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)

• 53.It is recommended that the BAF be expressed both as a function of total dry weight and, when required (i.e. for highly hydrophobic chemicals), as a function of the lipid content. Suitable methods should be used for determination of lipid content (some existing methods – e.g. (31) (58) – should be adapted for this purpose). These methods use a chloroform/methanol extraction technique. However, to avoid the use of chlorinated solvents, a modification of the Bligh and Dyer method (9) as described in (17) should be used. Since the various methods may not give identical values, it is important to give details of the method used. When possible, i.e. if sufficient worm tissue is available, the lipid analysis should ideally be made on the same sample or extract as the one used for analysis of the test chemical, since the lipids often have to be removed from the extract before it can be analysed chromatographically (49). Alternatively, control animals may be used to measure the lipid content, which can then be used to normalise BAF values. This latter approach reduces the contamination of equipment with the test chemical.

DATA AND REPORTING

Treatment of results

• 54.The uptake curve of the test chemical is obtained by plotting its concentration in/on the worms during the uptake phase against time on arithmetic scales. When the curve has reached a plateau, or steady state (see definitions in Appendix 1), the steady state bioaccumulation factor BAFss is calculated from:

Image omitted; see official source

• Ca is the concentration of test chemical in the test organism

• Cs is the concentration of test chemical in the soil

• 55.When no steady state is reached, the BAFK, based on the rate constants, should be determined instead of BAFss, as described below:

• — Determine the accumulation factor (BAFK) as the ratio ks/ke.

• — Uptake and elimination rates are preferably calculated simultaneously (see Equation 11 in Appendix 2)

• — The elimination rate constant (ke) is usually determined from the elimination curve (i.e. a plot of the concentration of the test item in the worms during the elimination phase). The uptake rate constant ks is then calculated given ke and a value of Ca which is derived from the uptake curve – See Appendix 2 for a description of these methods. The preferred method for obtaining BAFK and the rate constants, ks, and ke, is to use non-linear parameter estimation methods on a computer. If the elimination is obviously not first-order, then more complex models should be employed.

Test report

• 56.The test report should include the following information:

Test chemical:

• — Any available information on acute or long term toxicity (e.g. ECx, LCx„ NOEC) of the test chemical towards soil-dwelling oligochaetes;

• — purity, physical nature and, physicochemical properties e.g. log Kow, water solubility;

• — chemical identification data; source of the test item, identity and concentration of any solvent used;

• — if radiolabelled test chemical is used, the precise position of the labelled atoms, the specific radioactivity, and the radiochemical purity.

Test species:

• — scientific name, strain, source, any pre-treatment, acclimation, age, size-range, etc..

Test conditions:

• — test procedure used;

• — type and characteristics of illumination used and photoperiod(s);

• — test design (e.g. number and size of test vessels, soil mass and height of soil layer, number of replicates, number of worms per replicate, number of test concentrations, duration of uptake and elimination phases, sampling frequency);

• — rationale for the choice of test vessel material;

• — method of test item preparation and application as well as reasons for choosing a specific method;

• — the nominal test concentrations, the means of the measured values and their standard deviations in the test vessels, and the method by which these values were obtained;

• — source of the constituents of the artificial soil or – if natural media are used – origin of the soil, description of any pre-treatment, results of the controls (survival, biomass development, reproduction), soil characteristics (pH, total organic carbon content, particle size distribution (percent sand, silt, and clay), WHCmax, percent water content at start and at end of the test, and any other measurements made);

• — detailed information on the treatment of soil and worm samples, including details of preparation, storage, spiking procedures, extraction, and analytical procedures (and precision) for the test item in worms and soil, and lipid content (if measured), and recoveries of the test item.

Results:

• — mortality of the control worms and the worms in each test vessel and any observed abnormal behaviour (e.g. soil avoidance, lack of reproduction in a bioaccumulation test with enchytraeids);

• — the dry weight to wet weight ratio of the soil and the test organisms (useful for normalisation);

• — the wet weights of the worms at each sampling time; for earthworms, the wet weights at start of the test, and at each sampling time before and after gut purging;

• — the lipid content of the test organisms (if determined);

• — curves, showing the uptake and elimination kinetics of the test chemical in the worms, and the time to steady state;

• — Ca and Cs (with standard deviation and range, if appropriate) for all sampling times (Ca expressed in g kg^–1 wet and dry weight of whole body, Cs expressed in g kg^–1 wet and dry weight of soil). If a biota-soil accumulation factor (BSAF) is required (e.g. for comparison of results from two or more tests performed with animals of differing lipid content), Ca may additionally be expressed as g kg^–1 lipid content of the organism, and Cs may be expressed as g kg^–1 organic carbon (OC) of the soil;

• — BAF (expressed in kg soil·kg^–1 worm), soil uptake rate constant ks (expressed in g soil kg^–1 of worm day^–1), and elimination rate constant ke (expressed in day^–1); BSAF (expressed in kg soil OC kg^–1 worm lipid content) may be reported additionally;

• — if measured: percentages of parent chemical, metabolites, and bound residues (i.e. the percentage of test chemical that cannot be extracted with common extraction methods) detected in soil and test animals;

• — methods used for the statistical analyses of data.

Evaluation of results:

• — compliance of the results with the validity criteria as listed in paragraph 17;

• — unexpected or unusual results, e.g. incomplete elimination of the test chemical from the test animals.

