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)

• — Test conditions: — test procedure used (e.g. static, semi-static or flow-through); — test design (e.g. number, material and size of test chambers, water volume per vessel, sediment mass and volume per vessel, (for flow-through or semi-static procedures: water volume replacement rate), any aeration used before and during the test, number of replicates, number of worms per replicate at start of exposure, number of test concentrations, length of conditioning, equilibration and exposure periods, sampling frequency); — depth of sediment and overlying water; — method of test chemical pre-treatment and spiking/application; — the nominal test concentrations, details about the sampling for chemical analysis, and the analytical methods by which concentrations of the test chemical were obtained; — sediment characteristics as described in paragraphs 24 - 25, and any other measurements made; preparation of formulated sediment; — preparation of the test water (if reconstituted water is used) and characteristics (oxygen concentration, pH, conductivity, hardness, and any other measurements made) before the start of the test; — detailed information on feeding including type of food, preparation, amount and feeding regimen; — light intensity and photoperiod(s); — methods used for determination of all biological parameters (e.g. sampling, inspection, weighing of test organisms) and all abiotic parameters (e.g. water and sediment quality parameters); — volumes and/or weights of all samples for chemical analysis; — detailed information on the treatment of all samples for chemical analysis, including details of preparation, storage, spiking procedures, extraction, and analytical procedures (and precision) for the test chemical, and recoveries of the test chemical.

• — Results: — water quality within the test vessels (pH, temperature, dissolved oxygen concentration, hardness, ammonia concentrations, and any other measurements made); — total organic carbon content (TOC), dry weight to wet weight ratio, pH of the sediment, and any other measurements made; — total number, and if determined, number of complete and incomplete worms in each test chamber at the end of the test; — dry weight of the worms of each test chamber at the end of the test, and if measured, dry weight of a sub-sample of the worms at start of the test; — any observed abnormal behaviour in comparison to the controls (e.g., sediment avoidance, presence or absence of fecal pellets); — any observed mortalities; — estimates of toxic endpoints (e.g. ECx, NOEC and/or LOEC), and the statistical methods used for their determination; — the nominal test concentrations, the measured test concentrations and the results of all analyses made to determine the concentration of the test chemical in the test vessels; — any deviations from the validity criteria.

• — Evaluation of results: — compliance of the results with the validity criteria as listed in paragraph 13; — discussion of the results, including any influence on the outcome of the test resulting from deviations from this test method.

LITERATURE

• (1)EC (2003). Technical Guidance Document in support of Commission Directive 93/67/EEC on Risk Assessment for new notified substances, Commission Regulation (EC) No 1488/94 on Risk Assessment for existing substances and Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market; Part I — IV. Office for Official Publications of the EC (European Commission), Luxembourg.

• (2)OECD (1992a). Report of the OECD workshop on effects assessment of chemicals in sediment. OECD Monographs No. 60. Organisation for Economic Co-operation and Development (OECD), Paris.

• (3)ASTM International (2000). Standard guide for the determination of the bioaccumulation of sediment-associated contaminants by benthic invertebrates, E 1688-00a. In ASTM International 2004 Annual Book of Standards. Volume 11.05. Biological Effects and Environmental Fate; Biotechnology; Pesticides. ASTM International, West Conshohocken, PA.

• (4)ASTM International (2002). Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates, E1706-00. In ASTM International 2004 Annual Book of Standards. Volume 11.05. Biological Effects and Environmental Fate; Biotechnology; Pesticides. ASTM International, West Conshohocken, PA.

• (5)Phipps, G.L., Ankley, G.T., Benoit, D.A. and Mattson, V.R. (1993). Use of the aquatic Oligochaete Lumbriculus variegatus for assessing the toxicity and bioaccumulation of sediment-associated contaminants. Environ.Toxicol. Chem. 12, 269-279.

• (6)Chapter C.27 of this Annex, ‘Sediment-water chironomid toxicity test using spiked sediment’.

• (7)U.S. EPA (2000). Methods for measuring the toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates. Second Edition. EPA 600/R-99/064, U.S. Environmental Protection Agency, Duluth, MN, March 2000.

• (8)Environment Canada (1997). Test for Growth and Survival in Sediment using Larvae of Freshwater Midges (Chironomus tentans or Chironomus riparius). Biological Test Method. Report SPE 1/RM/32. December 1997.

• (9)Hill, I.R., Matthiessen, P., Heimbach, F. (eds), 1993, Guidance document on Sediment Toxicity Tests and Bioassays for freshwater and Marine Environments, From the SETAC-Europe Workshop On Sediment Toxicity Assessment, 8-10 November 1993, Renesse (NL).

• (10)BBA (1995). Long-term toxicity test with Chironomus riparius: Development and validation of a new test system. Edited by M. Streloke and H.Köpp. Berlin 1995.

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• (12)ASTM International (2004). Standard guide for collection, storage, characterisation, and manipulation of sediment for toxicological testing and for selection of samplers used to collect benthic invertebrates. American Society for Testing and Materials, E 1391-03.

