stock solution should preferably be prepared just before application to soil samples and should be kept closed in the dark at 4 °C. The storage time depends on the stability of the test substance and its concentration in the solution.
Only for poorly soluble substances (Sw < 10-4 g l-1), an appropriate solubilising agent may be needed when it is difficult to dissolve the test substance. This solubilising agent: (a) should be miscible with water such as methanol or acetonitrile; (b) its concentration should not exceed 1 % of the total volume of the stock solution and should constitute less than that in the solution of the test substance which will come in contact with the soil (preferably less than 0,1 %); and (c) should not be a surfactant or undergo solvolytic reactions with the test chemical. The use of a solubilising agent should be stipulated and justified in the reporting of the data.
Another alternative for poorly soluble substances is to add the test substance to the test system by spiking: the test substance is dissolved in an organic solvent, an aliquot of which is added to the system of soil and 0,01 M solution of CaCl2 in distilled or deionised water. The content of organic solvent in the aqueous phase should be kept as low as possible, normally not exceeding 0,1 %. Spiking from an organic solution may suffer from volume unreproducibility. Thus, an additional error may be introduced as the test substance and co-solvent concentration would not be the same in all tests.
1.8. PREREQUISITES FOR PERFORMING THE ADSORPTION/DESORPTION TEST
1.8.1. Analytical method
The key parameters that can influence the accuracy of sorption measurements include the accuracy of the analytical method in analysis of both the solution and adsorbed phases, the stability and purity of the test substance, the attainment of sorption equilibrium, the magnitude of the solution concentration change, the soil/solution ratio and changes in the soil structure during the equilibration process (35) (59-62). Some examples bearing upon the accuracy issues are given in Appendix 2.
The reliability of the analytical method used must be checked at the concentration range which is likely to occur during the test. The experimenter should feel free to develop an appropriate method with appropriate accuracy, precision, reproducibility, detection limits and recovery. Guidance on how to perform such a test is given by the experiment below.
An appropriate volume of 0,01 M CaCl2, e.g. 100 cm3, is agitated during 4 h with a weight of soil, e.g. 20 g, of high adsorbability, i.e. with high organic carbon and clay content; these weights and volumes may vary depending on analytical needs, but a soil/solution ratio of 1:5 is a convenient starting point. The mixture is centrifuged and the aqueous phase may be filtrated. A certain volume of the test substance stock solution is added to the latter to reach a nominal concentration within the concentration range which is likely to occur during the test. This volume should not exceed 10 % of the final volume of the aqueous phase, in order to change as little as possible the nature of the pre-equilibration solution. The solution is analysed.
One blank run consisting of the system soil + CaCl2 solution (without test substance) must be included, in order to check for artefacts in the analytical method and for matrix effects caused by the soil.
The analytical methods which can be used for sorption measurements include gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC), spectrometry (e.g. GC/mass spectrometry, HPLC/mass spectrometry) and liquid scintillation counting (for radiolabelled substances). Independent of the analytical method used, it is considered suitable if the recoveries are between 90 % and 110 % of the nominal value. In order to allow for detection and evaluation after partitioning has taken place, the detection limits of the analytical method should be at least two orders of magnitude below the nominal concentration.
The characteristics and detection limits of the analytical method available for carrying out adsorption studies play an important role in defining the test conditions and the whole experimental performance of the test. This method follows a general experimental path and provides recommendations and guidance for alternative solutions where the analytical method and laboratory facilities may impose limitations.
1.8.2. Selection of optimal soil/solution ratios
Selection of appropriate soil to solution ratios for sorption studies depends on the distribution coefficient Kd and the relative degree of adsorption desired. The change of the substance concentration in the solution determines the statistical accuracy of the measurement based on the form of adsorption equation and the limit of the analytical methodology, in detecting the concentration of the chemical in solution. Therefore, in general practice it is useful to settle on a few fixed ratios, for which the percentage adsorbed is above 20 %, and preferably > 50 % (62), while care should be taken to keep the test substance concentration in the aqueous phase high enough to be measured accurately. This is particularly important in the case of high adsorption percentages.
A convenient approach to selecting the appropriate soil/water ratios, is based on an estimate of the Kd value either by preliminary studies or by established estimation techniques (Appendix 3). Selection of an appropriate ratio can then be made based on a plot of soil/solution ratio versus Kd for fixed percentages of adsorption (Fig. 1). In this plot it is assumed that the adsorption equation is linear(3). The applicable relationship is obtained by rearranging equation (4) of the Kd in the form of equation (1):
>REFERENCE TO A GRAPHIC>
or in its logarithmic form assuming that
>REFERENCE TO A GRAPHIC>:
>REFERENCE TO A GRAPHIC>
>PIC FILE= "L_2001225EN.020701.TIF">
Fig. 1. Relationship between soil to solution ratios and Kd at various percentages of adsorbed test substance
Fig. 1 shows soil/solution ratios required as a function of Kd for different levels of adsorption. For example, with a soil/solution ratio of 1:5 and a Kd of 20, approximately 80 % adsorption would occur. To obtain 50 % adsorption for the same Kd, a 1:25 ratio must be used. This approach to selecting the appropriate soil/solution ratios gives the investigator the flexibility to meet experimental needs.