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Appendix 1

DEFINITIONS

Bioaccumulation is the increase in concentration of the test chemical in or on an organism relative to the concentration of the test chemical in the surrounding medium. Bioaccumulation results from both bioconcentration and biomagnification processes (see below).

Bioconcentration is the increase in concentration of the test chemical in or on an organism, resulting from the uptake of the chemical exclusively from the surrounding medium (i.e. via the body surface and ingested soil), relative to the concentration of the test chemical in the surrounding medium.

Biomagnification is the increase in concentration of the test chemical in or on an organism, resulting mainly from uptake from contaminated food or prey, relative to the concentration of the test chemical in the food or prey. Biomagnification can lead to a transfer or accumulation of the test item within food webs.

The elimination of a test chemical is the loss of this chemical from the test organism tissue by active or passive processes that occurs independently of presence or absence of the test item in the surrounding medium.

The bioaccumulation factor (BAF) at any time during the uptake phase of this bioaccumulation test is the concentration of test chemical in/on the test organism (Ca in g·kg^-1 dry weight of worm) divided by the concentration of the chemical in the surrounding medium (Cs as g·kg^-1 of dry weight of soil); the BAF has the units of kg soil·kg^-1 worm.

The steady state bioaccumulation factor (BAFss) is the BAF at steady state and does not change significantly over a prolonged period of time, the concentration of the test chemical in the surrounding medium (Cs as g.kg^-1 of dry weight of soil) being constant during this period of time.

Bioaccumulation factors calculated directly from the ratio of the soil uptake rate constant and the elimination rate constant (ks and ke, see below) are termed kinetic bioaccumulation factor (BAFK).

The biota-soil accumulation factor (BSAF) is the lipid-normalised concentration of the test chemical in/on the test organism divided by the organic carbon-normalised concentration of the test chemical in the soil at steady state. Ca is then expressed as g·kg^-1 lipid content of the organism, and Cs as g·kg^-1 organic content of the soil; the BSAF has the units of kg OC·kg^-1 lipid.

A plateau or steady state is defined as the equilibrium between the uptake and elimination processes that occur simultaneously during the exposure phase. The steady state is reached in the plot of BAF against time when the curve becomes parallel to the time axis and three successive analyses of BAF made on samples taken at intervals of at least two days are within 20 % of each other, and there are no statistically significant differences among the three sampling periods. For test chemicals which are taken up slowly, more appropriate intervals would be seven days (49).

The organic carbon-water partitioning coefficient (Koc) is the ratio of a chemical’s concentration in/on the organic carbon fraction of a soil and the chemical's concentration in water at equilibrium.

The octanol-water partitioning coefficient (Kow) is the ratio of a chemical’s solubility in n-octanol and water at equilibrium, also sometimes expressed as Pow. The logarithm of Kow (log Kow) is used as an indication of a chemical's potential for bioaccumulation by aquatic organisms.

The uptake or exposure phase is the time during which the test organisms are exposed to the test chemical.

The soil uptake rate constant (ks) is the numerical value defining the rate of increase in the concentration of the test item in/on the test organism resulting from uptake from the soil phase. ks is expressed in g soil kg^-1 of worm d^-1.

The elimination phase is the time, following the transfer of the test organisms from a contaminated medium to a medium free of the test item, during which the elimination (or the net loss) of the chemical from the test organisms is studied.

The elimination rate constant (ke) is the numerical value defining the rate of reduction in the concentration of the test item in/on the test organism, following the transfer of the test organisms from a medium containing the test item to a chemical-free medium; ke is expressed in d^-1.

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

Appendix 2

Calculation of uptake and elimination parameters

The main endpoint of a bioaccumulation test is the bioaccumulation factor, BAF. The measured BAF can be calculated by dividing the concentration in the test organism, Ca, by the concentration in the soil, Cs, at steady state. If the steady state is not reached during the uptake phase, the BAFK is calculated from the rate constants instead of BAFss. However, it should be noted if the BAF is based on steady state concentrations or not.

The usual means for obtaining the kinetic bioaccumulation factor (BAFK), the soil uptake rate constant (ks) and the elimination rate constant (ke) is to use non-linear parameter estimation methods on a computer, e.g. based on the models described in (68). Given a set of sequential time concentration data and the model equations:

Image omitted; see official source	0 < t < tc	[equation 1]

or

Image omitted; see official source	t > tc	[equation 2]

where:

Ca	=	concentration of chemical in worms [g kg-1 wet or dry weight]
---	---	---
ks	=	uptake rate constant in tissue [g soil kg-1 of worm d-1]
---	---	---
Cs	=	concentration of chemical in soil [g kg-1 of wet or dry weight]
---	---	---
ke	=	elimination rate constant [d-1]
---	---	---
tc	=	time at the end of the uptake phase,

these computer programs calculate values for BAFK, ks and ke.

When the background concentration in the non-exposed worms e.g. on day 0 differs significantly from zero (this may e.g. be the case for metals), this background concentration (Ca,0) should be included in these equations, to make them read:

Image omitted; see official source	0 < t < tc	[equation 3]

and

Image omitted; see official source	t > tc	[equation 4]

In cases where a significant decrease of the test chemical concentration in the soil is observed over time during the uptake phase, the following models can be used e.g. (67) (79):

Image omitted; see official source	[equation 5]

where:

Cs	=	concentration of chemical in the soil [g kg-1 wet or dry weight]
---	---	---
k0	=	degradation rate constant in soil [d-1]
---	---	---
C0	=	initial concentration of chemical in soil [g kg-1 of wet or dry weight]
---	---	---
Image omitted; see official source	0 < t < tc	[equation 6]
Image omitted; see official source	t > tc	[equation 7]

where:

Ca	=	concentration of chemical in worms [g kg-1 wet or dry weight]
---	---	---
ks	=	uptake rate constant in tissue [g soil kg-1 of worm d-1]
---	---	---
k0	=	degradation rate constant in soil [d-1]
---	---	---
ke	=	elimination rate constant [d-1]
---	---	---
tc	=	time at the end of the uptake phase.

