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)

• NOTE:Most surfactants are not single molecular species but are mixtures of isomers, homologues, etc. which degrade after different characteristic lag periods and at different kinetic rates resulting in ‘blurred’, extenuated curves, so that the 60 % pass value may not be reached within ‘the 10-d window’, even though each individual molecular species would reach > 60 % within 10 days if tested alone. This may be observed with other complex mixtures as well.

DESCRIPTION OF THE METHOD

Apparatus

• 15.Normal laboratory apparatus and:

• (a) Glass serum bottles, sealed with butyl rubber stoppers and crimp-on aluminium seals. The recommended size is ‘125 ml’ which have a total volume of around 160 ml (in this case the volume of each bottle should be known to be 160 ± 1 ml). A smaller size of vessel may be used when the results fulfil the conditions described in paragraph 66 and 67;

• (b) Carbon analyser or other instrument (e.g. gas chromatograph) for measuring inorganic carbon;

• (c) Syringes of high precision for gaseous and liquid samples;

• (d) Orbital shaker in a temperature-controlled environment;

• (e) A supply of CO2 free air — this can be prepared by passing air through soda lime granules or by using an 80 % N2/20 % 02 gas mixture (optional) (see paragraph 28);

• (f) Membrane-filtration device of 0,20–0,45 μm porosity (optional);

• (g) Organic carbon analyser (optional).

Reagents

• 16.Use analytical grade reagents throughout.

Water

• 17.Distilled or de-ionised water should be used containing ≤ 1 mg/l as total organic carbon. This represents ≤ 5 % of the initial organic carbon content introduced by the recommended dose of the test chemical.

Stock solutions for the mineral salts medium

• 18.The stock solutions and the mineral salts medium are similar to those in ISO 14593 (16) and C.4 ‘ready biodegradability’ tests (20). The use of a higher concentration of ammonium chloride (2,0 g/l instead of 0,5 g/l) should only be necessary in very exceptional cases, e.g. when the test chemical concentration is > 40 mg C/l. Stock solutions should be stored under refrigeration and disposed of after six months, or earlier if there is evidence of precipitation or microbial growth. Prepare the following stock solutions:

• (a) Potassium dihydrogen phosphate (KH2PO4) 8,50 g Dipotassium hydrogen phosphate (K2HPO4) 21,75 g Disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) 33,40 g Ammonium chloride (NH4Cl) 0,50 g Dissolve in water and make up to 1 litre. The pH of this solution should be 7,4 (± 0,2). If this is not the case, then prepare a new solution.

• (b) Calcium chloride dihydrate (CaCl2.2H2O) 36,40 g Dissolve in water and make up to 1 litre.

• (c) Magnesium sulphate heptahydrate (MgSO4.7H2O) 22,50 g Dissolve in water and make up to 1 litre.

• (d) Iron (III) chloride hexahydrate (FeCl3.6H20) 0,25 g Dissolve in water and make up to 1 litre and add one drop of concentrated HCl.

Preparation of mineral medium

• 19.Mix 10 ml of solution (a) with approximately 800 ml water (paragraph 17), then add 1 ml of solutions (b), (c) and (d) and make up to 1 litre with water (paragraph 17).

Other reagents

• 20.Concentrated ortho-phosphoric acid (H3PO4) (> 85 % mass per volume).

Sodium hydroxide solution 7M

• 21.Dissolve 280 g of sodium hydroxide (NaOH) in 1 litre of water (paragraph 17). Determine the concentration of DIC of this solution and consider this value when calculating the test result (see paragraphs 55 and 61), especially in the light of the validity criterion in paragraph 66 (b). Prepare a fresh solution if the concentration of DIC is too high.

Test chemical

• 22.Prepare a stock solution of a sufficiently water-soluble test chemical in water (paragraph 17) or in the test medium (paragraph 19) at a concentration preferably 100-fold greater than the final concentration to be used in the test; it may be necessary to adjust the pH of the stock solution. The stock solution should be added to the mineral medium to give a final organic carbon concentration of between 2 and 40 mg C/l, preferably 20 mg C/l. If concentrations lower than these are used, the precision obtained may be impaired. Soluble and insoluble liquid chemicals may be added to the vessels directly using high precision syringes. Poorly soluble and insoluble test chemicals may require special treatment (25). The choices are:

• (a) direct addition of known weighed amounts;

• (b) ultrasonic dispersion before addition;

• (c) dispersion with the aid of emulsifying agents to be required to establish whether they have any inhibitory or stimulatory effects on microbial activity before addition;

• (d) adsorption of liquid test chemicals, or a solution in a suitable volatile solvent, on to an inert medium or support (e.g. glass fibre filter), followed by evaporation of the solvent, if used, and direct addition of known amounts;

• (e) addition of known volume of a solution of the test chemical in an easily volatile solvent to an empty test vessel, followed by evaporation of the solvent.

• Agents or solvents used in (c), (d) and (e) have to be tested for any stimulatory or inhibitory effect on microbial activity (see paragraph 42(b).)

Reference chemical

• 23.Prepare a stock solution of the (soluble) reference chemical in water (paragraph 17) at a concentration preferably 100-fold greater than the final concentration to be used (20 mg C/l) in the test.

Inhibition check

• 24.Test chemicals frequently show no significant degradation under the conditions used in ready biodegradation assessments. One possible cause is that the test chemical is inhibitory to the inoculum at the concentration at which it is applied in the test. An inhibition check may be included in the test design to facilitate identification (in retrospect) of inhibition as a possible cause or contributory factor. Alternatively, the inhibition check may rule out such interferences and show that zero or slight degradation is attributable solely to non-amenability to microbial attack under the conditions of the test. In order to obtain information on the toxicity of the test chemical to (aerobic) micro-organisms, prepare a solution in the test medium containing the test chemical and the reference chemical (paragraph 19), each at the same concentrations as added, respectively (see paragraph 22 and 23).

Inoculum

• 25.The inoculum may be derived from a variety of sources: activated sludge; sewage effluent (non-chlorinated); surface waters and soils; or from a mixture of these (20). The biodegradative activity of the source should be checked by using a reference chemical. Whatever the source, micro-organisms previously exposed to the test chemical should not be used if the procedure is to be used as a test for ready biodegradability.

• Warning: Activated sludge, sewage and sewage effluent contain pathogenic organisms and must be handled with caution.

• 26.Based on experience, the optimal volume for the inoculum is that which:

• — is sufficient to give adequate biodegradative activity;

• — degrades the reference chemical by the stipulated percentage (see paragraph 66);

• — gives 10² to 10⁵ colony-forming units per millilitre in the final mixture;

• — normally gives a concentration of 4 mg/l suspended solids in the final mixture when activated sludge is used, concentrations up to 30 mg/l may be used but may significantly increase CO2 production of the blanks (26);

• — contributes less than 10 % of the initial concentration of organic carbon introduced by the test chemical;

• — is generally 1-10 ml of inoculum for 1 litre of test solution.

Activated sludge

• 27.Activated sludge is freshly collected from the aeration tank of a sewage treatment plant or laboratory-scale unit treating predominantly domestic sewage. If necessary, coarse particles should be removed by sieving (e.g. using a 1 mm² mesh sieve) and the sludge should be kept aerobic until used.

• 28.Alternatively, after removal of any coarse particles, settle or centrifuge (e.g. 1 100 × g for 10 minutes). Discard the supernatant liquid. The sludge may be washed in the mineral solution. Suspend the concentrated sludge in mineral medium to yield a concentration of 3-5 g suspended solids/l. Thereafter aerate until required.