• (13)Egeler, Ph., Meller, M., Schallnaß, H.J. & Gilberg, D. (2005). Validation of a sediment toxicity test with the endobenthic aquatic oligochaete Lumbriculus variegatus by an international ring test. In co-operation with R. Nagel and B. Karaoglan. Report to the Federal Environmental Agency (Umweltbundesamt Berlin), R&D No.: 202 67 429.

• (14)OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Environment, Health and Safety Publications, Series on Testing and Assessment No. 23.

• (15)Environment Canada (2003). Guidance Document on Statistical Methods for Environmental Toxicity Tests; fifth draft, March 2003; Report EPS 1/RM/___

• (16)Nikkilä A., Halme A., Kukkonen J.V.K. (2003). Toxicokinetics, toxicity and lethal body residues of two chlorophenols in the oligochaete worm, Lumbriculus variegatus, in different sediments. Chemosphere 51: 35-46.

• (17)Baily H.C., & Liu D.H.W. (1980). Lumbriculus variegatus, a Benthic Oligochaete, as a Bioassay Organism. p. 205-215. In J.C. Eaton, P.R. Parrish, and A.C. Hendricks (eds). Aquatic Toxicology, ASTM STP 707. American Society for Testing and Materials.

• (18)Chapman K. K., Benton M. J., Brinkhurst R. O. & Scheuerman P. R. (1999). Use of the aquatic oligochaetes Lumbriculus variegatus and Tubifex tubifex for assessing the toxicity of copper and cadmium in a spiked-artificial-sediment toxicity test. Environmental Toxicology. 14(2): 271-278.

• (19)Meyer J.S., Boese C.J. & Collyard S.A. (2002). Whole-body accumulation of copper predicts acute toxicity to an aquatic oligochaete (Lumbriculus variegatus) as pH and calcium are varied. Comp. Biochem. Physiol. Part C 133:99-109.

• (20)Schubauer-Berigan M.K., Dierkes J.R., Monson P.D. & Ankley G.T. (1993). pH-dependent toxicity of cadmium, copper, nickel, lead and zinc to Ceriodaphnia dubia, Pimephales promelas, Hyalella azteca and Lumbriculus variegatus. Environ. Toxciol. Chem. 12(7):1261-1266.

• (21)West, C.W., V.R. Mattson, E.N. Leonard, G.L. Phipps & G.T. Ankley (1993). Comparison of the relative sensitivity of three benthic invertebrates to copper-contaminated sediments from the Keweenaw Waterway. Hydrobiol. 262:57-63.

• (22)Ingersoll, C.G., Ankley, G.T., Benoit D.A., Brunson, E.L., Burton, G.A., Dwyer, F.J., Hoke, R.A., Landrum, P. F., Norberg-King, T. J. and Winger, P.V. (1995). Toxicity and bioaccumulation of sediment-associated contaminants using freshwater invertebrates: A review of methods and applications. Environ. Toxicol. Chem. 14, 1885-1894.

• (23)Kukkonen, J. and Landrum, P.F. (1994). Toxicokinetics and toxicity of sediment-associated Pyrene to Lumbriculus variegatus (Oligochaeta). Environ. Toxicol. Chem. 13, 1457-1468.

• (24)Leppänen, M.T. & Kukkonen, J.V.K. (1998a). Relationship between reproduction, sediment type and feeding activity of Lumbriculus variegatus (Müller): Implications for sediment toxicity testing. Environ. Toxicol. Chem. 17: 2196-2202.

• (25)Leppänen, M.T. & Kukkonen, J.V.K. (1998b). Factors affecting feeding rate, reproduction and growth of an oligochaete Lumbriculus variegatus (Müller). Hydrobiologia 377: 183-194.

• (26)Landrum, P.F., Gedeon, M.L., Burton, G.A., Greenberg. M.S., & Rowland, C.D. (2002). Biological Responses of Lumbriculus variegatus Exposed to Fluoranthene-Spiked Sediment. Arch. Environ. Contam. Toxicol. 42: 292-302.

• (27)Brunson, E.L., Canfield, T.J., Ingersoll, C.J. & Kemble, N.E. (1998). Assessing the bioaccumulation of contaminants from sediments of the Upper Mississippi river using field-collected oligochaetes and laboratory-exposed Lumbriculus variegatus. Arch. Environ. Contam. Toxicol. 35, 191-201.

• (28)Ingersoll, C.G., Brunson, E.L., Wang N., Dwyer, F.J., Ankley, G.T., Mount D.R., Huckins J., Petty. J. and Landrum, P. F. (2003). Uptake and depuration of non-ionic organic contaminants from sediment by the oligochaete, Lumbriculus variegatus. Environmental Toxicology and Chemistry 22, 872-885.

• (29)Rodriguez, P. & Reynoldson, T.B. (1999). Laboratory methods and criteria for sediment bioassessment. In: A. Mudroch, J.M. Azcue & P. Mudroch (eds.): Manual of Bioassessment of aquatic sediment quality. Lewis Publishers, Boca Raton, CRC Press LLC.

• (30)Liebig, M., Egeler, Ph. Oehlmann, J., & Knacker, Th. (2005). Bioaccumulation of ¹⁴C-17α-ethinylestradiol by the oligochaete Lumbriculus variegatus in artificial sediment. Chemosphere 59, 271-280.