When steady state is reached during the uptake phase (i.e. t = ∞), equation 1

Image omitted; see official source	0 < t < tc	[equation 1]

may be reduced to:

Image omitted; see official source

or

Image omitted; see official source	[equation 8]

Then ks/ke x Cs is an approach to the concentration of the test item in the worm tissue at steady state (Ca,ss).

The biota-soil accumulation factor (BSAF) can be calculated as follows:

Image omitted; see official source	[equation 9]

where foc is the fraction of soil organic carbon, and flip is the fraction of worm lipid, both preferably determined on samples taken from the test, and based either on dry weight or on wet weight, respectively.

The elimination kinetics can be modelled using the data from the elimination phase and applying the following model equation and a computer-based non-linear parameter estimation method. If the data points plotted against time indicate a constant exponential decline of the test item concentration in the animals, a one-compartment model (equation 9) can be used to describe the time course of elimination.

Image omitted; see official source	[equation 10]

Elimination processes sometimes appear to be biphasic, showing a rapid decline of Ca during the early phases, that changes to a slower loss of test items in the later phases of the elimination, e.g. (27) (68). The two phases can be interpreted by the assumption, that there are two different compartments in the organism, from which the test item is lost with different velocities. In these cases, specific LITERATURE should be studied e.g. (38) (39) (40) (78).

Using the model equations above, the kinetic parameters (ks and ke) may also be calculated in one run by applying the first order kinetics model to all data from both the uptake and elimination phase simultaneously. For a description of a method that may allow for such a combined calculation of uptake and elimination rate constants, references (41), (73) and (70) may be consulted.

Image omitted; see official source	[equation 11]

• Note: When uptake and elimination parameters are estimated simultaneously from the combined uptake and the elimination data, ‘m’ as shown in equation 11 is a descriptor that allows the computer program to assign the equation’s sub-terms to the data sets of the respective phase and to perform the evaluation correctly (m = 1 for uptake phase; m = 2 for elimination phase).

Nevertheless, these model equations should be used with caution, especially when changes in the test chemical's bioavailability, or (bio)degradation occur during the test (see e.g. (79)).

Appendix 3

EXAMPLES OF SCHEDULES FOR SOIL BIOACCUMULATION TESTS

Earthworm test

• (a)Uptake phase with 8 sampling dates used for calculation of kinetics

Day	Activity
– 6	Conditioning of the prepared soil for 48 h;
– 4	Spiking of the soil fraction with the test chemical solution; evaporating of any solvent; mixing of the soil constituents; distributing the soil to the test vessels; equilibration at test conditions for 4 days (3 weeks for metal-spiked soil);
– 3 to – 1	Separation of the test organisms from the culture for acclimation; preparation and moisturising of the soil constituents;
0	Measuring temperature, and soil pH; removing soil samples from treated vessels and solvent controls for determination of test chemical concentration; addition of food ration; weighing and randomised distribution of the worms to the test vessels; retaining of sufficient subsamples of worms for determination of analytical background values, wet and dry weight, and lipid content; weighing of all test vessels to control soil moisture; controlling air supply, if closed test system is used;
1	Controlling air supply, recording worm behaviour and temperature; taking soil and worm samples for determination of test item concentration;
2	Same as day 1;
3	Controlling air supply, worm behaviour and temperature;
4	Same as day 1;
5-6	Same as day 3;
7	Same as day 1; addition of food ration; control soil moisture by re-weighing the test vessels and compensate evaporated water;
8-9	Same as day 3;
10	Same as day 1;
11-13	Same as day 3;
14	Same as day 1; addition of food ration; control soil moisture by re-weighing the test vessels and compensate evaporated water;
15-16	Same as day 3;
17	Same as day 1;
18-20	Same as day 3;
21	Same as day 1; measuring temperature and soil pH; control soil moisture by re-weighing the test vessels; end of uptake phase; transfer worms from remaining exposed replicates to vessels containing clean soil for elimination phase (no gut-purging); sampling of soil and worms from solvent controls.
	Pre-exposure activities (equilibration phase) should be scheduled taking into account the properties of the test chemical.
	Activities described for day 3 should be performed daily (at least on workdays).

• (b)Elimination phase

Day	Activity
– 6	Preparation and moisturising of the soil constituents; conditioning of the prepared soil for 48 h;
– 4	Mixing of the soil constituents; distributing the soil to the test vessels; incubation at test conditions for 4 days;
0 (end of uptake phase)	Measuring temperature and soil pH; weighing and randomised distribution of the worms to the test vessels; addition of food ration; transfer worms from remaining exposed replicates to vessels containing clean soil; taking soil and worm samples after 4-6 h for determination of test chemical concentration;
1	Controlling air supply, recording worm behaviour and temperature; taking soil and worm samples for determination of test chemical concentration;
2	Same as day 1;
3	Controlling air supply, worm behaviour and temperature;
4	Same as day 1;
5-6	Same as day 3;
7	Same as day 1; addition of food ration; control soil moisture by re-weighing the test vessels and compensate evaporated water;
8-9	Same as day 3;
10	Same as day 1;
11-13	Same as day 3;
14	Same as day 1; addition of food ration; control soil moisture by re-weighing the test vessels and compensate evaporated water;
15-16	Same as day 3;
17	Same as day 1;
18-20	Same as day 3;
21	Same as day 1; measuring temperature and soil pH; control soil moisture by re-weighing the test vessels; sampling of soil and worms from solvent controls.
	Preparation of the soil prior to start of elimination phase should be done in the same manner as before the uptake phase.
	Activities described for day 3 should be performed daily (at least on workdays).