• 29.Sludge should be taken from a properly working conventional treatment plant. If sludge has to be taken from a high rate treatment plant, or is thought to contain inhibitors, it should be washed. Settle or centrifuge the re-suspended sludge after thorough mixing, discard the supernatant liquid and again suspend the washed sludge in a further volume of mineral medium. Repeat this procedure until the sludge is considered to be free from excess substrate or inhibitor.

• 30.After complete re-suspension is achieved, or with untreated sludge, withdraw a sample just before use for the determination of the dry weight of the suspended solids.

• 31.A further alternative is to homogenise activated sludge (3-5 g suspended solids/l). Treat the sludge in a Waring blender for 2 minutes at medium speed. Settle the blended sludge for 30 minutes or longer if required and decant liquid for use as inoculum at the rate of about 10 mg/l of mineral medium.

• 32.Still further reduction of the blank CO2 evolution can be achieved by aerating the sludge overnight with CO2-free air. Use 4 mg/l activated sludge solids as the concentration of the inoculum in this test (13).

Secondary sewage effluent

• 33.Alternatively, the inoculum can be derived from the secondary effluent of a treatment plant or laboratory-scale unit receiving predominantly domestic sewage. Maintain the sample under aerobic conditions and use on the day of collection, or pre-condition if necessary. The effluent should be filtered through a coarse filter to remove gross particulate matter and the pH value is measured.

• 34.To reduce its IC content, the filtrate is sparged with CO2-free air (paragraph 15-e) for 1 h while maintaining the pH at 6,5 using orthophosphoric acid (paragraph 20). The pH value is restored to its original value with sodium hydroxide (paragraph 21) and after settling for about 1 h a suitable volume of the supernatant is taken for inoculation. This sparging procedure reduces the IC content of the inoculum. For example, when the maximum recommended volume of filtered sparged effluent (100 ml) per litre was used as inoculum, the amount of IC present in blank control vessels was in the range 0,4 to 1,3 mg/l (14), representing 2-6,5 % of test chemical C at 20 mg C/l and 4-13 % at 10 mg C/l.

Surface waters

• 35.A sample is taken of an appropriate surface water. It should be kept under aerobic conditions and used on the day of collection. The sample should be concentrated, if necessary, by filtration or centrifugation. The volume of inoculum to be used in each test vessel should meet the criteria given in paragraph 26.

Soils

• 36.A sample is taken of an appropriate soil, collected to a depth of up to 20 cm below the soil surface. Stones, plant remains and invertebrates should be removed from the sample of soil before it is sieved through a 2 mm mesh (if the sample is too wet to sieve immediately, then partially air dry to facilitate sieving). It should be kept under aerobic conditions and used on the day of collection (If the sample is transported in a loosely-tied black polythene bag, it can be stored at 2 to 4 °C in the bag for up to one month).

Preconditioning of inoculum

• 37.Inoculum may be pre-conditioned to the experimental conditions, but not pre-adapted to the test chemical. Pre-conditioning can reduce the blank CO2 evolution. Pre-conditioning consists of aerating activated sludge after diluting in test medium to 30 mg/l with moist CO2-free air for up to 5-7 days at the test temperature.

TEST PROCEDURE

Number of bottles

• 38.The number of bottles (paragraph 15-a) needed for a test will depend on the frequency of analysis and the test duration.

• 39.It is recommended that triplicate bottles be analysed after a sufficient number of time intervals such that the 10-d window may be identified. Also at least five test bottles (paragraph 15-a) from sets (a), (b) and (c) (see paragraph 42) are analysed at the end of the test, to enable 95 % confidence intervals to be calculated for the mean percentage biodegradation value.

Inoculated medium

• 40.The inoculum is used at a concentration of 4 mg/l activated sludge dry solids. Prepare immediately before use sufficient inoculated medium by adding, for example, 2 ml suitably treated activated sludge (paragraphs 27 to 32) at 2 000 mg/l to 1 litre of mineral salts medium (paragraph 19). When secondary sewage effluent is to be used add up to 100 ml effluent (paragraph 33) to 900 ml mineral salts medium (paragraph 19) and dilute to 1 litre with medium.

Preparation of bottles

• 41.Aliquots of inoculated medium are dispensed into replicate bottles to give a headspace to liquid ratio of 1:2 (e.g. add 107 ml to 160 ml-capacity bottles). Other ratios may be used, but see the warning given in paragraph 11. When using either type of inoculum, care must be taken to ensure that the inoculated medium is adequately mixed to ensure that it is uniformly distributed to the test bottles.

• 42.Sets of bottles (paragraph 15a) are prepared to contain the following:

• (a) Test vessels (denoted FT) containing the test chemical;

• (b) Blank controls (denoted FB) containing only the test medium plus inoculum; any chemicals, solvents, agents or glass fibre filters used to introduce the test chemical into the test vessels must also be added;

• (c) Vessels (denoted FC) for checking the procedure containing the reference chemical;

• (d) If needed, vessels (denoted FI) for checking a possible inhibitory effect of the test chemical containing both the test chemical and reference chemical at the same concentrations (paragraph 24) as in bottles FT and FC, respectively;

• (e) Vessels (denoted FS) for checking a possible abiotic degradation as (a) plus 50 mg/l HgCl2 or sterilised by some other means (e.g. by autoclaving).

• 43.Water-soluble test chemicals and reference chemicals are added as aqueous stock solutions (paragraphs 22, 23 and 24) to give a concentration of 10 to 20 mg C/l.

• 44.Insoluble test chemicals and insoluble reference chemicals are added to bottles in a variety of ways (see paragraph 22a-e) according to the nature of the test chemical, either before or after addition of the inoculated medium, depending on the method of treatment of the test chemical. If one of the procedures given in paragraph 22a-e is used, then the blank bottles FB (paragraph 42b) should be treated in a similar fashion but excluding the test chemical or reference chemical.

• 45.Volatile test chemicals should be injected into sealed bottles (paragraph 47) using a micro syringe. The dose is calculated from the volume injected and the density of the test chemical.

• 46.Water should be added to vessels, where necessary, to give the same liquid volume in each vessel. It must be ensured that the headspace to liquid ratio (usually 1:2) and concentration of the test chemical are such that sufficient oxygen is available in the headspace to allow for complete biodegradation.

• 47.All bottles are then sealed for example, with butyl rubber septa and aluminium caps. Volatile tests chemicals should be added at this stage (paragraph 45). If the decrease in DOC concentration of the test solution is to be monitored and for time zero analyses to be performed for initial IC concentration (sterile controls, paragraph 42e) or other determinands, remove an appropriate sample from the test vessel. The test vessel and its contents are then discarded.

• 48.The sealed bottles are placed on a rotary shaker (paragraph 15d), with a shaking rate sufficient to keep the bottle contents well mixed and in suspension (e.g. 150 to 200 rpm), and incubated in the dark at 20 °C, to be kept within ± 1 °C.