Areas which are more difficult to deal with are those where the chemical is highly or very slightly adsorbed. Where low adsorption occurs, a 1:1 soil/solution ratio is recommended, although for some very organic soil types smaller ratios may be necessary to obtain a slurry. Care must be taken with the analytical methodology to measure small changes in solution concentration; otherwise the adsorption measurement will be inaccurate. On the other hand, at very high distribution coefficients Kd, one can go up to a 1:100 soil/solution ratio in order to leave a significant amount of chemical in solution. However, care must be taken to ensure good mixing, and adequate time must be allowed for the system to equilibrate. An alternative approach to deal with these extreme cases when adequate analytical methodology is missing, is to predict the Kd value applying estimation techniques based, for example, on Pow values (Appendix 3). This could be useful especially for low adsorbed/polar chemicals with Pow < 20 and for lipophilic/highly sorptive chemicals with Pow > 104.
1.9. PERFORMANCE OF THE TEST
1.9.1. Test conditions
All experiments are done at ambient temperature and, if possible, at a constant temperature between 20 °C and 25 °C.
Centrifugation conditions should allow the removal of particles larger than 0,2 μm from the solution. This value triggers the smallest sized particle that is considered as a solid particle, and is the limit between solid and colloid particles. Guidance on how to determine the centrifugation conditions is given in Appendix 4.
If the centrifugation facilities cannot guarantee the removal of particles larger than 0,2 μm, a combination of centrifugation and filtration with 0,2 μm filters could be used. These filters should be made of a suitable inert material to avoid any losses of the test substance on them. In any case, it should be proven that no losses of the test substance occur during filtration.
1.9.2. Tier 1 - Preliminary study
The purpose of conducting a preliminary study has already been given in section Scope. Guidance for setting up such a test is given with the experiment suggested below.
1.9.2.1. Selection of optimal soil/solution ratios
Two soil types and three soil/solution ratios (six experiments) are used. One soil type has high organic carbon and low clay content, and the other low organic carbon and high clay content. The following soil to solution ratios are suggested:
- 50 g soil and 50 cm3 aqueous solution of the test substance (ratio 1/1),
- 10 g soil and 50 cm3 aqueous solution of the test substance (ratio 1/5),
- 2 g soil and 50 cm3 aqueous solution of the test substance (ratio 1/25).
The minimum amount of soil on which the experiment can be carried out depends on the laboratory facilities and the performance of analytical methods used. However, it is recommended to use at least 1 g, and preferably 2 g, in order to obtain reliable results from the test.
One control sample with only the test substance in 0,01 M CaCl2 solution (no soil) is subjected to precisely the same steps as the test systems, in order to check the stability of the test substance in CaCl2 solution and its possible adsorption on the surfaces of the test vessels.
A blank run per soil with the same amount of soil and total volume of 50 cm3 0,01 M CaCl2 solution (without test substance) is subjected to the same test procedure. This serves as a background control during the analysis to detect interfering substances or contaminated soils.
All the experiments, included controls and blanks, should be performed at least in duplicate. The total number of the samples which should be prepared for the study can be calculated with respect to the methodology which will be followed.
Methods for the preliminary study and the main study are generally the same, exceptions are mentioned where relevant.
The air-dried soil samples are equilibrated by shaking with a minimum volume of 45 cm3 of 0,01 M CaCl2 overnight (12 h) before the day of the experiment. Afterwards, a certain volume of the stock solution of the test substance is added in order to adjust the final volume to 50 cm3. This volume of the stock solution added: (a) should not exceed 10 % of the final 50 cm3 volume of the aqueous phase in order to change as little as possible the nature of the pre-equilibration solution; and (b) should preferably result in an initial concentration of the test substance being in contact with the soil (C0) at least two orders of magnitude higher than the detection limit of the analytical method; this threshold safeguards the ability to perform accurate measurements even when strong adsorption occurs ( > 90 %) and to determine later the adsorption isotherms. It is also recommended, if possible, that the initial substance concentration (C0) not exceed half of its solubility limit.
An example of how to calculate the concentration of the stock solution (Cst) is given below. A detection limit of 0,01 μg cm-3 and 90 % adsorption are assumed; thus, the initial concentration of the test substance in contact with the soil should preferably be 1μg cm-3 (two orders of magnitude higher than the detection limit). Supposing that the maximum recommended volume of the stock solution is added, i.e. 5 to 45 cm3 0,01 M CaCl2 equilibration solution (= 10 % of the stock solution to 50 cm3 total volume of aqueous phase), the concentration of the stock solution should be 10 μg cm-3; this is three orders of magnitude higher than the detection limit of the analytical method.