Enchytraeid test

• (a)Uptake phase with 8 sampling dates used for calculation of kinetics

Day	Activity
– 6	Conditioning of the prepared soil for 48 h;
– 4	Spiking of the soil fraction with the test chemical solution; evaporating of any solvent; mixing of the soil constituents; distributing the soil to the test vessels; equilibration at test conditions for 4 days (3 weeks for metal-spiked soil);
– 3 to – 1	Separation of the test organisms from the culture for acclimation; preparation and moisturising of the soil constituents;
0	Measuring temperature, and soil pH; removing soil samples from treated vessels and solvent controls for determination of test chemical concentration; addition of food ration to soil; weighing and randomised distribution of the worms to the test vessels; retaining of sufficient subsamples of worms for determination of analytical background values, wet and dry weight, and lipid content; weighing of all test vessels to control soil moisture; controlling air supply, if closed test system is used;
1	Controlling air supply, recording worm behaviour and temperature; taking soil and worm samples for determination of test item concentration;
2	Same as day 1;
3	Controlling air supply, worm behaviour and temperature;
4	Same as day 1;
5-6	Same as day 3;
7	Same as day 1; addition of food ration to soil; control soil moisture by re-weighing the test vessels and compensate evaporated water;
9	Same as day 1;
10	Same as day 3;
11	Same as day 1;
12-13	Same as day 3;
14	Same as day 1; addition of food ration to soil; measuring temperature and soil pH; control soil moisture by re-weighing the test vessels; end of uptake phase; transfer worms from remaining exposed replicates to vessels containing clean soil for elimination phase (no gut-purging); sampling of soil and worms from solvent controls.
	Pre-exposure activities (equilibration phase) should be scheduled taking into account the properties of the test chemical.
	Activities described for day 3 should be performed daily (at least on workdays).

Appendix 4

Artificial soil – preparation and storage recommendations

Since natural soils from a particular source may not be available throughout the year, and indigenous organisms as well as the presence of micro-pollutants can influence the test, an artificial substrate, the artificial soil according to Chapter C.8 of this Annex, Toxicity for Earthworms (48), is recommended for use in this test. Several test species can survive, grow, and reproduce in this soil, and maximum standardisation as well as intra- and interlaboratory comparability of test and culture conditions are provided.

Soil constituents:

Peat:	10 %	Sphagnum-peat, in accordance with the OECD Guideline 207 (48);
Quartz sand:	70 %	Industrial quartz sand (air dried); grain size: more than 50 % of the particles should be in the range of 50-200 μm, but all particles should be ≤ 2 mm;
Kaolinite clay:	20 %	Kaolinite content ≥ 30 %;
Calcium carbonate:	≤ 1 %	CaCO3, pulverised, chemically pure.

As an option, the organic carbon content of the artificial soil may be reduced, e.g. by lowering the peat content to 4-5 % of dry soil and increasing the sand content accordingly. By such a reduction in organic carbon content, the possibilities of adsorption of test chemical to the soil (organic carbon) may be decreased, and the availability of the test chemical to the worms may increase (74). It has been demonstrated that Enchytraeus albidus and Eisenia fetida can comply with the validity criteria on reproduction when tested in field soils with lower organic carbon content, e.g. 2,7 % (33), (61), and there is experience that this can also be achieved in artificial soil with 5 % peat.

Preparation

The dry constituents of the soil are mixed thoroughly (e.g. in a large-scale laboratory mixer). This should be done about one week before starting the test. The mixed dry soil constituents should be moistened with deionised water at least 48 h before application of the test item in order to equilibrate/stabilise the acidity. For the determination of pH a mixture of soil and 1 M KCl solution in a 1:5 ratio is used. If the pH value is not within the required range (6,0 ± 0,5), a sufficient amount of CaCO3 is added to the soil, or a new batch of soil is prepared.

The maximum water holding capacity (WHC) of the artificial soil is determined according to ISO 11268-2 (35). At least two days before starting the test, the dry artificial soil is moistened by adding enough deionised or reconstituted water to obtain approximately half of the final water content. The final water content should be 40 % to 60 % of the maximum WHC. At the start of the test, the pre-moistened soil is divided into as many batches as the number of test concentrations and controls used for the test, and the moisture content is adjusted to 40-60 % of WHCmax by using the solution of the test item and/or by adding deionised or reconstituted water. The moisture content is determined at the beginning and at the end of the test (at 105 °C). It should be optimal for the species’ requirements (the moisture content can also be checked as follows: when the soil is gently squeezed in the hand, small drops of water should appear between the fingers).

Storage

The dry constituents of the artificial soil may be stored at room temperature until use. The prepared, pre-moistened soil may be stored in a cool place for up to three days prior to spiking; care should be taken to minimise evaporation of water. Soil spiked with the test item should be used immediately unless there is information indicating that the particular soil can be stored without affecting the toxicity and bioavailability of the test item. Samples of spiked soil may then be stored under the conditions recommended for the particular test item until analysis.