Sampling

• 49.The pattern of sampling will depend on the lag period and kinetic rate of biodegradation of the test chemical. Bottles are sacrificed for analysis on the day of sampling, which should be at least weekly or more frequently (e.g. twice per week) if a complete degradation curve is required. The requisite number of replicate bottles is taken from the shaker, representing FT, FB and FC and, if used FI and FS (see paragraph 42). The test normally runs for 28d. If the biodegradation curve indicates that a plateau has been attained before 28d, the test may be concluded earlier than 28d. Take samples from the five bottles reserved for the 28th day of the test for analysis and use the results to calculate the confidence limits or coefficient of variation of percentage biodegradation. Bottles representing the checks for inhibition and for abiotic degradation need not be sampled as frequently as the other bottles; day 1 and day 28 would be sufficient.

Inorganic carbon (IC) analysis

• 50.CO2 production in the bottles is determined by measuring the increase in the concentration of inorganic carbon (IC) during incubation. There are two recommended methods available for measuring the amount of IC produced in the test, and these are described immediately below. Since the methods can give slightly different results only one should be used in a test run.

• 51.Method (a) is recommended if the medium is likely to contain remnants of, for example, a glass-filter paper and/or insoluble test chemical. This analysis can be performed using a gas chromatograph if a carbon analyser is not available. It is important that the bottles should be at or close to the test temperature when the headspace gas is analysed. Method (b) can be easier for laboratories using carbon analysers to measure IC. It is important that the sodium hydroxide solution (paragraph 21) used to convert CO2 to carbonate is either freshly prepared or its IC content is known, so that this can be taken into account when calculating the test results (see paragraph 66-b.)

Method (a):   acidification to pH < 3

• 52.Before each batch of analyses, the IC analyser is calibrated using an appropriate IC standard (e.g. 1 % w/w CO2 in N2). Concentrated orthophosphoric acid (paragraph 20) is injected through the septum of each bottle sampled to lower the pH of the medium to < 3 (e.g. add 1 ml to 107 ml test medium). The bottles are placed back on the shaker. After shaking for one hour at the test temperature the bottles are removed from the shaker, aliquots (e.g. 1 ml) of gas are withdrawn from the headspace of each bottle and injected into the IC analyser. The measured IC concentrations are recorded as mg C/l.

• 53.The principle of this method is that after acidification to pH < 3 and equilibration at 20 °C, the equilibrium constant for the distribution of CO2 between the liquid and gaseous phases in the test bottles is 1,0 when measured as a concentration (13). This should be demonstrated for the test system at least once as follows:

• Set up bottles containing 5 and 10 mg/l as IC using a solution of anhydrous sodium carbonate (Na2 CO3) in CO2-free water prepared by acidifying water to pH 6,5 with concentrated ortho-phosphoric acid (paragraph 20), sparging overnight with CO2-free air and raising the pH to neutrality with alkali. Ensure that the ratio of the headspace volume to the liquid volume is the same as in the tests (e.g. 1:2). Acidify and equilibrate as described in paragraph 52, and measure the IC concentrations of both the headspace and liquid phases. Check that the two concentrations are the same within experimental error. If they are not, the operator should review the procedures. This check on the distribution of IC between liquid and gaseous phases need not be made every time the test is performed; it could presumably be made while performing the calibration.

• 54.If DOC removal is to be measured (water-soluble test chemicals only), samples should be taken of the liquid phase from separate (non-acidified) bottles, membrane-filtered and injected into the DOC analyser. These bottles can be used for other analyses as necessary, to measure primary biodegradation.

Method (b):   conversion of CO2 to carbonate

• 55.Before each batch of analyses, the IC analyser is calibrated using an appropriate standard — for example, a solution of sodium bicarbonate (NaHCO3) in CO2 free water (see paragraph 53) in the range 0 to 20 mg/l as IC. Sodium hydroxide solution (7M, paragraph 21) (e.g. 1 ml to 107 ml medium) is injected through the septum of each bottle sampled and the bottles are shaken for 1 h at the test temperature. Use the same NaOH solution on all bottles sacrificed on a particular day, but not necessarily on all sampling occasions throughout a test. If absolute blank IC values are required at all sampling occasions, IC determinations of the NaOH solution will be required each time it is used. The bottles are removed from the shaker and allowed to settle. Suitable volumes (e.g. 50 to 1 000 μl) of the liquid phase in each vessel are withdrawn by syringe. The samples are injected into the IC analyser and the concentrations of IC are recorded. It should be ensured that the analyser used is equipped properly to deal with the alkaline samples produced in this method.

• 56.The principle of this method is that after the addition of alkali and shaking, the concentration of IC in the headspace is negligible. This should be checked for the test system at least once by using IC standards, adding alkali and equilibrating, and measuring the concentration of IC in both the headspace and liquid phases (see paragraph 53). The concentration in the headspace should approach zero. This check on the virtually complete absorption of CO2 need not be made every time the test is performed.

• 57.If DOC removal is to be measured (water-soluble test chemicals only), samples should be taken of the liquid phase from separate bottles (containing no added alkali), membrane filtered and injected into the DOC analyser. These bottles can be used for other analyses, as necessary, to measure primary biodegradability.

DATA AND REPORTING

Calculating of results

• 58.Assuming 100 % mineralisation of the test chemical to CO2, the ThIC in excess of that produced in the blank controls equals the TOC added to each test bottle at the start of the test, that is:

Image omitted; see official source

• The total mass (mg) of inorganic carbon (TIC) in each bottle is:

Image omitted; see official source	Equation [1]

• where:

VL	=	volume of liquid in the bottle (litre);
---	---	---
CL	=	concentration of IC in the liquid (mg/l as carbon);
---	---	---
VH	=	volume of the headspace (litre);
---	---	---
CH	=	concentration of IC in the headspace (mg/l as carbon).

• The calculations of TIC for the two analytical methods used for measuring IC in this test are described below in paragraphs 60 and 61. Percentage biodegradation (% D) in each case is given by:

Image omitted; see official source	Equation [2]

• where:

TICt	=	mg TIC in test bottle at time t;
---	---	---
TICb	=	mean mg TIC in blank bottles at time t;
---	---	---
TOC	=	mg TOC added initially to the test vessel.

• The percentage biodegradation % D is calculated for the test (FT), reference (FC) and, if included inhibition monitoring control (FI) bottles from the respective amounts of TIC produced up to each sampling time.

• 59.If there has been a significant increase in the TIC content of the sterile controls (FS) over the test period, then it may be concluded that abiotic degradation of the test chemical has occurred and this must be taken into account in the calculation of D in Equation [2].

Acidification to pH < 3

• 60.Since acidification to pH < 3 and equilibration results in the equalisation of the concentration of TIC in the liquid and gaseous phases, only the concentration of IC in the gas phase needs to be measured. Thus, from Equation [1]

Image omitted; see official source , where VB = volume of the serum bottle.

Conversion of CO2 to carbonate

• 61.In this method calculations are performed as in Equation [1], but the negligible amount of IC in the gaseous phase is ignored, that is

Image omitted; see official source , and

Image omitted; see official source .

Expression of Results

• 62.A biodegradation curve is obtained by plotting percentage biodegradation, D, against time of incubation and if possible, the lag phase, biodegradation phase, 10-d window and plateau phase, that is the phase in which the maximal degradation has been reached and the biodegradation curve has levelled out, are indicated. If comparable results are obtained for parallel test vessels FT (< 20 % difference), a mean curve is plotted (see Appendix 2, Fig.1); if not, curves are plotted for each vessel. The mean value of the percentage biodegradation in the plateau phase is determined or the highest value is assessed (e.g. when the curve decreases in the plateau phase), but it is important to assess that in the latter case the value is not an outlier. Indicate this maximum level of biodegradation as ‘degree of biodegradation of the test chemical’ in the test report. If the number of test vessels was insufficient to indicate a plateau phase, the measured data of the last day of the test are used to calculate a mean value. This last value, the mean of five replicates, serves to indicate the precision with which the percentage biodegradation was determined. Also report the value obtained at the end of the 10-d window.