• (31)Brust, K., O. Licht, V. Hultsch, D. Jungmann & R. Nagel (2001). Effects of Terbutryn on Aufwuchs and Lumbriculus variegatus in Artificial Indoor Streams. Environ. Toxicol. Chemistry, Vol. 20, pp. 2000–2007.

• (32)Oetken, M., K.-U. Ludwichowski & R. Nagel (2000). Sediment tests with Lumbriculus variegatus and Chironomus riparius and 3,4-dichloroaniline (3,4-DCA) within the scope of EG-AltstoffV. By order of the Federal Environmental Agency (Umweltbundesamt Berlin), FKZ 360 12 001, March 2000.

• (33)Leppänen M.T. & Kukkonen J.V.K. (1998). Relative importance of ingested sediment and porewater as bioaccumulation routes for pyrene to oligochaete (Lumbriculus variegatus, Müller). Environ. Sci. Toxicol. 32, 1503-1508.

• (34)Dermott R. & Munawar M. (1992). A simple and sensitive assay for evaluation of sediment toxicity using Lumbriculus variegatus (Müller). Hydrobiologia 235/236: 407-414.

• (35)Drewes C.D. & Fourtner C.R. (1990). Morphallaxis in an aquatic oligochaete, Lumbriculus variegatus: Reorganisation of escape reflexes in regenerating body fragments. Develop. Biol. 138: 94-103.

• (36)Brinkhurst, R.O. (1971). A guide for the identification of British aquatic oligochaeta. Freshw. Biol. Assoc., Sci. Publ. No. 22.

• (37)Chapter C.1 of this Annex, Fish, Acute Toxicity Test.

• (38)OECD (1992c). Guidelines for Testing of Chemicals No. 210. Fish, Early-life Stage Toxicity Test. OECD, Paris.

• (39)Egeler, Ph., Römbke, J., Meller, M., Knacker, Th., Franke, C., Studinger, G. & Nagel, R. (1997). Bioaccumulation of lindane and hexachlorobenzene by tubificid sludgeworms (Oligochaeta) under standardised laboratory conditions. Chemosphere 35, 835-852.

• (40)Meller, M., P. Egeler, J. Roembke, H. Schallnass, R. Nagel and B. Streit. (1998). Short-term Toxicity of Lindane, Hexachlorobenzene and Copper Sulphate on Tubificid Sludgeworms (Oligochaeta) in Artificial Media. Ecotox. and Environ. Safety, 39, 10-20.

• (41)Egeler, Ph., Römbke, J., Knacker, Th., Franke, C. & Studinger, G. (1999). Workshop on ‘Bioaccumulation: Sediment test using benthic oligochaetes’, 26.-27.4.1999, Hochheim/Main, Germany. Report on the R+D-project No. 298 67 419, Umweltbundesamt, Berlin.

• (42)Suedel, B.C. and Rodgers, J.H. (1993). Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem. 13, 1163-1175.

• (43)Naylor, C. and C. Rodrigues. (1995). Development of a test method for Chironomus riparius using a formulated sediment. Chemosphere 31: 3291-3303.

• (44)Kaster, J.L., Klump, J.V., Meyer, J., Krezoski, J. & Smith, M.E. (1984). Comparison of defecation rates of Limnodrilus hoffmeisteri using two different methods. Hydrobiologia 11, 181-184.

• (45)Martinez-Madrid, M., Rodriguez, P., Perez-Iglesias, J.I. & Navarro, E. (1999). Sediment toxicity bioassays for assessment of contaminated sites in the Nervion river (Northern Spain). 2. Tubifex tubifex (Müller) reproduction sediment bioassay. Ecotoxicology 8, 111-124.

• (46)Environment Canada (1995). Guidance document on measurement of toxicity test precision using control sediments spiked with a reference toxicant. Environmental Protection Series Report EPS 1/RM/30.

• (47)Landrum, P.F. (1989). Bioavailability and toxicokinetics of polycyclic aromatic hydrocarbons sorbed to sediments for the amphipod Pontoporeia hoyi. Environ. Sci. Technol. 23, 588-595.

• (48)Brooke, L.T., Ankley, G.T., Call, D.J. & Cook, P.M. (1996). Gut content and clearance for three species of freshwater invertebrates. Environ. Toxicol. Chem. 15, 223-228.

• (49)Mount, D.R., Dawson, T.D. & Burkhard, L.P. (1999). Implications of gut purging for tissue residues determined in bioaccumulation testing of sediment with Lumbriculus variegatus. Environ. Toxicol. Chem. 18, 1244-1249.

• (50)OECD 2006. Current approaches in the statistical analysis of ecotoxicity data: A guidance to application. OECD Series on Testing and Assessment No. 54, OECD, Paris, France.

• (51)Liebig M., Meller M. & Egeler P. (2004). Sedimenttoxizitätstests mit aquatischen Oligochaeten — Einfluss verschiedener Futterquellen im künstlichen Sediment auf Reproduktion und Biomasse von Lumbriculus variegatus. Proceedings 5/2004: Statusseminar Sedimentkontakttests. March 24-25, 2004. BfG (Bundesanstalt für Gewässerkunde), Koblenz, Germany. pp. 107-119.

