Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

This magnification value is indicated for 3 μm fibres, for 6 μm fibres a magnification of × 5 000 may be more suitable.

For 3 μm fibres, see previous note.

This information is only given for the convenience of users. Other equivalent computer programmes may be used if they can be shown to produce the same results.

An upper limit is given by the necessity to achieve a complete separation phase after adjustments of the partitition equilibrium and before samples are taken out for analytical determinations. If proper care is taken, the upper limit can be extended to higher values of Pow

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 chemical. If overnight fasting is adopted, clinical biochemical determinations should be performed after the conduct of functional observations in week 4 of the study.

Statisticians who take a modelling approach such as using General Linear Models (GLMs) may approach the analysis in a different but comparable way but will not necessarily derive the traditional anova table, which dates back to algorithmic approaches to calculating the statistics developed in a pre-computer age.

Statisticians who take a modelling approach such as using General Linear Models (GLMs) may approach the analysis in a different but comparable way but will not necessarily derive the traditional ANOVA table, which dates back to algorithmic approaches to calculating the statistics developed in a pre-computer age.

Now known as serum alanine aminotransferase.

Now known as serum aspartate aminotransferase.

Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, OJ L 353/1, 31.12.2008

The corrosivity evaluation could be based on expert judgment using such evidence as: human and animal experience, existing (in vitro) data, e.g. chapter B.40 (10), B.40 bis (11) of this annex or OECD TG 435 (12), pH values, information from similar chemicals or any other pertinent data.

In the following tables reference is made to GHS (Globally Harmonised System of Classification and Labelling of Chemicals (GHS). The EU equivalent is Regulation (EC) No 1272/2008. In the case of Acute Inhalation Toxicity, the Regulation (EC) No 1272/2008 (9) does not implement Category 5.

In the following tables reference is made to GHS (Globally Harmonised System of Classification and Labelling of Chemicals (GHS). The EU equivalent is Regulation (EC) No 1272/2008. In the case of Acute Inhalation Toxicity, the Regulation (EC) No 1272/2008 (9) does not implement Category 5.

In the following tables reference is made to GHS (Globally Harmonised System of Classification and Labelling of Chemicals (GHS). The EU equivalent is Regulation (EC) No 1272/2008. In the case of Acute Inhalation Toxicity the Regulation (EC) No 1272/2008 (9) does not implement Category 5.

Statisticians who take a modelling approach such as using General Linear Models (GLMs) may approach the analysis in a different but comparable way but will not necessarily derive the traditional ANOVA table, which dates back to algorithmic approaches to calculating the statistics developed in a pre-computer age.

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 (pregnant) animals, disturbs lactation and nursing behaviour, and, particularly in feeding studies, may disturb the daily exposure to the test chemical. If overnight fasting is adopted, clinical biochemical determinations should be performed after the conduct of functional observations in week 4 of the study for the males. The dams should be retained for an additional day after the pups are removed on e.g. PND 13). Dams should be fasted overnight from lactation day 13-14 and terminal blood used for clinical chemistry parameters.

Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, OJ L 353/1, 31.12.2008

‘Acid derivative’ is a non-specific class designation and is broadly defined as a chemical produced from an acid either directly or by modification or partial substitution. This class includes anhydrides, halo acids, salts, and other types of chemicals.

The results of these inter laboratory tests and a Technical Corrigendum to ISO 6341 give an EC50 — 24 h of the potassium dichromate (K2Cr2O7) within the range 0,6 mg/l to 1,7 mg/l.

After use, solutions containing mercury salts should be treated to avoid dissemination of mercury in the environment.

Mabey and Mill recommend the use of borate or acetate buffers instead of phosphate (11).

Such information may come from other sources such as hydrolysis data of structurally similar compounds from the literature or from other preliminary, semi-quantitative hydrolysis tests with the test substance at an earlier development stage.