• 63.In the same way, a curve for the reference chemical, FC, is plotted and, if included, for the abiotic elimination check, FS and the inhibition control, FI.

• 64.The amounts of TIC present in the blank controls (FB) are recorded as are those in flasks FS (abiotic check), if these vessels were included in the test.

• 65.Calculate D for the FI vessels, based on the theoretical IC yield anticipated from only the reference component of the mixture. If, at day 28, [(DFC ([^E0184]) – DFI ([^E0185])/DFC)] × 100 > 25 %, it may be assumed that the test chemical inhibited the activity of the inoculum, and this may account for low values of DFT obtained under the conditions of the test. In this case the test could be repeated using a lower test concentration and preferably reducing the DIC in the inoculum and TIC formed in the blank controls, since the lower concentration will otherwise reduce the precision of the method. Alternatively, another inoculum may be used. If in flask FS (abiotic) a significant increase (> 10 %) in the amount of TIC is observed, abiotic degradation processes may have occurred.

Validity of results

• 66.A test is considered valid if:

• (a) the mean percentage degradation in vessels FC containing the reference chemical is > 60 % by the 14^th day of incubation; and

• (b) the mean amount of TIC present in the blank controls FB at the end of the test is < 3mg C/l.

• If these limits are not met, the test should be repeated with an inoculum from another source and/or the procedures used should be reviewed. For example, if high blank IC production is a problem the procedure given in paragraphs 27 to 32 should be followed.

• 67.If the test chemical does not reach 60 % ThIC and was shown not to be inhibitory (paragraph 65), the test could be repeated with increased concentration of inoculum (up to 30 mg/l activated sludge and 100 ml effluent/l) or inocula from other sources, especially if degradation had been in the range 20 to 60 %.

Interpretation of results

• 68.Biodegradation > 60 % ThIC within the 10-d window in this test demonstrates that the test chemical is readily biodegradable under aerobic conditions.

• 69.If the pass value of 60 % ThIC is not attained, determine the pH value in media in bottles which have not been made acid or alkaline; a value of less than 6,5 could indicate that nitrification had occurred. In such a case repeat the test with a buffer solution of higher concentration.

Test Report

• 70.Compile a table of % D for each test (FT), reference (FC) and, if included, inhibition control bottle (FI) for each day sampled. If comparable results are obtained for replicate bottles, plot a curve of mean % D against time. Record the amount of TIC in the blanks (FB) and in the sterile controls (FS) DOC and/or other determinands, and their percentage removal.

• 71.Determine the mean value of % D in the plateau phase, or use the highest value if the biodegradation curve decreases in the plateau phase, and report this as the ‘degree of biodegradation of the test chemical’. It is important to ensure that in the latter case the highest value is not an outlier.

• 72.The test report must include the following information:

Test chemical:

• — common name, chemical name, CAS number, structural formula and relevant physical-chemical properties;

• — purity (impurities) of test chemical.

Test conditions:

• — reference to this Test Method;

• — description of the test system used (e.g. volume of the vessel, head space to liquid ratio, method of stirring, etc.);

• — application of test chemical and reference chemical to test system: test concentration used and amount of carbon dosed into each test bottle, any use of solvents;

• — details of the inoculum used, any pre-treatment and pre-conditioning;

• — incubation temperature;

• — validation of the principle of IC analysis;

• — main characteristics of the IC analyser employed (and any other analytical methods used);

• — number of replicates.

Results:

• — raw data and calculated values of biodegradability in tabular form;

• — the graph of percentage degradation against time for the test and reference chemicals, the lag phase, degradation phase, 10-d window and slope;

• — percentage removal at plateau, at end of test, and after 10-d window;

• — reasons for any rejection of the test results;

• — any other facts that are relevant to the procedure followed;

• — discussion of results.

LITERATURE:

• (1) Chapter C.4 of this Annex Determination of ‘Ready’ Biodegradability — CO2 Evolution Test (Method C.4-C).

• (2) Sturm RN (1973). Biodegradability of Nonionic surfactants: screening test for predicting rate and ultimate biodegradation. J.A,.Oil Chem Soc. 50: 159-167.

• (3) Larson RJ (1979). Estimation of biodegradation potential of xenobiotic organic chemicals. Appl Env. Microbiol. 38: 1153-1161.

• (4) Larson RJ, Hansmann MA and Bookland EA (1996). Carbon dioxide recovery in ready biodegradability tests: mass transfer and kinetic constants, Chemosphere 33: 1195-1210.

• (5) ISO 9439 (1990; revised 1999). Water Quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium — Carbon dioxide evolution Test (Sturm).

• (6) US EPA (1996). Fate, Transport and Transformation Test Guideline. 835.3110 Carbon dioxide evolution test. Office, Prevention Pesticides and Toxic Substances Washington, DC.

• (7) US EPA (1996). Fate, Transport and Transformation Test Guideline. 835. 3100. Aerobic aquatic biodegradation. Office, Prevention Pesticides and Toxic Substances Washington, DC.

• (8) Gledhill WE (1975). Screening test for assessment of biodegradability: Linear alkyl benzene sulfonate. Appl Microbiol. 30: 922-929.

• (9) Weytjens D, Van Ginneken I and Painter HA (1994). The recovery of carbon dioxide in the Sturm test for ready biodegradability. Chemosphere 28: 801-812.

• (10) Ennis DM and Kramer A (1975). A rapid microtechnique for testing biodegradability of nylons and polyamides. J. Food Sci. 40: 181-185.

• (11) Ennis DM, Kramer A, Jameson CW, Mazzoccki PH and Bailey PH (1978). Appl. Env. Microbiol. 35: 51-53.

• (12) Boatman RJ, Cunningham SL and Ziegler DA (1986). A method for measuring the biodegradation of organic chemicals, Env. Toxicol. Chem. 5: 233-243.

• (13) Struijs J and Stoltenkamp J (1990). Head space determination of evolved carbon dioxide in a biodegradability screening test. Ecotox. Env. Safety 19: 204-211.

• (14) Birch RR and Fletcher RJ (1991). The application of dissolved inorganic carbon measurements to the study of aerobic biodegradability. Chemosphere 23: 507-524.

• (15) Birch RR, Biver C, Campagna R, Gledhill WE, Pagga U, Steber J, Reust H, and Bontinck WJ (1989). Screening of chemicals for anaerobic biodegradation. Chemosphere 19: 1527-1550.

• (16) ISO 14593, (1999) Water Quality — Evaluation of ultimate aerobic biodegradability of organic compounds in an aerobic medium-method by analysis of inorganic carbon in sealed vessels (CO2 headspace test).

• (17) Battersby NS (1997). The ISO headspace CO2 biodegradation test, Chemosphere 34: 1813-1822.

• (18) US EPA (1996). Fate, Transport and Transportation. 835.3120. Sealed vessel carbon dioxide production test. Office, Prevention Pesticides and Toxic Substance, Washington, DC.

• (19) Battersby NS, Ciccognani D, Evans MR, King D, Painter HA, Peterson DR and Starkey M (1999). An ‘inherent’ biodegradability test for oil products: description and results of an international ring test. Chemosphere 38: 3219-3235.