The pH of the aqueous phase should be measured before and after contact with the soil since it plays an important role in the whole adsorption process, especially for ionisable substances.
The mixture is shaken until adsorption equilibrium is reached. The equilibrium time in soils is highly variable, depending on the chemical and the soil; a period of 24 h is generally sufficient (77). In the preliminary study, samples may be collected sequentially over a 48 h period of mixing (for example at 4, 8, 24, 48 h). However, times of analysis should be considered with flexibility with respect to the work schedule of the laboratory.
There are two options for the analysis of the test substance in the aqueous solution: (a) the parallel method and (b) the serial method. It should be stressed that, although the parallel method is experimentally more tedious, the mathematical treatment of the results is simpler (Appendix 5). However, the choice of the methodology to be followed, is left to the experimenter who will need to consider the available laboratory facilities and resources.
(a) Parallel method: samples with the same soil/solution ratio are prepared, as many as the time intervals at which it is desired to study the adsorption kinetics. After centrifugation and if so wished filtration, the aqueous phase of the first tube is recovered as completely as possible and is measured after, for example, 4 h, that of the second tube after 8 h, that of the third after 24, etc.
(b) Serial method: only a duplicate sample is prepared for each soil/solution ratio. At defined time intervals the mixture is centrifuged to separate the phases. A small aliquot of the aqueous phase is immediately analysed for the test substance; then the experiment continues with the original mixture. If filtration is applied after centrifugation, the laboratory should have facilities to handle filtration of small aqueous aliquots. It is recommended that the total volume of the aliquots taken not exceed 1 % of the total volume of the solution, in order not to change significantly the soil/solution ratio and to decrease the mass of solute available for adsorption during the test.
The percentage adsorption Ati is calculated at each time point (ti) on the basis of the nominal initial concentration and the measured concentration at the sampling time (ti), corrected for the value of the blank. Plots of the Ati versus time (Fig. 1 Appendix 5) are generated in order to estimate the achievement of equilibrium plateau(4). The Kd value at equilibrium is also calculated. Based on this Kd value, appropriate soil/solution ratios are selected from Fig.1, so that the percentage adsorption reaches above 20 % and preferably > 50 % (61). All the applicable equations and principles of plotting are given in the section on Data and reporting and in Appendix 5.
1.9.2.2. Determination of adsorption equilibration time and of the amount of test substance adsorbed at equilibrium
As already mentioned, plots of Ati or Caqads versus time permit estimation of the achievement of the adsorption equilibrium and the amount of test substance adsorbed at equilibrium. Figs. 1 and 2 in the Appendix 5 show examples of such plots. Equilibration time is the system needs to reach a plateau.
If, with a particular soil, no plateau but a steady increase is found, this may be due to complicating factors such as biodegradation or slow diffusion. Biodegradation can be shown by repeating the experiment with a sterilised sample of the soil. If no plateau is achieved even in this case, the experimenter should search for other phenomena that could be involved in his specific studies; this could be done with appropriate modifications of the experiment conditions (temperature, shaking times, soil/solution ratios). It is left to the experimenter to decide whether to continue the test procedure in spite of a possible failure to achieve an equilibrium.
1.9.2.3. Adsorption on the surface of the test vessel and stability of the test substance
Some information on the adsorption of the test substance on the surface of test vessels, as well as its stability, can be derived by analysing the control samples. If a depletion more than the standard error of the analytical method is observed, abiotic degradation and/or adsorption on the surface of the test vessel could be involved. Distinction between these two phenomena could be achieved by thoroughly washing the walls of the vessel with a known volume of an appropriate solvent and subjecting the wash solution to analysis for the test substance. If no adsorption on the surface of the test vessels is observed, the depletion demonstrates abiotic unstability of the test substance. If adsorption is found, changing the material of the test vessels is necessary. However, data on the adsorption on the surface of the test vessels gained from this experiment cannot be directly extrapolated to soil/solution experiment. The presence of soil will affect this adsorption.
Additional information on the stability of the test substance can be derived by determination of the parental mass balance over time. This means that the aqueous phase, extracts of soil and test vessel walls are analysed for the test substance. The difference between the mass of the test chemical added and the sum of the test chemical masses in the aqueous phase, extracts of the soil and test vessel walls is equal to the mass degraded and/or volatilised and/or not extracted. In order to perform a mass balance determination, the adsorbion equilibrium should have been reached within the period of the experiment.