Appendix 5

Species of terrestrial oligochaetes recommended for testing bioaccumulation from soil

Earthworms

The recommended test species is Eisenia fetida (Savigny 1826), belonging to the family Lumbricidae. Since 1972 it is divided into two subspecies (Eisenia fetida and Eisenia andrei (10)). According to Jaenike (36), they are true, separate species. Eisenia fetida is easily recognised by its bright intersegmental yellow stripes whereas Eisenia andrei has a uniform, dark red colour. Originating probably from the region of the Black Sea, they are distributed worldwide today, especially in anthropogenically modified habitats like compost heaps. Both can be used for ecotoxicological as well as bioaccumulation tests.

Eisenia fetida and Eisenia andrei are commercially available, e.g. as fish bait. In comparison to other lumbricid earthworms, they have a short life-cycle, reaching maturity within ca. 2-3 months (at room temperature). Their optimum temperature is approximately at 20-24 °C. They prefer relatively moist substrates with a nearly neutral pH and a high content of organic material. Since these species have been widely used in standardised ecotoxicological tests for about 25 years, their culturing is well established (48) (77).

Both species can be bred in a wide range of animal wastes. The breeding medium recommended by ISO (35) is a 50:50 mixture of horse or cattle manure and peat. The medium should have a pH value of about 6 to 7 (regulated with calcium carbonate), a low ionic conductivity (less than 6 mS/cm or less than 0,5 % salt concentration) and should not be contaminated excessively with ammonia or animal urine. Also, a commercial gardening soil free of additives, or artificial soil according to OECD (48), or a 50:50 mixture of both can be used. The substrate should be moist but not too wet. Breeding boxes of 10 litre to 50 litre volume are suitable.

To obtain worms of standard age and mass, it is best to start the culture with cocoons. Therefore, adult worms are added to a breeding box containing fresh substrate to produce cocoons. Practical experience has shown that a population density of approximately 100 adult worms per kg substrate (wet weight) leads to good reproduction rates. After 28 days, the adult worms are removed. The earthworms hatched from the cocoons are used for testing when mature after at least 2 months but less than 12 months.

Worms of the species described above can be considered healthy if they move through the substrate, do not try to leave the substrate, and reproduce continuously. Very slow motioning or a yellow posterior end (in the case of Eisenia fetida) indicates substrate exhaustion. In this case, fresh substrate and/or a lower number of animals per box is recommended.

Additional selected references

Gerard BM (1964). Synopsis of the British fauna. No 6 Lumbricidae. Linnean Soc. London, 6: 1-58.

Graff O (1953). Die Regenwürmer Deutschlands. Schr. Forsch. Anst. Landwirtsch. 7: 1-81.

Römbke J, Egeler P, Füll C (1997). Literaturstudie über Bioakkumulationstests mit Oligochaeten im terrestrischen Medium. Bericht für das UBA F + E 206 03 909, 86 S.

Rundgren S (1977). Seasonality of emergence in lumbricids in southern Sweden. Oikos 28: 49-55.

Satchell JE (1955). Some aspects of earthworm ecology. Soil Zoology (Kevan): 180-201.

Sims RW and Gerard BM (1985). A synopsis of the earthworms. Linnean Soc. London 31: 1-171.

Tomlin AD (1984). The earthworm bait market in North America. In: Earthworm Ecology — from Darwin to vermiculture. Satchell, J.E. (ed.), Chapman & Hall, London. 331-338 pp.

Enchytraeids

The recommended test species is Enchytraeus albidus Henle 1837 (white potworm). Enchytraeus albidus is one of the biggest (up to 15 mm) species of the annelid oligochaete family Enchytraeidae and it is worldwide distributed e.g. (8). Enchytraeus albidus is found in marine, limnic and terrestrial habitats, mainly in decaying organic matter (seaweed, compost) and rarely in meadows (42). This broad ecological tolerance and some morphological variations indicate that there might be different races for this species.

Enchytraeus albidus is commercially available, sold as food for fish. It should be checked whether the culture is contaminated by other, usually smaller species (60). If contamination occurs, all worms should be washed with water in a Petri dish. Large adult specimens of Enchytraeus albidus are then selected (by using a stereomicroscope) to start a new culture. All other worms are discarded. Its life cycle is short as maturity is reached between 33 days (at 18 °C) and 74 days (at 12 °C). Only cultures which have been kept in the laboratory for at least 5 weeks (one generation) without problems should be used for a test.

Other species of the Enchytraeus genus are also suitable, especially Enchytraeus luxuriosus. This species is a true soil inhabitant, which has been newly described in (65). If other species of Enchytraeus are used, they should be clearly identified and the rationale for the selection of the species should be reported.

Enchytraeus crypticus (Westheide & Graefe 1992) is a species belonging to the same group as Enchytraeus luxuriosus. It has not been found to exist with certainty in the field, having only been described from earthworm cultures and compost heaps (Römbke 2003). Its original ecological requirements are therefore not known. However, recent laboratory studies in various field soils have confirmed that this species has a broad tolerance towards soil properties like pH and texture (Jänsch et al. 2005). In recent years, this species has often been used in ecotoxicological studies because of the simplicity of its breeding and testing, e.g. Kuperman et al. 2003). However, it is small (3-12 mm; 7 mm on average (Westheide & Müller 1996), and this makes handling more difficult compared with Enchytraeus albidus. When using this species instead of Enchytraeus albidus, the size of the test vessel can but needs not to be smaller. In addition, it should be considered that this species reproduces very rapidly having a generation time of less than 20 days at 20 ± 2 °C (Achazi et al. 1999) and even quicker at higher temperatures.