• (20) Chapter C.4 of this Annex, Determination of ‘Ready’ Biodegradability.

• (21) OECD (1988). OECD Ring-test of methods for determining ready biodegradability: Chairman’s report (M. Hashimoto; MITI) and final report (M. Kitano and M. Takatsuki; CITI). Paris.

• (22) Chapter C.11 of this Annex, Activated sludge respiration inhibition test.

• (23) Struijs J, Stoltenkamp-Wouterse MJ and Dekkers ALM (1995). A rationale for the appropriate amount of inoculum in ready biodegradability tests. Biodegradation 6: 319-327.

• (24) EU (1999). Ring-test of the ISO Headspace CO2 method: application to surfactants: Surfactant Ring Test-1, Report EU4697, Water Research Centre, May 1999, Medmenham, SL7 2HD, UK.

• (25) ISO 10634 (1996) Water Quality — Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium.

Appendix 1

ABBREVIATIONS AND DEFINITIONS

IC : Inorganic carbon

ThCO2 : Theoretical carbon dioxide (mg) is the quantity of carbon dioxide calculated to be produced from the known or measured carbon content of the test chemical when fully mineralised; also expressed as mg carbon dioxide evolved per mg test chemical.

DOC : Dissolved organic carbon is the organic carbon present in solution or that which passes through a 0,45 micrometre filter or remains in the supernatant after centrifuging at approx. 4 000 g (about 40 000 m sec^-2) for 15 min.

DIC : Dissolved inorganic carbon

ThIC : Theoretical inorganic carbon

TIC : Total inorganic carbon

Readily biodegradable : An arbitrary classification of chemicals which have passed certain specified screening tests for ultimate biodegradability; these tests are so stringent that it is assumed that such chemicals will rapidly and completely biodegrade in aquatic environments under aerobic conditions.

10-d window : The 10 days immediately following the attainment of 10 % biodegradation.

Inherent biodegradability : A classification of chemicals for which there is unequivocal evidence of biodegradation (primary or ultimate) in any test of biodegradability.

Ultimate aerobic biodegradation : The level of degradation achieved when the test chemical is totally utilised by micro-organisms resulting in the production of carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass).

Mineralisation : Mineralisation is the complete degradation of an organic chemical to CO2 and H2O under aerobic conditions, and CH4, CO2 and H2O under anaerobic conditions.

Lag phase : The time from the start of a test until acclimatization and/or adaptation of the degrading microorganisms is achieved and the biodegradation degree of a test chemical or organic matter has increased to a detectable level (e.g. 10 % of the maximum theoretical biodegradation, or lower, dependent on the accuracy of the measuring technique).

Degradation phase : The time from the end of the lag period to the time when 90 % of the maximum level of degradation has been reached.

Plateau phase : Plateau phase is the phase in which the maximal degradation has been reached and the biodegradation curve has levelled out.

Test chemical : Any substance or mixture tested using this Test Method.

Appendix 2

Example of a biodegradation curve

Figure 1

Biodegradation of 1-octanol in the CO2 headspace test

Image omitted; see official source

Glossary

Biodegradation:

Degradation phase:

Maximum level of biodegradation:

Plateau phase:

10-d(ay) window:

Test time (days):

C. 30.   BIOACCUMULATION IN TERRESTRIAL OLIGOCHAETES

INTRODUCTION

• 1.This Test Method is equivalent to OECD Test Guideline (TG) 317 (2010). Among the Test Methods relating to environmental fate, the Bioconcentration: Flow-through Fish Test (chapter C.13 of this Annex (49)) and the Bioaccumulation in Sediment-dwelling Benthic Oligochaetes (53) were published in 1996 and 2008 respectively. The extrapolation of aquatic bioaccumulation data to terrestrial organisms like earthworms is difficult, if possible at all. Model calculations based on a test chemical’s lipophilicity, e.g. (14) (37), are currently used for the assessment of bioaccumulation of chemicals in soil, as e.g. in the EU Technical Guidance Document (19). The need for a compartment-specific test method has already been addressed, e.g. (55). Such a method is especially important for the evaluation of secondary poisoning in terrestrial food chains (4). Several national test methods address the issue of bioaccumulation in organisms other than fish e.g. (2) and (72). A method on the measurement of bioaccumulation from contaminated soils in earthworms (Eisenia fetida, Savigny) and potworms has been developed by the American Society for Testing and Materials (3). An internationally accepted method for the determination of bioaccumulation in spiked soil will improve the risk assessment of chemicals in terrestrial ecosystems e.g. (25) (29).

• 2.Soil-ingesting invertebrates are exposed to soil bound chemicals. Among these animals, terrestrial oligochaetes play an important role in the structure and function of soils (15) (20). Terrestrial oligochaetes live in soil and partly at the soil surface (especially the litter layer); they frequently represent the most abundant species in terms of biomass (54). By bioturbation of the soil and by serving as prey these animals can have a strong influence on the bioavailability of chemicals to other organisms like invertebrates (e.g. predatory mites and beetles; e.g. (64)) or vertebrate (e.g. foxes and gulls) predators (18) (62). Some species of terrestrial oligochaetes currently used in ecotoxicological testing are described in Appendix 5.

• 3.The ASTM Standard Guide for Conducting Laboratory Soil Toxicity or Bioaccumulation Tests with the Lumbricid Earthworm Eisenia fetida and the Enchytraeid Potworm Enchytraeus albidus (3) provides many essential and useful details for the performance of the present soil bioaccumulation Test Method. Further documents that are referred to in this Test Method are chapter C.13 of this Annex, Bioconcentration: Flow-through Fish Test (49) and OECD TG 315: Bioaccumulation in Sediment-dwelling Benthic Oligochates (53). Practical experience with soil bioaccumulation studies and publications from LITERATURE e.g. (1) (5) (11) (12) (28) (40) (43) (45) (57) (59) (76) (78) (79) are also major sources of information for this Test Method.

• 4.This Test Method is mostly applicable to stable, neutral organic chemicals, which tend to adsorb to soils. Testing for bioaccumulation of soil-associating, stable metallo-organic compounds may be possible with this Test Method. It is also applicable to metals and other trace elements.

PRE-REQUISITE

• 5.Tests for measuring the bioaccumulation of a chemical in terrestrial oligochaetes have been performed with heavy metals (see e.g. (63)) and persistent, organic chemicals having log Kow values between 3,0 and 6,0, e.g. (40). Such tests also apply to:

• — Chemicals that show a log Kow of more than 6,0 (super-hydrophobic chemicals);

• — Chemicals which belong to a class of organic chemicals known to have the potential to bioaccumulate in living organisms, e.g. surface active or highly adsorptive chemicals;

• — Chemicals that indicate the potential for bioaccumulation from structural features, e.g. analogues of chemicals with known bioaccumulation potential; and

• — Metals.

• 6.Information on the test chemical such as common name, chemical name (preferably IUPAC name), structural formula, CAS registry number, purity, safety precautions, proper storage conditions and analytical methods should be obtained before beginning the study. In addition, the following information should be known:

• (a) solubility in water;

• (b) octanol-water partition coefficient, Kow;

• (c) soil-water partition coefficient, expressed as Koc;

• (d) vapour pressure;

• (e) degradability (e.g. in soil, water);

• (f) known metabolites.