Additional literature on statistical procedures:

Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. Amer. Statist. Ass. J. 50, 1096-1121.

Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482-491.

Finney, D.J. (1971). Probit Analysis (3rd ed.), pp. 19-76. Cambridge Univ. Press.

Finney, D.J. (1978). Statistical Method in Biological Assay. Charles Griffin & Company Ltd, London.

Hamilton, M.A., R.C. Russo and R.V. Thurston. (1977). Trimmed Spearman-Karber Method for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol. 11(7), 714-719; Correction: Environ. Sci. Technol. 12 (1998), 417.

Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scand. J. Statist. 6, 65-70.

Sokal, R.R. and F.J. Rohlf. (1981) Biometry. The principles and practice of statistics in biological research. 2nd edition. W.H. Freeman and Company. New York.

Miller, R.G., Jr. (1986). Beyond ANOVA, basics of applied statistics. John Wiley & Sons. New York.

Shapiro S.S. & Wilk M.B (1965). An analysis of variance test for normality (complete samples). Biometrika 52: 591-611.

Williams, D.A. (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27, 103-117.

Williams, D.A. (1972). The comparison of several dose levels with a zero dose control. Biometrics 28, 519 531.

Appendix 1

Definitions

For the purpose of this test method the following definitions are used:

A chemical means a substance or a mixture.

The conditioning period is used to stabilise the microbial component of the sediment and to remove e.g. ammonia originating from sediment components; it takes place prior to spiking of the sediment with the test chemical. Usually, the overlying water is discarded after conditioning.

The ECx is the concentration of the test chemical in the sediment that results in X % (e.g. 50 %) effect on a biological parameter within a stated exposure period.

The equilibration period is used to allow for distribution of the test chemical between the solid phase, the pore water and the overlying water; it takes place after spiking of the sediment with the test chemical and prior to addition of the test organisms.

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

Formulated sediment or reconstituted, artificial or synthetic sediment, is a mixture of materials used to mimic the physical components of a natural sediment.

The Lowest Observed Effect Concentration (LOEC) is the lowest tested concentration of a test chemical at which the chemical is observed to have a significant toxic effect (at p ≤ 0,05) when compared with the control. However, all test concentrations above the LOEC must have an effect equal to or greater than those observed at the LOEC. If these two conditions cannot be satisfied, a full explanation must be given for how the LOEC (and hence the NOEC) has been selected.

The No Observed Effect Concentration (NOEC) is the test concentration immediately below the LOEC which, when compared with the control, has no statistically significant effect (p ≤ 0,05), within a given exposure period.

The octanol-water partitioning coefficient (Kow; also sometimes expressed as Pow) is the ratio of the solubility of a chemical in n-octanol and water at equilibrium and represents the lipophilicity of a chemical (Chapter A.24 of this Annex). The Kow or its logarithm of Kow (log Kow) is used as an indication of the potential of a chemical for bioaccumulation by aquatic organisms.

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

Overlying water is the water covering the sediment in the test vessel.

Pore water or interstitial water is the water occupying space between sediment or soil particles.

Spiked sediment is sediment to which test chemical has been added.

Test chemical means any substance or mixture tested using this test method.

Appendix 2

Composition of the recommended reconstituted water

(adopted from Chapter C.1 of this Annex (1))

• (a) Calcium chloride solution

• Dissolve 11,76 g CaCl2·2H2O in deionised water; make up to 1 l with deionised water

• (b) Magnesium sulphate solution

• Dissolve 4,93 g MgSO4·7H2O in deionised water; make up to 1 l with deionised water

• (c) Sodium bicarbonate solution

• Dissolve 2,59 g NaHCO3 in deionised water; make up to 1 l with deionised water

• (d) Potassium chloride solution

• Dissolve 0,23 g KCl in deionised water; make up to 1 l with deionised water

All chemicals must be of analytical grade.

The conductivity of the distilled or deionised water should not exceed 10 μScm^{– 1}.

25 ml each of solutions (a) to (d) are mixed and the total volume made up to 1 l with deionised water. The sum of the calcium and magnesium ions in these solutions is 2,5 mmol/l.

The proportion Ca:Mg ions is 4:1 and Na:K ions 10:1. The acid capacity KS4.3 of this solution is 0,8 mmol/l.

Aerate the dilution water until oxygen saturation is achieved, then store it for approximately two days without further aeration before use.

REFERENCE

• (1)Chapter C.1 of this Annex, Fish Acute Toxicity Test.

Appendix 3

Physical-chemical characteristics of an acceptable dilution water

Component	Concentrations
Particulate matter	< 20 mg/l
Total organic carbon	< 2 μg/l
Unionised ammonia	< 1 μg/l
Residual chlorine	< 10 μg/l
Total organophosphorous pesticides	< 50 ng/l
Total organochlorine pesticides plus polychlorinated biphenyls	< 50 ng/l
Total organic chlorine	< 25 ng/l
Adopted from OECD (1992) (1)	Adopted from OECD (1992) (1)

REFERENCE

• (1)OECD (1992). Guidelines for Testing of Chemicals No. 210. Fish, Early-life Stage Toxicity Test. OECD, Paris.