Enchytraeids of the species Enchytraeus albidus (as well as other Enchytraeus species) can be bred in large plastic boxes (e.g. 30 × 60 × 10 cm or 20 × 12 × 8 cm which is suitable for culture of worms of small size) filled with a mixture of artificial soil and commercially available, uncontaminated garden soil free of additives. Compost material should be avoided since it could contain toxic chemicals like heavy metals. Fauna should be removed from the breeding soil before use by three times deep-freezing. Pure artificial soil can also be used but the reproduction rate could be slower compared to that obtained with mixed substrates. The substrate should have a pH of 6,0 ± 0,5. The culture is kept in an incubator at a temperature of 15 ± 2 °C without light. In any case, a temperature higher than 23 °C should be avoided. The artificial/natural soil moisture should be moist but not wet. When the soil is gently pressed by hand, only small drops of water should appear. In any case, anoxic conditions should be avoided (e.g. if a lid is used, the number of lid holes should be high enough to provide sufficient exchange of air). The breeding soil should be aerated by carefully mixing it once per week.

The worms should be fed at least once per week ad libitum with rolled oats which are placed into a cavity on the soil surface and covered with soil. If food from the last feeding date remains in the container, the amount of food given should be adjusted accordingly. If fungi grow on the remaining food, it should be replaced by a new quantity of rolled oats. In order to stimulate reproduction, the rolled oats may be supplemented with commercially available, vitamin amended protein powder every two weeks. After three months, the animals are transferred to a freshly prepared culture or breeding substrate. The rolled oats, which have to be stored in sealed vessels, should be autoclaved or heated before use in order to avoid infections by flour mites (e.g. Glyzyphagus sp., Astigmata, Acarina) or predacious mites (e.g. Hypoaspis (Cosmolaelaps) miles, Gamasida, Acarina). After disinfecting, the food is ground up so that it can easily be strewn on the soil surface. Another possible food source is baker’s yeast or the fish food TetraMin^®.

In general, the culturing conditions are sufficient if worms do not try to leave the substrate, move quickly through the soil, exhibit a shiny outer surface without soil particles clinging to it, are more or less whitish coloured, and if worms of different ages are visible. Actually, worms can be considered healthy if they reproduce continuously.

Additional selected references

Achazi RK, Fröhlich E, Henneken M, Pilz C (1999). The effect of soil from former irrigation fields and of sewage sludge on dispersal activity and colonizing success of the annelid Enchytraeus crypticus (Enchytraeidae, Oligochaeta). Newsletter on Enchytraeidae 6: 117-126.

Jänsch S, Amorim MJB, Römbke J (2005). Identification of the ecological requirements of important terrestrial ecotoxicological test species. Environ. Reviews 13: 51-83.

Kuperman RG, Checkai RT, Simini M, Phillips CT, Kolakowski JE, Kurnas CW, Sunahara GI (2003). Survival and reproduction of Enchytraeus crypticus (Oligochaeta, Enchytraeidae) in a natural sandy loam soil amended with the nitro-heterocyclic explosives RDX and HMX. Pedobiologia 47: 651-656.

Römbke J (2003). Ecotoxicological laboratory tests with enchytraeids: A review. Pedobiologia 47: 607-616.

Westheide W and Graefe U (1992). Two new terrestrial Enchytraeus species (Oligochaeta, Annelida). J. Nat. Hist. 26: 479-488.

Westheide W and Müller MC (1996). Cinematographic documentation of enchytraeid morphology and reproductive biology. Hydrobiologia 334: 263-267.

C.31.   TERRESTRIAL PLANT TEST: SEEDLING EMERGENCE AND SEEDLING GROWTH TEST

INTRODUCTION

• 1.This test method is equivalent to OECD Test Guideline (TG) 208 (2006). Test methods are periodically reviewed in the light of scientific progress and applicability to regulatory use. This updated test method is designed to assess potential effects of chemicals on seedling emergence and growth. As such it does not cover chronic effects or effects on reproduction (i.e. seed set, flower formation, fruit maturation). Conditions of exposure and properties of the chemical to be tested must be considered to ensure that appropriate test methods are used (e.g. when testing metals/metal compounds the effects of pH and associated counter ions should be considered) (1). This test method does not address plants exposed to vapours of chemicals. The test method is applicable to the testing of general chemicals, biocides and crop protection products (also known as plant protection products or pesticides). It has been developed on the basis of existing methods (2) (3) (4) (5) (6) (7). Other references pertinent to plant testing were also considered (8) (9) (10). Definitions used are given in Appendix 1.

PRINCIPLE OF THE TEST

• 2.The test assesses effects on seedling emergence and early growth of higher plants following exposure to the test chemical in the soil (or other suitable soil matrix). Seeds are placed in contact with soil treated with the test chemical and evaluated for effects following usually 14 to 21 days after 50 % emergence of the seedlings in the control group. Endpoints measured are visual assessment of seedling emergence, dry shoot weight (alternatively fresh shoot weight) and in certain cases shoot height, as well as an assessment of visible detrimental effects on different parts of the plant. These measurements and observations are compared to those of untreated control plants.