The mass balance is performed on both soils and for one soil/solution ratio per soil that gives a depletion above 20 % and preferably > 50 % at equilibrium. When the ratio-finding experiment is completed with the analysis of the last sample of the aqueous phase after 48 h, the phases are separated by centrifugation and, if so wished, filtration. The aqueous phase is recovered as much as possible, and a suitable extraction solvent (extraction coefficient of at least 95 %) is added to the soil to extract the test substance. At least two successive extractions are recommended. The amount of test substance in the soil and test vessel extracts is determined and the mass balance is calculated (equation 10, Data and reporting). If it is less than 90 %, the test substance is considered to be unstable in the time scale of the test. However, studies could still be continued, taking into account the unstability of the test substance; in this case it is recommended to analyse both phases in the main study.
1.9.3. Tier 2 - Adsorption kinetics at one concentration of the test substance
Five soils are used, selected from Table 1. There is an advantage to including some or all of the soils used in the preliminary study, if appropriate, among these five soils. In this case, Tier 2 has not to be repeated for the soils used in preliminary study.
The equilibration time, the soil/solution ratio, the weight of the soil sample, the volume of the aqueous phase in contact with the soil and concentration of the test substance in the solution are chosen based on the preliminary study results. Analysis should preferably be done approximately after 2, 4, 6, 8 (possibly also 10) and 24 h contact time; the agitation time may be extended to a maximum of 48 h in case a chemical requires longer equilibration time with respect to ratio-finding results. However, times of analysis could be considered with flexibility.
Each experiment (one soil and one solution) is done at least in duplicate to allow estimation of the variance of the results. In every experiment one blank is run. It consists of the soil and 0,01 M CaCl2 solution, without test substance, and of weight and volume, respectively, identical to those of the experiment. A control sample with only the test substance in 0,01 M CaCl2 solution (without soil) is subjected to the same test procedure, serving to safeguard against the unexpected.
The percentage adsorption is calculated at each time point Ati and/or time interval AΔti (according to the need) and is plotted versus time. The distribution coefficient Kd at equilibrium, as well as the organic carbon normalised adsorption coefficient Koc (for non-polar organic chemicals), are also calculated.
Results of the adsorption kinetics test
The linear Kd value is generally accurate to describe sorptive behaviour in soil (35) (78) and represents an expression of inherent mobility of chemicals in soil. For example, in general chemicals with Kd <= 1 cm3 g-1 are considered to be qualitatively mobile. Similarly, a mobility classification scheme based on Koc values has been developed by MacCall et al. (16). Additionally, leaching classification schemes exist based on a relationship between Koc and DT-50(5) (32) (79).
Also, according to error analysis studies (61), Kd values below 0,3 cm3 g-1 cannot be estimated accurately from a decrease in concentration in the aqueous phase, even when the most favourable (from point of view of accuracy) soil/solution ratio is applied, i.e. 1:1. In this case analysis of both phases, soil and solution, is recommended.
With respect to the above remarks, it is recommended that the study of the adsorptive behaviour of a chemical in soil and its potential mobility be continued by determining Freundlich adsorption isotherms for these systems, for which an accurate determination of Kd is possible with the experimental protocol followed in this test method. Accurate determination is possible if the value which results by multiplying the Kd with the soil/solution ratio is > 0,3, when measurements are based on concentration decrease in the aqueous phase (indirect method), or > 0,1, when both phases are analysed (direct method) (61).
1.9.4. Tier 3 - Adsorption isotherms and desorption kinetics/desorption isotherms
1.9.4.1. Adsorption isotherms
Five test substance concentrations are used, covering preferably two orders of magnitude; in the choice of these concentrations the water solubility and the resulting aqueous equilibrium concentrations should be taken into account. The same soil/solution ratio per soil should be kept along the study. The adsorption test is performed as described above, with the only difference that the aqueous phase is analysed only once at the time necessary to reach equilibrium as determined before in Tier 2. The equilibrium concentrations in the solution are determined and the amount adsorbed is calculated from the depletion of the test substance in the solution or with the direct method. The adsorbed mass per unit mass of soil is plotted as a function of the equilibrium concentration of the test substance (see Data and reporting).
Results from the adsorption isotherms experiment
Among the mathematical adsorption models proposed so far, the Freundlich isotherm is the one most frequently used to describe adsorption processes. More detailed information on the interpretation and importance of adsorption models is provided in the references (41) (45) (80) (81) (82).
Note:
It should be mentioned that a comparison of KF (Freundlich adsorption coefficient) values for different substances is only possible if these KF values are expressed in the same units (83).
1.9.4.2. Desorption kinetics
The purpose of this experiment is to investigate whether a chemical is reversibly or irreversibly adsorbed on a soil. This information is important, since the desorption process also plays an important role in the behaviour of a chemical in field soil. Moreover, desorption data are useful inputs in the computer modelling of leaching and dissolved run-off simulation. If a desorption study is desired, it is recommended that the study described below be carried out on each system for which an accurate determination of Kd in the preceding adsorption kinetics experiment was possible.
Likewise with the adsorption kinetics study, there are two options to proceed with the desorption kinetics experiment: (a) the parallel method and (b) the serial method. The choice of methodology to be followed is left to the experimenter who will need to consider the available laboratory facilities and resources.