Appendix 4

Recommended artificial sediment — guidance on preparation and storage

Sediment constituents

Constituent	Characteristics	% of sediment dry weight
Peat	Sphagnum moss peat, degree of decomposition: ‘medium’, air dried, no visible plant remains, finely ground (particle size ≤ 0,5 mm)	5 ± 0,5
Quartz sand	Grain size: ≤ 2 mm, but > 50 % of the particles should be in the range of 50-200 μm	75 - 76
Kaolinite clay	Kaolinite content ≥ 30 %	20 ± 1
Food source	e.g. Urtica powder (Folia urticae), leaves of Urtica dioica (stinging nettle), finely ground (particle size ≤ 0,5 mm); in accordance with pharmacy standards, for human consumption; in addition to dry sediment	0,4 - 0,5 %
Organic carbon	Adjusted by addition of peat and sand	2 ± 0,5
Calcium carbonate	CaCO3, pulverised, chemically pure, in addition to dry sediment	0,05 - 1
Deionised Water	Conductivity ≤ 10 μS/cm, in addition to dry sediment	30 - 50

Note: If elevated ammonia concentrations are expected, e.g. if the test chemical is known to inhibit nitrification, it may be useful to replace 50 % of the nitrogen-rich urtica powder by cellulose (e.g., α-Cellulose powder, chemically pure, particle size ≤ 0,5 mm; (1) (2)).

Preparation

The peat is air dried and ground to a fine powder. A suspension of the required amount of peat powder in deionised water is prepared using a high-performance homogenising device. The pH of this suspension is adjusted to 5,5 ± 0,5 with CaCO3. The suspension is conditioned for at least two days with gentle stirring at 20 ± 2 °C, to stabilise pH and establish a stable microbial component. pH is measured again and should be 6,0 ± 0,5. Then the peat suspension is mixed with the other constituents (sand and kaolin clay) and deionised water to obtain an homogeneous sediment with a water content in a range of 30–50 per cent of dry weight of the sediment. The pH of the final mixture is measured again and is adjusted to 6,5 to 7,5 with CaCO3 if necessary. However, if ammonia development is expected, it may be useful to keep the pH of the sediment below 7,0 (e.g. between 6,0 and 6,5). Samples of the sediment are taken to determine the dry weight and the organic carbon content. If ammonia development is expected, the formulated sediment may be conditioned for seven days under the same conditions which prevail in the subsequent test (e.g. sediment-water ratio 1 : 4, height of sediment layer as in test vessels) before it is spiked with the test chemical, i.e. it should be topped with water, which should be aerated. At the end of the conditioning period, the overlying water should be removed and discarded. Thereafter, the spiked quartz sand is mixed with the sediment for each treatment level, the sediment is distributed to the replicate test vessels, and topped with the test water. The vessels are then incubated at the same conditions which prevail in the subsequent test. This is where the equilibration period starts. The overlying water should be aerated.

The chosen food source should be added prior to or during spiking the sediment with the test chemical. It can be mixed initially with the peat suspension (see above). However, excessive degradation of the food source prior to addition of the test organisms — e.g. in case of long equilibration period — can be avoided by keeping the time period between food addition and start of exposure as short as possible. In order to ensure that the food is spiked with the test chemical, the food source should be mixed with the sediment not later than on the day the test chemical is spiked to the sediment.

Storage

The dry constituents of the artificial sediment may be stored in a dry, cool place or at room temperature. The prepared sediment spiked with the test chemical should be used in the test immediately. Samples of spiked sediment may be stored under the conditions recommended for the particular test chemical until analysis.

REFERENCES

• (1)Egeler, Ph., Meller, M., Schallnaß, H.J. & Gilberg, D. (2005). Validation of a sediment toxicity test with the endobenthic aquatic oligochaete Lumbriculus variegatus by an international ring test. In co-operation with R. Nagel and B. Karaoglan. Report to the Federal Environmental Agency (Umweltbundesamt Berlin), R&D No.: 202 67 429.

• (2)Liebig M., Meller M. & Egeler P. (2004). Sedimenttoxizitätstests mit aquatischen Oligochaeten — Einfluss verschiedener Futterquellen im künstlichen Sediment auf Reproduktion und Biomasse von Lumbriculus variegatus. Proceedings 5/2004: Statusseminar Sedimentkontakttests. March 24-25, 2004. BfG (Bundesanstalt für Gewässerkunde), Koblenz, Germany. pp. 107-119.

Appendix 5

Culture methods for Lumbriculus variegatus

Lumbriculus variegatus (MÜLLER), Lumbriculidae, Oligochaeta is an inhabitant of freshwater sediments and is widely used in ecotoxicological testing. It can easily be cultured under laboratory conditions. An outline of culture methods is given in the following.

Culture methods

Culture conditions for Lumbriculus variegatus are outlined in detail in Phipps et al. (1993) (1), Brunson et al. (1998) (2), ASTM (2000) (3), U.S. EPA (2000) (4). A short summary of these conditions is given below. A major advantage of L. variegatus is its quick reproduction, resulting in rapidly increasing biomass in laboratory cultured populations (e.g. (1), (3), (4), (5)).