If the plot of the log-transformed data vs. time does not indicate a linear function (equated with a first-order reaction rate), then the use of equation [3] is not appropriate for determining the hydrolysis rate constant of the test compound.

The pH values reported in these tables have been calculated from the potential measurements using Sörensen's standard equations (1909). The corresponding pH values are 0,04 units higher than the tabulated values.

Council Directive 91/271/EEC of 21 May 1991 concerning urban waste-water treatment (OJ L 135, 30.5.1991, p. 40).

See Appendix 1 for definitions and units

Sometimes denoted by P

See Appendix 1 for definitions and units

TLC: thin layer chromatography; HPLC: high pressure liquid chromatography; GC: gas chromatography

In some regulatory frameworks analysis of metabolites may be obligatory when certain conditions are met (cf. paragraph 65).

In general, measured concentrations in water during the uptake phase should be at least an order of magnitude above the limit of quantification so that more than one half-life of body burden can be measured in the depuration phase of the study.

See Appendix 1 for definitions and units

For most test substances, there should ideally be no detections in the control water. Background concentrations should only be relevant to naturally occurring materials (e.g., some metals) and substances that are ubiquitous in the environment.

If first order kinetics is obviously not obeyed, more complex models should be employed (see references in Appendix 5 and advice from a biostatistician sought.

Uptake may be limited by low exposure concentrations because of low water solubility in the bioconcentration test, whereas far higher exposure concentrations can be achieved with the dietary test.

For multi-constituent substances, UVCBs and mixtures, the water solubility of each relevant component should be considered to determine the appropriate exposure concentrations.

TOC includes organic carbon from particles and dissolved organic carbon, i.e. TOC = POC + DOC.

Although not generally recommended, if a solvent or solubilising agent is used the organic carbon originating from this agent should be added to the organic carbon from the test substance to evaluate the concentration of organic carbon in the test vessels.

If the lipid content is not analysed in the same fish as the test substance, fish should at least be of the similar weight, and (if relevant) the same sex.

This alternative is only valid if the fish in all test groups are held in similar group sizes, fish are removed according to the same pattern and fed in the same way. This ensures that fish growth in all test groups is similar, if the tested concentration is below the toxic range. If the growth is similar, also the lipid content is expected to be similar. A different growth in the control would indicate a substance effect and invalidate the study.

In addition to weight, total length should be recorded because comparison of the extent of length increase during the test is a good indicator of whether an adverse effect has occurred.

A t-test on growth rate constants can be performed, to test whether growth differs between control and test groups, or an F-test in case of analysis of variance. If needed, an F-test or likelihood ratio test can be used to assist in the choice of the appropriate growth model (OECD monograph 54, (32).

These percentages assume that the analytical methods are reliable and the half life is < 14 days. If the analytical methods are less reliable or the half life is (greatly) increased these numbers will become larger.

The minimised test may in fact be used to demonstrate rapid metabolism when it is known that rapid metabolism is likely.

When only two data points are measured, estimates of the confidence limits for BCFKm can be made using bootstrap methods. When intermediate data points are also available confidence limits for BCFKm can be calculated as in the full test.

See Appendix 1 for definitions and units

For the purpose of Regulation (EC) No 1907/2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (OJ L 396, 30.12.2006, p. 1), this issue is addressed in the ‘Guidance on Information Requirements and Chemical Safety Assessment’, chapter R.7c, R.7.10.3.1; R.7.10.4.1; and figure R7.10-2.

For most test substances, there should ideally be no detections in the control water. Background concentrations should only be relevant to naturally occurring materials (e.g., some metals) and substances that are ubiquitous in the environment.

As the BMF is defined as the ratio of the concentration of a substance in an organism to that in the organism's food at steady-state, lipid is taken into account by correcting for the contents of lipid in the organism and in the food, hence it is described more accurately as a ‘correction’. This approach differs from ‘normalisation’ to a set organism lipid content as is done in the aqueous exposure bioconcentration test.