(a) Parallel method: for each soil which is chosen to proceed with the desorption study, samples with the same soil/solution ratio are prepared, as many as the time intervals at which it is desired to study the desorption kinetics. Preferably, the same time intervals as in the adsorption kinetics experiment should be used; however, the total time may be extended as appropriate in order the system to reach desorption equilibrium. In every experiment (one soil, one solution) one blank is run. It consists of the soil and 0,01 M CaCl2 solution, without test substance, and of weight and volume, respectively, identical to those of the experiment. As a control sample the test substance in 0,01 M CaCl2 solution (without soil) is subjected to the same test procedure. All the mixtures of the soil with the solution is agitating until to reach adsorption equilibrium (as determined before in Tier 2). Then, the phases are separated by centrifugation and the aqueous phases are removed as much as possible. The volume of solution removed is replaced by an equal volume of 0,01 M CaCl2 without test substance and the new mixtures are agitated again. The aqueous phase of the first tube is recovered as completely as possible and is measured after, for example, 2 h, that of the second tube after 4 h, that of the third after 6 h, etc. until the desorption equilibrium is reached.
(b) Serial method: after the adsorption kinetics experiment, the mixture is centrifuged and the aqueous phase is removed as much as possible. The volume of solution removed is replaced by an equal volume of 0,01 M CaCl2 without test substance. The new mixture is agitated until the desorption equilibrium is reached. During this time period, at defined time intervals, the mixture is centrifuged to separate the phases. A small aliquot of the aqueous phase is immediately analysed for the test substance; then, the experiment continues with the original mixture. The volume of each individual aliquot should be less than 1 % of the total volume. The same quantity of fresh 0,01 M CaCl2 solution is added to the mixture to maintain the soil to solution ratio, and the agitation continues until the next time interval.
The percentage desorption is calculated at each time point (Dti) and/or time interval (DΔti) (according to the needs of the study) and is plotted versus time. The desorption coefficient of Kdes at equilibrium is also calculated. All applicable equations are given in Data and reporting and Appendix 5.
Results from desorption kinetics experiment
Common plots of the percentage desorption Dti and adsorption Ati versus time, allow estimation of the reversibility of the adsorption process. If the desorption equilibrium is attained even within twice the time of the adsorption equilibrium, and the total desorption is more than 75 % of the amount adsorbed, the adsorption is considered to be reversible.
1.9.4.3. Desorption isotherms
Freundlich desorption isotherms are determined on the soils used in the adsorption isotherms experiment. The desorption test is performed as described in the section Desorption kinetics, with the only difference that the aqueous phase is analysed only once, at desorption equilibrium. The amount of the test substance desorbed is calculated. The content of test substance remaining adsorbed on soil at desorption equilibrium is plotted as a function of the equilibrium concentration of the test substance in solution (see Data and reporting and Appendix 5).
2. DATA AND REPORTING
The analytical data are presented in tabular form (see Appendix 6). Individual measurements and averages calculated are given. Graphical representations of adsorption isotherms are provided. The calculations are made as described below.
For the purpose of the test, it is considered that the weight of 1 cm3 of aqueous solution is 1 g. The soil/solution ratio may be expressed in units of w/w or w/vol with the same figure.
2.1. ADSORPTION
The adsorption (Ati) is defined as the percentage of substance adsorbed on the soil related to the quantity present at the beginning of the test, under the test conditions. If the test substance is stable and does not adsorb significantly to the container wall, Ati is calculated at each time point ti, according to the equation:
>REFERENCE TO A GRAPHIC>
where:
Ati= adsorption percentage at the time point ti (%);
>REFERENCE TO A GRAPHIC>
= mass of the test substance adsorbed on the soil at the time ti (μg);
m0= mass of the test substance in the test tube, at the beginning of the test (μg).
Detailed information on how to calculate the percentage of adsorption Ati for the parallel and serial methods is given in Appendix 5.
The distribution coefficient Kd is the ratio between the content of the substance in the soil phase and the mass concentration of the substance in the aqueous solution, under the test conditions, when adsorption equilibrium is reached.
>REFERENCE TO A GRAPHIC>
where:
>REFERENCE TO A GRAPHIC>
= content of the substance adsorbed on the soil at adsorption equilibrium (μg g-1)
>REFERENCE TO A GRAPHIC>
= mass concentration of the substance in the aqueous phase at adsorption equilibrium (μg cm-3). This concentration is analytically determined taking into account the values given by the blanks
>REFERENCE TO A GRAPHIC>
= mass of the substance adsorbed on the soil at adsorption equilibrium (μg)
>REFERENCE TO A GRAPHIC>
= mass of the substance in the solution at adsorption equilibrium (μg)
msoil= quantity of the soil phase, expressed in dry mass of soil (g)
v0= initial volume of the aqueous phase in contact with the soil (cm3).