The worms can be cultured in large aquaria (57 - 80 l) at 23 °C with a 16 L:8 D photoperiod (100 – 1 000 lx) using daily renewed natural water (45 - 50 l per aquarium). The substrate is prepared by cutting unbleached brown paper towels into strips, which may then be blended with culture water for a few seconds to result in small pieces of paper substrate. This substrate can then directly be used in the Lumbriculus culture aquaria by covering the bottom area of the tank, or be stored frozen in deionised water for later use. New substrate in the tank will generally last for approximately two months.

Each worm culture is started with 500 – 1 000 worms, and fed a 10 ml suspension containing 6 g of trout starter food 3 times per week under renewal or flow-through conditions. Static or semi-static cultures should receive lower feeding rates to prevent bacterial and fungal growth. .

Under these conditions the number of individuals in the culture generally doubles in approximately 10 to 14 d.

Alternatively Lumbriculus variegatus can also be cultured in a system consisting of a layer of quartz sand as used for the artificial sediment (1 - 2 cm depth), and reconstituted water. Glass or stainless steel containers with a height of 12 to 20 cm can be used as culture vessels. The water body should be gently aerated (e.g. 2 bubbles per second) via a pasteur pipette positioned approx. 2 cm above the sediment surface. To avoid accumulation e.g. of ammonia, the overlying water should be exchanged using a flow-through system, or, at least once a week, manually. The oligochaetes can be held at room temperature with a photo period of 16 hours light (intensity 100 – 1 000 lx) and 8 hours dark. In the semi-static culture (water renewal once per week), the worms are fed with TetraMin twice a week (e.g. 0,6 - 0,8 mg per cm² of sediment surface), which can be applied as a suspension of 50 mg TetraMin per ml de-ionized water.

Lumbriculus variegatus can be removed from the cultures e.g. by transferring substrate with a fine mesh net, or organisms using a fire polished wide mouth (approximately 5 mm diameter) glass pipette, to a separate beaker. If substrate is co-transferred to this beaker, the beaker containing worms and substrate is left overnight under flow-through conditions, which will remove the substrate from the beaker, while the worms remain at the bottom of the vessel. They can then be introduced to newly prepared culture tanks, or processed further for the test as outlined in (3) and (4), or in the following.

An issue to be regarded critically when using L. variegatus in sediment tests is its reproduction mode (architomy or morphallaxis, e.g. (6)). This asexual reproduction results in two fragments, which do not feed for a certain period until the head or tail part is regenerated (e.g., (7), (8)). This means that in L. variegatus exposure via ingestion of contaminated sediment does not take place continuously.

Therefore, a synchronisation should be performed to minimise uncontrolled reproduction and regeneration, and subsequent high variation in test results. Such variation can occur, when some individuals, which have fragmented and therefore do not feed for a certain time period, are less exposed to the test chemical than other individuals, which do not fragment during the test (9), (10), (11). 10 to 14 days before the start of exposure, the worms should be artificially fragmented (synchronisation). Large (adult) worms, which preferably do not show signs of recent morphallaxis should be selected for synchronisation. These worms can be placed onto a glass slide in a drop of culture water, and dissected in the median body region with a scalpel. Care should be taken that the posterior ends are of similar size. The posterior ends should then be left to regenerate new heads in a culture vessel containing the same substrate as used in the culture and reconstituted water until the start of exposure. Regeneration of new heads is indicated when the synchronised worms are burrowing in the substrate (presence of regenerated heads may be confirmed by inspecting a representative subsample under a binocular microscope). The test organisms are thereafter expected to be in a similar physiological state. This means, that when reproduction by morphallaxis occurs in synchronised worms during the test, virtually all animals are expected to be equally exposed to the spiked sediment. Feeding of the synchronised worms should be done once as soon as the worms are starting to burrow in the substrate, or 7 d after dissection. The feeding regimen should be comparable to the regular cultures, but it may be advisable to feed the synchronised worms with the same food source as is to be used in the test. The worms should be held at test temperature, at 20 ± 2 °C. After regenerating, intact complete worms, which are actively swimming or crawling upon a gentle mechanical stimulus, should be used for the test. Injuries or autotomy in the worms should be prevented, e.g. by using pipettes with fire polished edges, or stainless steel dental picks for handling these worms.

Sources of starter cultures for Lumbriculus variegatus (addresses in the U.S. adopted from (4))

Europe	Europe
ECT Oekotoxikologie GmbH Böttgerstr. 2-14 D-65439 Flörsheim/Main Germany	Bayer Crop Science AG Development — Ecotoxicology Alfred-Nobel-Str. 50 D-40789 Monheim Germany
University of Joensuu Laboratory of Aquatic Toxicology Dept. of Biology Yliopistokatu 7, P.O. Box 111 FIN-80101 Joensuu Finland	Dresden University of Technology Institut für Hydrobiologie Fakultät für Forst-, Geo- und Hydrowissenschaften Mommsenstr. 13 D-01062 Dresden Germany
C.N.R.- I.R.S.A. Italian National Research Council Water Research Institute Via Mornera 25 I-20047 Brugherio MI
U.S.A.	U.S.A.
U.S. Environmental Protection Agency Mid-Continent Ecological Division 6201 Congdon Boulevard Duluth, MN 55804	Michigan State University Department of Fisheries and Wildlife No. 13 Natural Resources Building East Lansing, MI 48824-1222
U.S. Environmental Protection Agency Environmental Monitoring System Laboratory 26 W. Martin Luther Dr. Cincinnati, OH 45244	Wright State University Institute for Environmental Quality Dayton, OH 45435
Columbia Environmental Research Center U.S. Geological Survey 4200 New Haven Road Columbia, MO 65201	Great Lakes Environmental Research Laboratory, NOAA 2205 Commonwealth Boulevard Ann Arbor, MI 48105-1593