Total length should also be recorded during the test as it is a good indicator of whether an adverse effect has occurred.

HCB is listed in Annexes A and C to the Stockholm Convention, and in Annexes I and III of Regulation (EC) No 850/2004 on persistent organic pollutants (OJ L 158, 30.4.2004, p. 7)

For rapid growth during the uptake phase the true feeding rate will decrease below that set at the beginning of exposure.

In an aqueous exposure study, a 14-day half-life would correspond to a BCF of ca. 10 000 L/kg using fish of 1 g with a corresponding uptake rate of about 500 L/kg/d (according to the equation of Sijm et al (46)).

Since the actual internal concentrations can only be determined after the test has been performed, an estimate of the expected internal concentration is needed (e.g. based on the expected BMF and the concentration in the food; cf. Equation A5.8 in Appendix 5).

The presence of the test substance in the test medium as a result of excretion by the fish or leaching from food may not be totally avoidable. Therefore one option is to measure the substance concentration in water at the end of the uptake period, especially if a semi-static set up is used, to help to establish whether any aqueous exposure has occurred.

This approach is specific to the dietary study, distinct from the procedure followed in the aqueous exposure, hence the word ‘correction’ has been used rather than ‘normalisation’ to prevent confusion — see also footnote in paragraph 106.

A t-test on growth rate constants can be performed, to test whether growth differs between control and test groups, or an F-test in case of analysis of variance. If needed, an F-test or likelihood ratio test can be used to assist in the choice of the appropriate growth model (OECD monograph 54, (32).

A foodstuff analysis technique for protein, lipid, crude fibre and ash content; this information is usually available from the feed supplier.

As with every empirical relationship, it should be verified that the test substance falls within the applicability domain of the relationship

The weight of fish at the end of the uptake phase can be estimated from previous study data or knowledge of the test species' likely increase in size from a typical test starting weight over a typical uptake duration (e.g. 28 days).

In most programs that allow a linear regression, also standard errors and confidence interval (CI) of the estimates are given, e.g. in Microsoft Excel using the Data Analysis tool pack.

In contrast with the linear regression method, using this formula will not yield a standard error for k2.

In contrast with a linear fitting procedure, this method will usually not yield a standard error or confidence interval for the estimated k1.

It should be realised that the uncertainty in the k2 estimate is not used properly in the bioaccumulation model when this is essentially regarded as constant when fitting k1 in the sequential fit method. The resulting BCF uncertainty will therefore be different between the simultaneous and sequential fitting methods.

In the wild the route leading to greatest exposure in aqueous environments is likely to be through ingestion for very hydrophobic substances and so an estimated BCF is not strictly representative of such a substance's bioaccumulation potential.

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, 231-236.

Image omitted; see official source

Plots of the concentration of the test substance in the aqueous phase

DT-50: degradation time for 50 % of the test substance.

Other daphnids may be used provided they meet the validity criteria as appropriate (the validity criterion relating to the reproductive output in the controls should be relevant for all species). If other daphnid are used they should be clearly identified and their use justified.

Accidental mortality: non chemical related mortality caused by an accidental incidence (i.e. known cause)

Inadvertent mortality: non chemical related mortality with no known cause

For example, if the test substance contains one ring, labelling on this ring is required; if the test substance contains two or more rings, separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable information on formation of transformation products.

Water retention characteristic of a soil can be measured as field capacity, as water holding capacity or as water suction tension (pF). For explanations see Appendix 1. It should be reported in the test report whether water retention characteristics and bulk density of soils were determined in undisturbed field samples or in disturbed (processed) samples.

Recent research results indicate that soils from temperate zones can also be stored at – 20 ^oC for more than three months (28)(29) without significant losses of microbial activity.

The soil should neither be too wet nor too dry to maintain adequate aeration and nutrition of soil microflora. Moisture contents recommended for optimal microbial growth range from 40-60 % water holding capacity (WHC) and from 0,1 -0,33 bar (6). The latter range is equivalent to a pF-range of 2,0 -2,5 . Typical moisture contents of various soil types are given in Appendix 2.