• 3.Depending on the expected route of exposure, the test chemical is either incorporated into the soil (or possibly into artificial soil matrix) or applied to the soil surface, which properly represents the potential route of exposure to the chemical. Soil incorporation is done by treating bulk soil. After the application the soil is transferred into pots, and then seeds of the given plant species are planted in the soil. Surface applications are made to potted soil in which the seeds have already been planted. The test units (controls and treated soils plus seeds) are then placed under appropriate conditions to support germination/growth of plants.

• 4.The test can be conducted in order to determine the dose-response curve, or at a single concentration/rate as a limit test according to the aim of the study. If results from the single concentration/rate test exceed a certain toxicity level (e.g. whether effects greater than x % are observed), a range-finding test is carried out to determine upper and lower limits for toxicity followed by a multiple concentration/rate test to generate a dose-response curve. An appropriate statistical analysis is used to obtain effective concentration ECx or effective application rate ERx (e.g. EC25, ER25, EC50, ER50) for the most sensitive parameter(s) of interest. Also, the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) can be calculated in this test.

INFORMATION ON THE TEST CHEMICAL

• 5.The following information is useful for the identification of the expected route of exposure to the chemical and in designing the test: structural formula, purity, water solubility, solubility in organic solvents, 1-octanol/water partition coefficient, soil sorption behaviour, vapour pressure, chemical stability in water and light, and biodegradability.

VALIDITY OF THE TEST

• 6.In order for the test to be considered valid, the following performance criteria must be met in the controls:

• — the seedling emergence is at least 70 %;

• — the seedlings do not exhibit visible phytotoxic effects (e.g. chlorosis, necrosis, wilting, leaf and stem deformations) and the plants exhibit only normal variation in growth and morphology for that particular species;

• — the mean survival of emerged control seedlings is at least 90 % for the duration of the study;

• — environmental conditions for a particular species are identical and growing media contain the same amount of soil matrix, support media, or substrate from the same source.

REFERENCE CHEMICAL

• 7.A reference chemical may be tested at regular intervals, to verify that performance of the test and the response of the particular test plants and the test conditions have not changed significantly over time. Alternatively, historical biomass or growth measurement of controls could be used to evaluate the performance of the test system in particular laboratories, and can serve as an intra-laboratory quality control measure.

DESCRIPTION OF THE METHOD

Natural soil — Artificial substrate

• 8.Plants may be grown in pots using a sandy loam, loamy sand, or sandy clay loam that contains up to 1,5 percent organic carbon (approx. 3 percent organic matter). Commercial potting soil or synthetic soil mix that contains up to 1,5 percent organic carbon may also be used. Clay soils should not be used if the test chemical is known to have a high affinity for clays. Field soil should be sieved to 2 mm particle size in order to homogenise it and remove coarse particles. The type and texture, % organic carbon, pH and salt content as electronic conductivity of the final prepared soil should be reported. The soil should be classified according to a standard classification scheme (11). The soil could be pasteurised or heat treated in order to reduce the effect of soil pathogens.

• 9.Natural soil may complicate interpretation of results and increase variability due to varying physical/chemical properties and microbial populations. These variables in turn alter moisture-holding capacity, chemical-binding capacity, aeration, and nutrient and trace element content. In addition to the variations in these physical factors, there will also be variation in chemical properties such as pH and redox potential, which may affect the bioavailability of the test chemical (12) (13) (14).

• 10.Artificial substrates are typically not used for testing of crop protection products, but they may be of use for the testing of general chemicals or where it is desired to minimize the variability of the natural soils and increase the comparability of the test results. Substrates used should be composed of inert materials that minimize interaction with the test chemical, the solvent carrier, or both. Acid washed quartz sand, mineral wool and glass beads (e.g. 0,35 to 0,85 mm in diameter) have been found to be suitable inert materials that minimally absorb the test chemical (15), ensuring that the chemical will be maximally available to the seedling via root uptake. Unsuitable substrates would include vermiculite, perlite or other highly absorptive materials. Nutrients for plant growth should be provided to ensure that plants are not stressed through nutrient deficiencies, and where possible this should be assessed via chemical analysis or by visual assessment of control plants.

Criteria for selection of test species

• 11.The species selected should be reasonably broad, e.g., considering their taxonomic diversity in the plant kingdom, their distribution, abundance, species specific life-cycle characteristics and region of natural occurrence, to develop a range of responses (8) (10) (16) (17) (18) (19) (20). The following characteristics of the possible test species should be considered in the selection:

• — the species have uniform seeds that are readily available from reliable standard seed source(s) and that produce consistent, reliable and even germination, as well as uniform seedling growth;

• — plant is amenable to testing in the laboratory, and can give reliable and reproducible results within and across testing facilities;

• — the sensitivity of the species tested should be consistent with the responses of plants found in the environment exposed to the chemical;

• — they have been used to some extent in previous toxicity tests and their use in, for example, herbicide bioassays, heavy metal screening, salinity or mineral stress tests or allelopathy studies indicates sensitivity to a wide variety of stressors;

• — they are compatible with the growth conditions of the test method;

• — they meet the validity criteria of the test.

• Some of the historically most used test species are listed in Appendix 2 and potential non-crop species in Appendix 3.

• 12.The number of species to be tested is dependent on relevant regulatory requirements, therefore it is not specified in this test method.

Application of the test chemical

• 13.The chemical should be applied in an appropriate carrier (e.g. water, acetone, ethanol, polyethylene glycol, gum Arabic, sand). Mixtures (formulated products or formulations) containing active ingredients and various adjuvants can be tested as well.