• 7.Radiolabelled or non-radiolabelled test chemicals can be used. However, to facilitate analysis it is recommended to use a radiolabelled test chemical. The decision will be made based on the detection limits or a requirement to measure parent test chemical and metabolites. If a radiolabelled test chemical is used and total radioactive residues are measured, it is important that the radiolabelled residues in both the soil and the test organisms are characterised for percentages of parent test chemical and labelled non-parent, e.g. in samples taken at steady state or at the end of the uptake phase, to allow a bioaccumulation factor (BAF) calculation for the parent test chemical and for the soil metabolites of concern (see paragraph 50). The method described here may have to be modified, e.g. to provide sufficient biomass, for measuring non-radiolabelled organic test chemical or metals. When total radioactive residues are measured (by liquid scintillation counting following extraction, combustion or tissue solubilisation), the bioaccumulation factor is based on the parent test chemical and metabolites. The BAF calculation should preferably be based on the concentration of the parent test chemical in the organisms and total radioactive residues. Subsequently, the biota-soil accumulation factor (BSAF), normalized to the lipid content of worm and organic carbon content (OC) of soil should be calculated from the BAF for reasons of comparability between results from different bioaccumulation tests.

• 8.Toxicity of the test chemical to the species used in the test should be known, e.g. an effect concentration (ECx) or lethal concentration (LCx) for the time of the uptake phase (e.g. (19)). The selected concentration of the test chemical should preferably be about 1 % of its acute asymptotic LC50, and at least 10-fold higher than its detection limit in soil by the analytical method used. If available, preference should be given to toxicity values derived from long-term studies on sublethal endpoints (51) (52). If such data are not available, an acute toxicity test will provide useful information (see e.g. (23)).

• 9.An appropriate analytical method of known accuracy, precision, and sensitivity for the quantification of the chemical in the test solutions, in the soil, and in the biological material should be available, together with details of sample preparation and storage as well as material safety data sheets. Analytical detection limits of the test item in soil and worm tissue should also be known. If a 14C-labelled test chemical is used, the specific radioactivity (i.e. Bq mol-1) and the percentage of radioactivity associated with impurities should be known. The specific radioactivity of the test chemical should be high enough to facilitate analysis, and the test concentrations used should not elicit toxic effects.

• 10.The test can be performed with an artificial soil or with natural soils. Information on characteristics of the natural soil used, e.g. origin of soil or its constituents, pH, organic carbon content, particle size distribution (percent sand, silt, and clay), and water holding capacity (WHC), should be known before the start of the test (3) (48).

PRINCIPLE OF THE TEST

• 11.The parameters which characterise the bioaccumulation of a test chemical include the bioaccumulation factor (BAF), the uptake rate constant (ks) and the elimination rate constant (ke). Definitions are provided in Appendix 1.

• 12.The test consists of two phases: the uptake (exposure) phase and the elimination (post-exposure) phase. During the uptake phase, replicated groups of worms are exposed to soil, which has been spiked with the test chemical. In addition to the test animals, groups of control worms are held under identical conditions without the test chemical. The dry weight and lipid content of the test organisms are measured. This can be done using worms of the control group. Analytical background values (blank) can be obtained by analysing samples of the control worms and soil. For the elimination phase, the worms are transferred to a soil free of the test chemical. An elimination phase is always required unless uptake of the test chemical during the exposure phase has been insignificant. An elimination phase provides information on the rate at which the test chemical is excreted by the test organisms (e.g. (27)). If a steady state has not been reached during the uptake phase, the determination of the kinetic parameters – kinetic bioaccumulation factor BAFk, uptake and elimination rate constant(s) – should preferably be based on simultaneous fitting of the results of the uptake and elimination phases. The concentration of the test chemical in/on the worms is monitored throughout both phases of the test.

• 13.During the uptake phase, measurements are made at sampling times up to 14 days (enchytraeids) or 21 days (earthworms) until the steady state is reached (11) (12) (67). The steady state occurs when a plot of the concentration in worms against time is parallel to the time axis, and three successive concentration analyses made on samples taken at intervals of at least two days do not vary more than ± 20 % of each other based on statistical comparisons (e.g. analysis of variance, regression analysis).

• 14.The elimination phase consists of transferring the test organisms to vessels containing the same substrate without the test chemical. During the elimination phase, measurements are made at sampling times during 14 days (enchytraeids) or 21 days (earthworms) unless earlier analytical determination showed 90 % reduction of the test chemical residues in worms. The concentration of the test chemical in the worms at the end of the elimination phase is reported as non-eliminated residues. The steady state bioaccumulation factor (BAFss) is calculated preferably both as the ratio of the concentration in worms (Ca) and in the soil (Cs) at apparent steady state, and as a kinetic bioaccumulation factor, BAFK, as the ratio of the rate constant of uptake from soil (ks) and the elimination rate constant (ke) (see Appendix 1 for definitions) assuming first-order kinetics (see Appendix 2 for calculations). If first-order kinetics is obviously not applicable, other models should be employed.

• 15.The uptake rate constant, the elimination rate constant (or constants, where other models are involved), the kinetic bioaccumulation factor (BAFK), and where possible, the confidence limits of each of these parameters are calculated from computerised model equations (see Appendix 2 for guidance). The goodness of fit of any model can be determined from e.g. the correlation coefficient or the coefficient of determination (coefficients close to one indicate a good fit) or chi-squared. Also the size of the standard error or confidence limit around the estimated parameters may be indicative of the goodness of fit of the model.

• 16.To reduce variability in test results for test chemicals with high lipophilicity, bioaccumulation factors should be expressed in relation to lipid content and organic carbon content (kg soil organic carbon (OC) kg-1 worm lipid content). This approach is based on the fact that for some chemical classes, there is a clear relationship between the potential for bioaccumulation and lipophilicity; this has been well established for fish (47). There is a relationship between the lipid content of fish and the bioaccumulation of such chemicals. For benthic organisms, similar correlations have been found e.g. (30) (44). Likewise for terrestrial oligochaetes this correlation has been demonstrated e.g. (5) (6) (7) (14). If sufficient worm tissue is available, the lipid content of the test animals can be determined on the same biological material as the one used to determine the concentration of the test chemical. Alternatively, control animals can be used to measure the lipid content.

VALIDITY OF THE TEST

• 17.For a test to be valid the following criteria should be fulfilled for both controls and treatments:

• — At the end of the test, the overall mortality during uptake and elimination phase should not exceed 10 % (earthworms) or 20 % (enchytraeids) of the total number of the introduced worms.

• — For Eisenia fetida and Eisenia andrei, the mean mass loss as measured at the end of the uptake and at the end of the elimination phase should not exceed 20 % compared to the initial fresh weight (f.w.) at start of each phase.

DESCRIPTION OF THE METHOD

Test species

• 18.Several species of terrestrial oligochaetes are recommended for bioaccumulation testing. The most commonly used species Eisenia fetida or Eisenia andrei (Lumbricidae), or Enchytraeus albidus, Enchytraeus crypticus, or Enchytraeus luxuriosus (Enchytraeidae)) are described in Appendix 5.