REFERENCES

• (1)Phipps, G.L., Ankley, G.T., Benoit, D.A. and Mattson, V.R. (1993). Use of the aquatic Oligochaete Lumbriculus variegatus for assessing the toxicity and bioaccumulation of sediment-associated contaminants. Environ.Toxicol. Chem. 12, 269-279.

• (2)Brunson, E.L., Canfield, T.J., Ingersoll, C.J. & Kemble, N.E. (1998). Assessing the bioaccumulation of contaminants from sediments of the Upper Mississippi river using field-collected oligochaetes and laboratory-exposed Lumbriculus variegatus. Arch. Environ. Contam. Toxicol. 35, 191-201.

• (3)ASTM International (2000). Standard guide for the determination of the bioaccumulation of sediment-associated contaminants by benthic invertebrates, E 1688-00a. In ASTM International 2004 Annual Book of Standards. Volume 11.05. Biological Effects and Environmental Fate; Biotechnology; Pesticides. ASTM International, West Conshohocken, PA.

• (4)U.S. EPA (2000). Methods for measuring the toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates. Second Edition. EPA 600/R-99/064, U.S. Environmental Protection Agency, Duluth, MN, March 2000.

• (5)Kukkonen, J. and Landrum, P.F. (1994). Toxicokinetics and toxicity of sediment-associated Pyrene to Lumbriculus variegatus (Oligochaeta). Environ. Toxicol. Chem. 13, 1457-1468.

• (6)Drewes C.D. & Fourtner C.R. (1990). Morphallaxis in an aquatic oligochaete, Lumbriculus variegatus: Reorganisation of escape reflexes in regenerating body fragments. Develop. Biol. 138: 94-103.

• (7)Leppänen, M.T. & Kukkonen, J.V.K. (1998a). Relationship between reproduction, sediment type and feeding activity of Lumbriculus variegatus (Müller): Implications for sediment toxicity testing. Environ. Toxicol. Chem. 17: 2196-2202.

• (8)Leppänen, M.T. & Kukkonen, J.V.K. (1998b). Factors affecting feeding rate, reproduction and growth of an oligochaete Lumbriculus variegatus (Müller). Hydrobiologia 377: 183-194.

• (9)Brust, K., O. Licht, V. Hultsch, D. Jungmann & R. Nagel (2001). Effects of Terbutryn on Aufwuchs and Lumbriculus variegatus in Artificial Indoor Streams. Environ. Toxicol. Chemistry, Vol. 20, pp. 2000–2007.

• (10)Oetken, M., K.-U. Ludwichowski & R. Nagel (2000). Sediment tests with Lumbriculus variegatus and Chironomus riparius and 3,4-dichloroaniline (3,4-DCA) within the scope of EG-AltstoffV. By order of the Federal Environmental Agency (Umweltbundesamt Berlin), FKZ 360 12 001, March 2000.

• (11)Leppänen M.T. & Kukkonen J.V.K. (1998). Relative importance of ingested sediment and porewater as bioaccumulation routes for pyrene to oligochaete (Lumbriculus variegatus, Müller). Environ. Sci. Toxicol. 32, 1503-1508.

Appendix 6

Summary of the ring test results

‘Sediment Toxicity Test with Lumbriculus variegatus

’**

Table 1

Results of individual ring test runs: Mean worm numbers in the controls and solvent controls at the end of the test; SD = standard deviation; CV = coefficient of variation

	mean worm number in the controls	SD	CV (%)	n	mean worm number in the solvent controls	SD	CV (%)	n
	32,3	7,37	22,80	3	39,0	3,61	9,25	3
	40,8	6,55	16,05	6	36,0	5,29	14,70	3
	41,5	3,54	8,52	2	38,5	7,05	18,31	4
	16,3	5,99	36,67	6	30,8	6,70	21,80	4
	24,3	10,69	43,94	3	26,3	3,06	11,60	3
	28,5	8,29	29,08	4	30,7	1,15	3,77	3
	28,3	3,72	13,14	6	28,8	2,56	8,89	6
	25,3	5,51	21,74	3	27,7	1,53	5,52	3
	23,8	2,99	12,57	4	21,3	1,71	8,04	4
	36,8	8,80	23,88	6	35,0	4,20	11,99	6
	33,0	3,58	10,84	6	33,5	1,73	5,17	4
	20,7	2,73	13,22	6	15,0	6,68	44,56	4
	42,0	7,07	16,84	6	43,7	0,58	1,32	3
	18,2	3,60	19,82	6	21,7	4,04	18,65	3
	32,0	3,95	12,34	6	31,3	4,79	15,32	4
interlaboratory mean	29,59		20,10		30,61		13,26
SD	8,32		10,03		7,57		10,48
n	15				15
min	16,3				15,0
max	42,0				43,7
CV (%)	28,1				24,7