The relation between Aeq and Kd is given by:
>REFERENCE TO A GRAPHIC>
where:
Aeq= percentage of adsorption at adsorption equilibrium, %.
The organic carbon normalised adsorption coefficient Koc relates the distribution coefficient Kd to the content of organic carbon of the soil sample:
>REFERENCE TO A GRAPHIC>
where:
% oc= percentage of organic carbon in the soil sample (g g-1).
Koc coefficient represents a single value which characterises the partitioning mainly of non-polar organic chemicals between organic carbon in the soil or sediment and water. The adsorption of these chemicals is correlated with the organic content of the sorbing solid (7); thus, Koc values depend on the specific characteristics of the humic fractions which differ considerably in sorption capacity, due to differences in origin, genesis, etc.
2.1.1. Adsorption isotherms
The Freundlich adsorption isotherms equation relates the amount of the test substance adsorbed to the concentration of the test substance in solution at equilibrium (equation 8).
The data are treated as under "Adsorption" and, for each test tube, the content of the test substance adsorbed on the soil after the adsorption test (>REFERENCE TO A GRAPHIC>, elsewhere denoted as x/m) is calculated. It is assumed that equilibrium has been attained and that
>REFERENCE TO A GRAPHIC>
represents the equilibrium value:
>REFERENCE TO A GRAPHIC>
The Freundlich adsorption equation is shown in (8):
>REFERENCE TO A GRAPHIC>
or in the linear form:
>REFERENCE TO A GRAPHIC>
where:
KFads= Freundlich adsorption coefficient; its dimension is cm3 g-1 only if 1/n = 1; in all other cases, the slope 1/n is introduced in the dimension of
>REFERENCE TO A GRAPHIC>
n= regression constant; 1/n generally ranges between 0,7-1,0, indicating that sorption data is frequently slightly non-linear.
Equations (8) and (9) are plotted and the values of KFads and 1/n are calculated by regression analysis using equation 9. The correlation coefficient r2 of the log equation is also calculated. An example of such plots is given in Fig. 2.
>PIC FILE= "L_2001225EN.021301.TIF">
Fig. 2. Freundlich adsorption plot, normal and linearised
2.1.2. Mass balance
The mass balance (MB) is defined as the percentage of substance which can be analytically recovered after an adsorption test versus the nominal amount of substance at the beginning of the test.
The treatment of data will differ if the solvent is completely miscible with water. In the case of water-miscible solvent, the treatment of data described under "Desorption" may be applied to determine the amount of substance recovered by solvent extraction. If the solvent is less miscible with water, the determination of the amount recovered has to be made.
The mass balance MB for the adsorption is calculated as follows; it is assumed that the term (mE) corresponds to the sum of the test chemical masses extracted from the soil and surface of the test vessel with an organic solvent:
>REFERENCE TO A GRAPHIC>
where:
MB= mass balance (%)
mE= total mass of test substance extracted from the soil and walls of the test vessel in two steps (μg)
C0= initial mass concentration of the test solution in contact with the soil (μg cm-3)
Vrec= volume of the supernatant recovered after the adsorption equilibrium (cm-3).
2.2. DESORPTION
The desorption (D) is defined as the percentage of the test substance which is desorbed, related to the quantity of substance previously adsorbed, under the test conditions:
>REFERENCE TO A GRAPHIC>
where:
Dti= desorption percentage at a time point ti (%)
>REFERENCE TO A GRAPHIC>
= mass of the test substance desorbed from soil at a time point ti (μg)
>REFERENCE TO A GRAPHIC>
= mass of the test substance adsorbed on soil at adsorption equilibrium (μg).
Detailed information on how to calculate the percentage of desorption Dti for the parallel and serial methods is given in Appendix 5.
The apparent desorption coefficient (Kdes) is, under the test conditions, the ratio between the content of the substance remaining in the soil phase and the mass concentration of the desorbed substance in the aqueous solution, when desorption equilibrium is reached:
>REFERENCE TO A GRAPHIC>
where:
Kdes= desorption coefficient (cm3 g-1)
>REFERENCE TO A GRAPHIC>
= total mass of the test substance desorbed from soil at desorption equilibrium (μg)
VT= total volume of the aqueous phase in contact with the soil during the desorption kinetics test (cm3).
Guidance for calculating the
>REFERENCE TO A GRAPHIC>
is given in Appendix 5 under the heading "Desorption".
Remark:
If the adsorption test which was preceded, was performed with the parallel method the volume VT in equation 12 is considered to be equal to V0.
2.2.1. Desorption isotherms
The Freundlich desorption isotherms equation relates the content of the test substance remaining adsorbed on the soil to the concentration of the test substance in solution at desorption equilibrium (equation 16).