Aerobic conditions are dominant in surface soils and even in sub-surface soils as shown in an EU sponsored research project [K. Takagi et al. (1992). Microbial diversity and activity in subsoils: Methods, field site, seasonal variation in subsoil temperatures and oxygen contents. Proc. Internat. Symp. Environm. Aspects Pesticides Microbiol., 270-277, 17-21 August 1992, Sigtuna, Sweden]. Anaerobic conditions may only occur occasionally during flooding of soils after heavy rainfalls or when paddy conditions are established in rice fields.

Aerobic studies might be terminated much before 120 days provided that ultimate transformation pathway and ultimate mineralisation are clearly reached at that time. Termination of the test is possible after 120 days, or when at least 90 % of the test substance is transformed, but only if at least 5 % CO2 is formed.

Calculation of the initial concentration on an area basis using the following equation:

Mückenhausen, E., (1975) Die Bodenkunde und ihre geologischen, geomorphologischen, mineralogischen und petrologischen Grundlagen. DLG-Verlag, Frankfurt, Main.

Guth, J.A., (1980) The study of transformations. In Interactions between Herbicides and the Soil (R.J. Hance, Ed.), Academic Press, p. 123-157.

Guth, J.A., (1981) Experimental approaches to studying the fate of pesticides in soil. In Progress in Pesticide Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, p. 85-114.

Anderson, J.P.E., (1975) Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und Festlegung von Diallat im Boden. Z. PflKrankh Pflschutz, Sonderheft VII, p. 141-146.

For example, if the substance contains one ring, labelling on this ring is required; if the test substance contains two or more rings, separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable information on formation of transformation products.

As these alkaline absorption solutions also absorb the carbon dioxide from the ventilation air and that formed by respiration in aerobic experiments, they have to be exchanged in regular intervals to avoid their saturation and thus loss of their absorption capacity.

(Clay + silt) is the mineral fraction of the sediment with particle size of < 50 μm.

Recent studies have shown that storage at 4 ^oC can lead to a decrease of the organic carbon content of the sediment which may possibly result in a decrease of microbial activity (34).

Test with a second concentration can be useful for chemicals that reach surface waters by different entry routes resulting in significantly different concentrations, as long as the lower concentration can be analysed with sufficient accuracy.

In cases where rapid re-oxidation of anaerobic transformation products may readily occur, anaerobic conditions should be maintained during sampling and analysis.

The percentage degradation in Vessels FC containing the reference substance.

The percentage degradation in Vessels FI.

Final Preparation: The formulated product containing the active chemical (active ingredient) sold in commerce.

The recommended size is 0,1 litre to 1 litre.

The use of gas-tight silicone septa is recommended. It is further recommended that the gas-tightness of caps, especially butyl rubber septa, be tested because several commercially available septa are not sufficiently gas-tight against methane and some septa do not stay tight when they are pierced with a needle under the conditions of the test.

The meter should be used and calibrated at regular intervals, according to the manufacturer's instructions. If a pressure-meter of the prescribed quality is used e.g. capsulated with a steel membrane, no calibration is necessary in the laboratory. It should be calibrated by a licensed institute at the recommended intervals. The accuracy of the calibration can be checked in the laboratory with a one-point measurement at 1 × 10⁵ Pa against a pressure-meter with a mechanical display. When this point is measured correctly, the linearity will also be unaltered. If other measurement devices are used (without certified calibration by the manufacturer), conversion is recommended over the total range at regular intervals (Appendix 2).

This applies to the experimental set-up and experimental conditions whereby the volumes of gas produced — from control blanks and from vessels indicating 70 - 80 % inhibition — may be estimated with acceptable margins of error.