Apparatus

• 19.Care should be taken to avoid the use of materials, for all parts of the equipment, which can dissolve, adsorb the test chemical or leach other chemicals, and have an adverse effect on the test animals. Standard rectangular or cylindrical vessels, made of chemically inert material and of suitable capacity can be used in compliance with the loading rate, i.e. the number of test worms. Stainless steel, plastic or glass may be used for any equipment having contact with the test media. The test vessels should be appropriately covered to prevent escaping of the worms, while allowing sufficient air supply. For chemicals with high adsorption coefficients, such as synthetic pyrethroids, silanised glass may be required. In these situations the equipment will have to be discarded after use (49). Radiolabelled test items and volatile chemicals should be prevented from escaping. Traps (e.g. glass gas washing bottles) should be employed containing suitable absorbents to retain any residues evaporating from the test vessels.

Soil

• 20.The test soil should be of a quality that will allow the survival and preferably the reproduction of the test organisms for the duration of the acclimation and test periods without them showing any abnormal appearance or behaviour. The worms should burrow in the soil.

• 21.The artificial soil described in the chapter C.8 of this Annex (48) is recommended for use as the substrate in the tests. Preparation of the artificial soil for use in the bioaccumulation tests and recommendations for the storage of artificial soil are given in Appendix 4. Air-dried artificial soil may be stored at room temperature until use.

• 22.However, natural soils from unpolluted sites may serve as test and/or culture soil. Natural soils should be characterised at least by origin (collection site), pH, organic carbon content, particle size distribution (percent sand, silt, and clay), maximum water holding capacity (WHCmax), and percent water content (3). Analysis of the soil or its constituents for micro-pollutants prior to use should provide useful information. If field soil from agricultural land is used, it should not have been treated with crop protection products or with manure from treated animals as fertilizers for at least one year and with organic fertilizers for at least six months prior to sampling (50). Manipulation procedures for natural soils prior to use in ecotoxicological tests with oligochaetes in the laboratory are described in (3). For natural soils the storage time in the laboratory should be kept as short as possible.

Application of the test chemical

• 23.The test chemical is incorporated into the soil. The physicochemical properties of the test chemical should be taken into consideration. A water-soluble test chemical should be completely dissolved in water before it be mixed with the soil. The recommended spiking procedure for poorly water-soluble test chemical involves coating of one or more of the (artificial) soil constituents with the test chemical. For example, the quartz sand, or a portion thereof, can be soaked with a solution of the test chemical in a suitable organic solvent, which is then slowly evaporated to dryness. The coated fraction can then be mixed into the wet soil. The major advantage of this procedure is that no solvent is introduced into the soil. When a natural soil is used, the test chemical may be added by spiking an air-dried portion of the soil as described above for the artificial soil, or by stirring the test chemical into the wet soil, with subsequent evaporating step if a solubilising agent is used. In general, the contact of wet soil with solvents should be avoided as far as possible. The following should be considered (3):

• — If a solvent other than water is used, it should be one that is water-miscible and/or can be driven off (for example, evaporated), leaving only the test chemical on the soil.

• — If a solvent control is used, there is no need for negative control. The solvent control should contain the highest concentration of solvent added to the soil and should use solvent from the same batch used to make the stock solution. Toxicity and volatility of the solvent, and solubility of the test chemical in the chosen solvent should be the main criteria used for the selection of a suitable solubilising agent.

• 24.For chemicals that are poorly soluble in water and in organic solvents, 2,0-2,5 g of finely ground quartz sand per test vessel can be mixed with the quantity of test chemical, e.g. using mortar and pestle, to obtain the desired test concentration. This mixture of quartz sand and test chemical is added to the pre-moistened soil and thoroughly mixed with an appropriate amount of de-ionised water to obtain the required moisture content. The final mixture is distributed to the test vessels. The procedure is repeated for each test concentration, and an appropriate control with 2,0-2,5 g of finely ground quartz sand per test vessel is also prepared.

• 25.The concentration of the test chemical in the soil should be determined after spiking. The homogenous distribution of the test chemical into the soil should be verified before introducing the test organisms. The method used for spiking, and the reasons for choosing a specific spiking procedure should be reported (24).

• 26.Equilibrium between the soil and the pore-water phase should ideally be established before adding the organisms; a time period of four days at 20 °C is recommended. For many poorly water-soluble organic chemicals the time required to reach a true equilibrium between adsorbed and dissolved fractions can be counted in days or months. Depending on the purpose of the study, for example when the environmental conditions are to be mimicked, the spiked soil may be ‘aged’ for a longer period, e.g. for metals three weeks at 20 °C (22).

Culturing of the test organisms

• 27.Worms should be preferably kept in permanent laboratory culture. Guidance on laboratory culture methods for Eisenia fetida and Eisenia andrei, and Enchytraeid species, is provided in Appendix 5 (see also (48) (51) (52)).

• 28.The worms used in the tests should be free from observable diseases, abnormalities and parasites.

PERFORMANCE OF THE TEST

• 29.The test organisms are exposed to the test chemical during the uptake phase. The uptake phase should be of 14 days (enchytraeids) or 21 days (earthworms) unless it is demonstrated that steady state has been reached.

• 30.For the elimination phase, the worms are transferred to a soil free of test chemical. The first sample should be taken at 4-24 h after the start of elimination phase. Examples of sampling schedules for a 21-day uptake phase and a 21-day elimination phase are given in Appendix 3.

Test organisms

• 31.For many species of terrestrial enchytraeids the individual weight is very low (e.g. 5-10 mg wet weight per individual for Enchytraeus albidus and less for Enchytraeus crypticus or Enchytraeus luxuriosus); in order to perform the weight measurements and chemical analysis, it may be necessary to pool the worms of the replicate test vessels (i.e. all the worms of a replicate vessel will be used for obtaining one analytical tissue result). 20 individual enchytraeids are added to each replicate, and at least three replicates should be used. If the analytical detection limit of the test chemical is high, more worms may be necessary. For test species with higher individual weight (Eisenia fetida and Eisenia andrei), replicate vessels containing one individual can be used.

• 32.The earthworms used in a test should be of similar weight (e.g. Eisenia fetida and Eisenia andrei should have an individual weight of 250-600 mg). Enchytraeids (e.g. Enchytraeus albidus) should have a length of approximately 1 cm. All worms used in a particular test should come from the same source, and should be adult animals with clitellum (see Appendix 5). Since the weight and age of an animal might have an effect on the BAF-values (e.g. due to varying lipid content and/or presence of eggs), these parameters should be recorded accurately and taken into account in the interpretation of results. In addition, cocoons can be deposited during the exposure period, which will also have an impact on the BAF values. It is recommended that a sub-sample of the test worms be weighed before the test in order to estimate the mean wet and dry weights.

• 33.A high soil-to-worm ratio should be used in order to minimise the decrease of the test chemical concentration in the soil during the uptake phase. For Eisenia fetida and Eisenia andrei a minimum amount of 50 g dry weight (d.w.) of soil per worm, and for enchytraeids, a minimum of 10-20 g d.w. of soil per test vessel are recommended. The vessels should contain a soil layer of 2-3 cm (enchytraeids) or 4-5 cm (earthworms).

• 34.The worms used in a test are removed from the culture (e.g. enchytraeids by using jeweller’s tweezers). Adult animals are transferred to non-treated test soil for acclimation, and fed (see paragraph 36). If the test conditions differ from the culture conditions, an acclimation phase of 24-72 h should be sufficient to adapt the worms to the test conditions. After acclimation, earthworms are rinsed by transfer to glass dishes (e.g. petri dishes) containing clean water, and subsequently weighed before they are added to the test soil. Prior to weighing, excess water should be removed from the worms by gently touching them against the edge of the dish or by blotting them cautiously dry by using a slightly moistened paper towel.