Incorporation into soil/artificial substrate

• 14.Chemicals which are water soluble or suspended in water can be added to water, and then the solution is mixed with soil with an appropriate mixing device. This type of test may be appropriate if exposure to the chemical is through soil or soil pore-water and that there is concern for root uptake. The water-holding capacity of the soil should not be exceeded by the addition of the test chemical. The volume of water added should be the same for each test concentration, but should be limited to prevent soil agglomerate clumping.

• 15.Chemicals with low water solubility should be dissolved in a suitable volatile solvent (e.g. acetone, ethanol) and mixed with sand. The solvent can then be removed from the sand using a stream of air while continuously mixing the sand. The treated sand is mixed with the experimental soil. A second control is established which receives only sand and solvent. Equal amounts of sand, with solvent mixed and removed, are added to all treatment levels and the second control. For solid, insoluble test chemicals, dry soil and the chemical are mixed in a suitable mixing device. Hereafter, the soil is added to the pots and seeds are sown immediately.

• 16.When an artificial substrate is used instead of soil, chemicals that are soluble in water can be dissolved in the nutrient solution just prior to the beginning of the test. Chemicals that are insoluble in water, but which can be suspended in water by using a solvent carrier, should be added with the carrier, to the nutrient solution. Water-insoluble chemicals, for which there is no non-toxic water-soluble carrier available, should be dissolved in an appropriate volatile solvent. The solution is mixed with sand or glass beads, placed in a rotary vacuum apparatus, and evaporated, leaving a uniform coating of chemical on sand or beads. A weighed portion of beads should be extracted with the same organic solvent and the chemical assayed before the potting containers are filled.

Surface application

• 17.For crop protection products, spraying the soil surface with the test solution is often used for application of the test chemical. All equipment used in conducting the tests, including equipment used to prepare and administer the test chemical, should be of such design and capacity that the tests involving this equipment can be conducted in an accurate way and it will give a reproducible coverage. The coverage should be uniform across the soil surfaces. Care should be taken to avoid the possibilities of chemicals being adsorbed to or reacting with the equipment (e.g. plastic tubing and lipophilic chemicals or steel parts and elements). The test chemical is sprayed onto the soil surface simulating typical spray tank applications. Generally, spray volumes should be in the range of normal agricultural practice and the volumes (amount of water etc. should be reported). Nozzle type should be selected to provide uniform coverage of the soil surface. If solvents and carriers are applied, a second group of control plants should be established receiving only the solvent/carrier. This is not necessary for crop protection products tested as formulations.

Verification of test chemical concentration/rate

• 18.The concentrations/rates of application must be confirmed by an appropriate analytical verification. For soluble chemicals, verification of all test concentrations/rates can be confirmed by analysis of the highest concentration test solution used for the test with documentation on subsequent dilution and use of calibrated application equipment (e.g., calibrated analytical glassware, calibration of sprayer application equipment). For insoluble chemicals, verification of compound material must be provided with weights of the test chemical added to the soil. If demonstration of homogeneity is required, analysis of the soil may be necessary.

PROCEDURE

Test design

• 19.Seeds of the same species are planted in pots. The number of seeds planted per pot will depend upon the species, pot size and test duration. The number of plants per pot should provide adequate growth conditions and avoid overcrowding for the duration of the test. The maximum plant density would be around 3 - 10 seeds per 100 cm² depending to the size of the seeds. As an example, one to two corn, soybean, tomato, cucumber, or sugar beet plants per 15 cm container; three rape or pea plants per 15 cm container; and 5 to 10 onion, wheat, or other small seeds per 15 cm container are recommended. The number of seeds and replicate pots (the replicate is defined as a pot, therefore plants within the same pot do not constitute a replicate) should be adequate for optimal statistical analysis (21). It should be noted that variability will be greater for test species using fewer large seeds per pot (replicate), when compared to test species where it is possible to use greater numbers of small seeds per pot. By planting equal seed numbers in each pot this variability may be minimized.

• 20.Control groups are used to assure that effects observed are associated with or attributed only to the test chemical exposure. The appropriate control group should be identical in every respect to the test group except for exposure to the test chemical. Within a given test, all test plants including the controls should be from the same source. To prevent bias, random assignment of test and control pots is required.

• 21.Seeds coated with an insecticide or fungicide (i.e. ‘dressed’ seeds) should be avoided. However, the use of certain non-systemic contact fungicides (e.g. captan, thiram) is permitted by some regulatory authorities (22). If seed-borne pathogens are a concern, the seeds may be soaked briefly in a weak 5 % hypochlorite solution, then rinsed extensively in running water and dried. No remedial treatment with other crop protection product is allowed.

Test conditions

• 22.The test conditions should approximate those conditions necessary for normal growth for the species and varieties tested (Appendix 4 provides examples of test condition). The emerging plants should be maintained under good horticultural practices in controlled environment chambers, phytotrons, or greenhouses. When using growth facilities these practices usually include control and adequately frequent (e.g. daily) recording of temperature, humidity, carbon dioxide concentration, light (intensity, wave length, photosynthetically active radiation) and light period, means of watering, etc., to assure good plant growth as judged by the control plants of the selected species. Greenhouse temperatures should be controlled through venting, heating and/or cooling systems. The following conditions are generally recommended for greenhouse testing:

• — temperature: 22 °C ± 10 °C;

• — humidity: 70 % ± 25 %;

• — photoperiod: minimum 16 hour light;

• — light intensity: 350 ± 50 μE/m²/s. Additional lighting may be necessary if intensity decreases below 200 μE/m²/s, wavelength 400 - 700 nm except for certain species whose light requirements are less.