For each test tube, the content of the substance remaining adsorbed on soil at desorption equilibrium is calculated as follows:
>REFERENCE TO A GRAPHIC>
>REFERENCE TO A GRAPHIC>
is defined as:
>REFERENCE TO A GRAPHIC>
where:
>REFERENCE TO A GRAPHIC>
= content of the test substance remaining adsorbed on the soil at desorption equilibrium (μg g-1)
>REFERENCE TO A GRAPHIC>
= mass of substance determined analytically in the aqueous phase at desorption equilibrium (μg)
>REFERENCE TO A GRAPHIC>
= mass of the test substance left over from the adsorption equilibrium due to incomplete volume replacement (μg)
>REFERENCE TO A GRAPHIC>
= mass of the substance in the solution at adsorption equilibrium (μg)
>REFERENCE TO A GRAPHIC>
VrF= volume of the solution taken from the tube for the measurement of the test substance, at desorption equilibrium (cm3)
VR= volume of the supernatant removed from the tube after the attainment of adsorption equilibrium and replaced by the same volume of a 0,01 M CaCl2 solution (cm3).
The Freundlich desorption equation is shown in (16):
>REFERENCE TO A GRAPHIC>
or in the linear form:
>REFERENCE TO A GRAPHIC>
where:
>REFERENCE TO A GRAPHIC>
= Freundlich desorption coefficient
n= regression constant
>REFERENCE TO A GRAPHIC>
= mass concentration of the substance in the aqueous phase at desorption equilibrium (μg cm-3).
The equations 16 and 17 can be plotted and the value of KFdes and 1/n are calculated by regression analysis using the equation 17.
Remark:
If the Freundlich adsorption or desorption exponent 1/n is equal to 1, the Freundlich adsorption or desorption binding constant (KFads and KFdes) will be equal to the adsorption or desorption equilibrium constants (Kd and Kdes) respectively, and plots of Cs vs Caq will be linear. If the exponents are not equal to 1, plots of Cs vs Caq will be non-linear and the adsorption and desorption constants will vary along the isotherms.
2.2.2. Testreport
The test report should include the following information:
- Complete identification of the soil samples used including:
- geographical reference of the site (latitude, longitude),
- date of sampling,
- use pattern (e.g. agricultural soil, forest, etc.),
- depth of sampling,
- sand/silt/clay content,
- pH values (in 0,01 M CaCl2),
- organic carbon content,
- organic matter content,
- nitrogen content,
- C/N ratio,
- cation exchange capacity (mmol/kg),
- all information relating to the collection and storage of soil samples,
- where appropriate, all relevant information for the interpretation of the adsorption/desorption of the test substance,
- reference of the methods used for the determination of each parameter,
- information on the test substance as appropriate,
- temperature of the experiments,
- centrifugation conditions,
- analytical procedure used to analyse the test substance,
- justification for any use of solubilising agent for the preparation of the stock solution of the test substance,
- explanations of corrections made in the calculations, if relevant,
- data according to the form sheet (Appendix 6) and graphical presentations,
- all information and observations helpful for the interpretation of the test results.
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(53) ISO/DIS 10381-1 Soil Quality - Sampling - Part 1: Guidance on the design of sampling programmes.
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(55) ISO/DIS 10381-3 Soil Quality - Sampling - Part 3: Guidance on safety of sampling.
(56) ISO/DIS 10381-4 Soil Quality - Sampling - Part 4: Guidance on the investigation of natural and cultivated soils.
(57) ISO/DIS 10381-5 Soil Quality - Sampling - Part 5: Guidance on the investigation of soil contamination of urban and industrial sites.
(58) ISO 10381-6, 1993: Soil Quality - Sampling - Part 6: Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory.
(59) Green R. E., and Yamane V. K., (1970), "Precision in pesticide adsorption measurements". Soil Sci. Am. Proc., 34, pp. 353-354.
(60) Grover R., and Hance R. J. (1970), "Effect of ratio of soil to water on adsorption of linuron and atrazine". Soil Sci., pp. 109-138.
(61) Boesten, J. J. T. I, "Influence of soil/liquid ratio on the experimental error of sorption coefficients in pesticide/soil system". Pest. Sci. 1990, 30, pp. 31-41.
(62) Boesten, J. J. T. I. "Influence of soil/liquid ratio on the experimental error of sorption coefficients in relation to OECD guideline 106". Proceedings of 5th international workshop on environmental behaviour of pesticides and regulatory aspects, Brussels, 26-29 April 1994.
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(78) Koskinen W. C., and Harper S. S., (1990), "The retention processes: mechanisms" in Pesticides in the Soil Environment: Processes, Impacts and Modelling (ed. H.H. Cheng). Soil Sci. Soc. Am. Book Series, No. 2, Madison, Wisconsin.
(79) Cohen S. Z., Creeger S. M., Carsel R. F., and Enfield C. G. (1984), "Potential pesticide contamination of groundwater from agricultural uses", in Treatment and Disposal of Pesticide Wastes, pp.297-325, ACS Symp. Ser. 259, American Chemical Society, Washington, DC.