Papillary processes normally appear only in adult males and are found on fin rays from the second to the seventh or eighth counting from the posterior end of the anal fin (Fig.1 and 2). However, processes rarely appear on the first fin ray from the posterior end of the anal fin. This SOP covers the measurement of processes on the first fin ray (the fin ray number refers to the order from the posterior end of the anal fin in this SOP).

Homogenisation buffer:

At 20 °C under standard atmospheric pressure the ASV in freshwater equals 9,1 mg/l (60 % equals 5,46 mg/l)

Homogenisation buffer:

Battelle AP4.6.04 (1,18 mg/ml (AAA)), purified according to: Denslow, N.D., Chow, M.C., Kroll, K.J., Green, L. (1999). Vitellogenin as a biomarker of exposure for estrogen or estrogen mimics. Ecotoxicology 8: 385-398.

Hank's Buffered Salt Solution (HBSS):

Mercury (II) chloride (HgCl2) is a very toxic substance which should be handled with suitable precautions. Aqueous wastes containing this chemical should be disposed of appropriately; they should not be discharged into the waste water system.

Mercury (II) chloride (HgCl2) is a very toxic substance which should be handled with suitable precautions. Aqueous wastes containing this chemical should be disposed of appropriately; they should not be discharged directly into the waste water system.

Digested sludge is a mixture of the settled phases of sewage and activated sludge, which have been incubated in an anaerobic digester at about 35 °C to reduce biomass and odour problems and to improve the dewater-ability of the sludge. It consists of an association of anaerobic fermentative and methanogenic bacteria producing carbon dioxide and methane (11).

The recommended size is 0,1 litre to 1 litre.

The use of gas-tight silicone septa is recommended. It is further recommended that the gas-tightness of caps, especially butyl rubber septa, be tested because several commercially available septa are not sufficiently gas-tight against methane and some septa do not stay tight when they are pierced with a needle under the conditions of the test.

The device should be used and calibrated at regular intervals, according to the manufacturer's instructions. If a pressure-meter of the prescribed quality is used e.g. capsulated with a steel membrane, no calibration is necessary in the laboratory. The accuracy of the calibration can be checked at the laboratory with a one-point measurement at 1 × 10⁵ Pa against a pressure-meter with a mechanical display. When this point is measured correctly, the linearity will also be unaltered. If other measurement devices are used (without certified calibration by the manufacturer), calibration is recommended over the total range at regular intervals.

This should be re-evaluated if adsorptive and insoluble reference chemicals are included.

Column leaching studies with crop protection products may provide mobility information on a test chemical and its transformation products and may supplement batch sorption studies.

The amount to be applied to cylindrical soil columns can be calculated by the following formula:

This simulates an extremely high rainfall. The average yearly rainfall, for example, in Central Europe is of the order of 800-1 000 mm.

Examples of bulk densities for disturbed soils are as follows:

Typical leachate volumes range from 230-260 ml corresponding to approx. 92-104 % of total artificial rain applied (251 ml) when using soil columns of 4 cm diameter and 30 cm length.

More than one major transformation product may be formed in soil which also may appear at different time points during a transformation study. In such cases, it may be necessary to conduct leaching studies with aged residues of different age.

Papillary processes normally appear only in adult males and are found on fin rays from the second to the seventh or eighth counting from the posterior end of the anal fin (Fig.1 and 2). However, processes rarely appear on the first fin ray from the posterior end of the anal fin. This SOP covers the measurement of processes on the first fin ray (the fin ray number refers to the order from the posterior end of the anal fin in this SOP).

Homogenisation buffer:

Carl von Linné (* May, 23th, 1707 in Råshult/Älmhult; † January, 10th, 1778 in Uppsala).

In accordance to Article 4.1 of Directive 2004/37/EC of the European Parliament and of the Council of 29 April 2004 on the protection of workers from the risks related to exposure to carcinogens or mutagens at work (Sixth individual Directive within the meaning of Article 16(1) of Council Directive 89/391/EEC) (OJ L 158, 30.4.2004, p. 50).