• 35.Burrowing behaviour of the test organisms should be observed and recorded. In tests with earthworms, the animals (control and treatments) normally burrow in the soil within a period of a few hours; this should be checked no later than 24 h after addition of the worms to the test vessels. If the earthworms fail to burrow in the soil (e.g. more than 10 % over more than half of the uptake phase), this indicates that either the test conditions are not appropriate or the test organisms are not healthy. In such a case the test should be stopped and repeated. Enchytraeids mainly live in the interstitial pores of the soil, and frequently their integument may be only partly in contact with the surrounding substrate; exposure of burrowing and non-burrowing enchytraeids is assumed to be equivalent and non-burrowing of the enchytraeids does not necessarily require the repetition of the test.

Feeding

• 36.Feeding should be envisaged when a soil with low total organic carbon content is used. When an artificial soil is used, a weekly feeding rate (i.e. the worms should be fed once a week) of 7 mg of dried dung per g soil dry weight is recommended for earthworms, and a weekly rate of 2-2,5 mg of ground oat flakes per g soil dry weight is recommended for enchytraeids (11). The first food ration should be mixed with the soil immediately before the test organisms are added. Preferably the same type of food like in the cultures should be used (see Appendix 5).

Light and temperature

• 37.The tests should be carried out under a controlled 16/8 hours light/dark cycle, preferably 400 to 800 lx in the area of the test vessels (3). The test temperature should be 20 ± 2 °C throughout the test.

Test concentrations

• 38.A single concentration is used. Situations where additional concentration(s) is(are) required should be justified. If toxicity (ECx) of the test chemical is close to the analytical detection limit, the use of radiolabelled test chemical with high specific radioactivity is recommended. For metals, the concentration should be above the background level in tissue and soil.

Replicates

• 39.For the kinetic measurements (uptake and elimination phase), the minimum number of treated replicate vessels should be three per sampling point. The total number of replicates prepared should be sufficient to cover all sampling times during the uptake and the elimination phase.

• 40.For the biological observations and measurements (e.g. dry-to-wet weight ratio, lipid content) and for the analysis of background concentrations in worms and soil, at least 12 replicate vessels of a negative control (four sampled at start, four at end of uptake, and four at end of elimination) should be provided if no solvent other than water is used. If any solubilising agent is used for application of the test chemical, a solvent control (four replicate vessels should be sampled at start, four at the end of the uptake phase, and four at the end of the elimination phase) containing all constituents except for test item should be run in addition to the treated replicates. In this case, four additional replicate vessels of a negative control (no solvent) may also be provided for optional sampling at the end of the uptake phase. These replicates can be compared biologically with the solvent control in order to gain information on a possible influence of the solvent on the test organisms. It is recommended establishing a sufficient number of additional reserve replicate vessels (e.g. eight) for treatment and control(s).

Frequency of soil quality measurements

• 41.Soil pH, soil moisture content and the temperature (continuously) in the test room should be measured at the start and end of the uptake and elimination phases. Once per week the soil moisture content should be controlled by weighing the test vessels and comparing actual weights with initial weights at test start. Water losses should be compensated by adding deionised water.

Sampling and analysis of worms and soil

• 42.An example of schedule for the uptake and elimination phases in earthworm and enchytraeid bioaccumulation tests is given in Appendix 3.

• 43.The soil is sampled from the test vessels for the determination of test chemical concentration before inserting the worms, and during the uptake and elimination phases. During the test the concentrations of test chemical are determined in the worms and the soil. In general, total soil concentrations are measured. As an option, concentrations in pore water may be measured; in such case, rationale and appropriate methods should be provided prior to initiation of a study, and included in the report.

• 44.The worms and soil are sampled at least at six occasions during the uptake and the elimination phases. If the stability of a test chemical is demonstrated, the number of soil analyses can be reduced. It is recommended analysing at least three replicates at the beginning and at the end of the uptake phase. If the concentration in soil measured at the end of the uptake phase deviates from the initial concentration by more than 30 %, the soil samples taken at other dates should also be analysed.

• 45.Remove the worms of a given replicate from the soil at each sampling time (e.g. after spreading the soil of the replicate on a shallow tray and picking the worms using soft jewellers’ tweezers), rinse them quickly with water in a shallow glass or steel tray. Remove excess water (see paragraph 34). Transfer the worms carefully to a pre-weighed vessel, weigh them instantly, including gut content.

• 46.The earthworms (Eisenia sp.) should then be allowed to purge their gut overnight e.g. on a moist filter paper in a covered petri dish (see paragraph 34). After purging, the weight of the worms should be determined in order to assess a possible decrease in biomass during the test (see validity criteria in paragraph 17). Weighing and tissue analysis of Enchytraeids is carried out without purging, as this is technically difficult due to the small size of these worms. After final weight determination, the worms should be killed immediately, using the most appropriate method (e.g. using liquid nitrogen, or freezing at temperatures below – 18 °C).

• 47.During the elimination phase, the worms replace contaminated gut contents with clean soil. This means, measurements in un-purged worms (enchytraeids in this context) sampled immediately before the elimination phase include contaminated gut soil. For aquatic oligochaetes it is assumed that after the initial 4-24 h of the elimination phase, most of the contaminated gut content has been replaced by clean sediment e.g. (46). Similar findings have been reported for earthworms in studies on the accumulation of radiolabelled cadmium and zinc (78). In the non-purged enchytraeids, the concentration of this first sample of the elimination phase may be considered as the tissue concentration after gut purge. To account for dilution of the test item concentration by uncontaminated soil during the elimination phase, the weight of the gut content may be estimated from worm wet weight/worm ash weight or worm dry weight/worm ash weight ratios.

• 48.The soil and worm samples should be preferably analysed immediately after removal (i.e. within 1-2 days) in order to prevent degradation or other losses, and it is recommended calculating the approximate uptake and elimination rates as the test proceeds. If the analysis is delayed, the samples should be stored by an appropriate method, e.g. by deep-freezing (≤ – 18 °C).

• 49.It should be checked that the precision and reproducibility of the chemical analysis, as well as the recovery of the test chemical from soil and worm samples are satisfactory for the given method; the extraction efficiency, the limit of detection (LOD) and the limit of quantification (LOQ) should be reported. Likewise it should be checked that the test chemical is not detectable in the control vessels in concentrations higher than background. When the concentration of the test chemical in the test organism Ca is > 0 in the control worms, this should be included in the calculation of the kinetic parameters (see Appendix 2). All samples should be handled throughout the test to minimise contamination and loss (e.g. resulting from adsorption of the test chemical on the sampling device).

• 50.When working with radiolabelled test chemicals, it is possible to analyse parent and metabolites. Quantification of parent test chemical and metabolites at steady state or at the end of the uptake phase provides important information. The samples should then be ‘cleaned up’ so that the parent test chemical can be quantified separately. If single metabolites exceed 10 % of total radioactivity in the analysed sample(s), the identification of these metabolites is recommended.

• 51.The overall recovery, and the recovery of test chemical in worms, soil, and if used, in traps containing absorbents to retain evaporated test chemical, should be recorded and reported.

• 52.Pooling of the individuals sampled from a given test vessel is acceptable for enchytraeid worms which are smaller than earthworms. If pooling involves the reduction of the number of replicates, this limits the statistical procedures which can be applied to the data. If a specific statistical procedure and power are required, then an adequate number of replicate test vessels should be included in the test to accommodate the desired pooling, procedure and power.