(80) Giles C. H., (1970), "Interpretation and use of sorption isotherms" in Sorption and Transport Processes in Soils. S.C.I. Monograph No. 37, pp. 14-32.
(81) Giles, C. H.; McEwan J. H.; Nakhwa, S.N. and Smith, D, (1960), "Studies in adsorption: XI. A system of classification of solution adsorption isotherms and its use in the diagnosis of adsorption mechanisms and in measurements of pesticides surface areas of soils". J. Chem. Soc., pp. 3973-93.
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APPENDIX 1
TESTING SCHEME
>PIC FILE= "L_2001225EN.022102.TIF">
APPENDIX 2
INFLUENCE OF ACCURACY OF ANALYTICAL METHOD AND CONCENTRATION CHANGE ON ACCURACY OF ADSORPTION RESULTS
>PIC FILE= "L_2001225EN.022202.TIF">
APPENDIX 3
ESTIMATION TECHNIQUES FOR Kd
1. Estimation techniques permit prediction of Kd based on correlations with, for example, Pow values (12) (39) (63-68), water solubility data (12) (19) (21) (39) (68-73), or polarity data derived by application of HPLC on reversed phase (74-76). As shown in Tables 1 and 2, is the Koc or Kom that are calculated from these equations and then, indirectly, the Kd from the equations:
>REFERENCE TO A GRAPHIC>
>REFERENCE TO A GRAPHIC>
2. The concept of these correlations is based on two assumptions: (1) it is the organic matter of the soil that mainly influences the adsorption of a substance; and (2) the interactions involved are mainly non-polar. As a result, these correlations: (1) are not, or are only to some extent, applicable to polar substances, and (2) are not applicable in cases where the organic matter content of the soil is very small (12). In addition, although satisfactory correlations have been found between Pow and adsorption (19), the same cannot be said for the relationship between water solubility and extent of adsorption (19) (21); so far the studies are very contradictory.
3. Some examples of correlations between the adsorption coefficient and the octanol-water partition coefficient, as well as water solubility are given in Tables 1 and 2, respectively.
>TABLE>
>TABLE>
APPENDIX 4
CALCULATIONS FOR DEFINING THE CENTRIFUGATION CONDITIONS
1. The centrifugation time is given by the following formula, assuming spherical particles:
>REFERENCE TO A GRAPHIC>
For simplification purposes, all parameters are described in non-SI units (g, cm).
where:
ω= rotational speed (=2 π rpm/60), rad s-1
rpm= revolutions per minute
η= viscosity of solution, g s-1 cm-1
rp= particle radius, cm
ρs= soil density, g cm-3
ρaq= solution density, g cm-3
Rt= distance from the centre of centrifuge rotor to top of solution in centrifuge tube, cm
Rb= distance from the centre of centrifuge rotor to bottom in centrifuge tube, cm
Rb-Rt= length of the soil/solution mixture in the centrifuge tube, cm.
In general practice, double the calculated times is used to ensure complete separation.
2. The equation (1) can be simplified further if we consider the viscosity (η) and the density (ρaq) of the solution as equal to the viscosity and density of water at 25 °C; thus, η = 8,95 × 10-3 g s-1 cm-1 and ρaq = 1,0 g, cm-3.
Then, the centrifugation time is given by the equation (2):
>REFERENCE TO A GRAPHIC>
3. From the equation 2 it becomes apparent that two parameters are important in defining the centrifugation condition, i.e. time (t) and speed (rpm), in order to achieve separation of particles with a specific size (in our case 0,1 μm radius): (1) the density of the soil and (2) the length of the mixture in the centrifuge tube (Rb-Rt), i.e. the distance which a soil particle covers from the top of the solution to the bottom of the tube; obviously, for a fixed volume the length of the mixture in the tube will depend on the square of the radius of the tube.
4. Fig. 1 presents variations in the centrifugation time (t) versus centrifugation speed (rpm) for different soil densities (ρs) (Fig. 1a) and different lengths of the mixture in the centrifuge tubes (Fig. 1b). From Fig. 1a the influence of the soil density appears obvious; for example, for a classical centrifugation of 3000 rpm the centrifugation time is approximately 240 min. for 1,2 g cm3 soil density, while it is only 50 min. for 2,0 g cm3. Similarly, from Fig. 1b, for a classical centrifugation of 3000 rpm the centrifugation time is approximately 50 min. for a length of the mixture of 10 cm and only 7 min. for a length of 1 cm. However, it is important to find an optimal relation between centrifugation which requires the less length possible and easy handling for the experimenter in separating the phases after centrifugation.
5. Moreover, when defining the experimental conditions for the separation of soil/solution phases, it is important to consider the possible existence of a third "pseudo-phase", the colloids. These particles, with a size less than 0,2 μm, can have an important impact on the whole adsorption mechanism of a substance in a soil suspension. When centrifugation is performed as described above, colloids r