Table 2

Results of individual ring test runs: Mean total dry weights of worms per replicate in the controls and solvent controls at the end of the test; SD = standard deviation; CV = coeff. of variation

	total dry weight of worms per replicate (controls)	SD	CV (%)	n	total dry weight of worms per replicate (solvent controls)	SD	CV (%)	n
	24,72	6,31	25,51	3	27,35	4,08	14,93	3
	30,17	2,04	6,75	6	33,83	10,40	30,73	3
	23,65	3,61	15,25	2	28,78	4,68	16,28	4
	12,92	6,83	52,91	6	24,90	6,84	27,47	4
	21,31	4,17	19,57	3	25,87	5,30	20,49	3
	22,99	4,86	21,16	4	24,64	5,09	20,67	3
	18,91	1,91	10,09	6	19,89	1,77	8,89	6
	24,13	1,63	6,75	3	25,83	2,17	8,41	3
	22,15	3,18	14,34	4	22,80	2,60	11,40	4
	35,20	8,12	23,07	6	31,42	8,45	26,90	6
	41,28	5,79	14,02	6	41,42	4,37	10,55	4
	15,17	5,78	38,09	6	10,50	3,42	32,53	4
	35,69	8,55	23,94	6	38,22	1,23	3,21	3
	19,57	5,21	26,65	6	28,58	6,23	21,81	3
	29,40	2,16	7,34	6	31,15	2,70	8,67	4
interlaboratory mean	25,15		20,36		27,68		17,53
SD	7,87		12,56		7,41		9,10
n	15				15
min	12,9				10,5
max	41,3				41,4
CV (%)	31,3				26,8

Table 3

Toxicity of PCP: Summary of endpoints in the ring test; interlaboratory means for EC50, NOEC and LOEC; SD = standard deviation; CV = coefficient of variation

biological parameter		Inter- laboratory mean (mg/kg)	min	max	Inter- laboratory factor	SD	CV (%)	geometr. mean (mg/kg)
total number of worms	EC50	23,0	4,0	37,9	9,4	10,7	46,3	19,9
total number of worms	NOEC	9,9	2,1	22,7	10,7	7,2	72,3	7,6
total number of worms	LOEC	27,9	4,7	66,7	14,2	19,4	69,4	20,9
total number of worms	MDD (%)	22,5	7,1	39,1
total dry weight of worms	EC50	20,4	7,3	39,9	5,5	9,1	44,5	18,2
total dry weight of worms	NOEC	9,3	2,1	20,0	9,4	6,6	70,4	7,4
total dry weight of worms	LOEC	25,7	2,1	50,0	23,5	16,8	65,5	19,4
total dry weight of worms	MDD (%)	24,8	10,9	44,7
mortality/survival	LC50	25,3	6,5	37,2	5,7	9,4	37,4	23,1
mortality/survival	NOEC	16,5	2,1	40,0	18,8	10,3	62,4	12,8
mortality/survival	LOEC	39,1	4,7	66,7	14,2	18,1	46,2	32,6
reproduction (increase of number of worms per replicate)	EC50	20,0	6,7	28,9	4,3	7,6	37,9	18,3
reproduction (increase of number of worms per replicate)	NOEC	7,9	2,1	20,0	9,4	5,2	66,0	6,4
reproduction (increase of number of worms per replicate)	LOEC	22,5	2,1	50,0	23,5	15,4	68,6	16,0
reproduction (increase of number of worms per replicate)	MDD (%)	29,7	13,9	47,9
growth (biomass increase per replicate)	EC50	15,3	5,7	29,9	5,2	7,1	46,5	13,7
growth (biomass increase per replicate)	NOEC	8,7	2,1	20,0	9,4	6,0	68,1	6,9
growth (biomass increase per replicate)	LOEC	24,0	2,1	50,0	23,5	15,7	65,5	17,3
growth (biomass increase per replicate)	MDD (%)	32,2	13,6	65,2
MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.	MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.	MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.	MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.	MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.	MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.	MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.	MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.	MDD: minimum detectable difference from the control values during hypothesis testing; used as a measure of statistical power.

REFERENCE

Egeler, Ph., Meller, M., Schallnaß, H.J. & Gilberg, D. (2005). Validation of a sediment toxicity test with the endobenthic aquatic oligochaete Lumbriculus variegatus by an international ring test. In co-operation with R. Nagel and B. Karaoglan. Report to the Federal Environmental Agency (Umweltbundesamt Berlin), R&D No.: 202 67 429.

C.36.   PREDATORY MITE (HYPOASPIS (GEOLAELAPS) ACULEIFER) REPRODUCTION TEST IN SOIL

The full description of this test method has been deleted. The equivalent international test method appears in Table 3 of Part 0.

C.37.   21-DAY FISH ASSAY: A SHORT-TERM SCREENING FOR OESTROGENIC AND ANDROGENIC ACTIVITY, AND AROMATASE INHIBITION

INTRODUCTION

• 1.This test method is equivalent to OECD test guideline (TG) 230 (2009). The need to develop and validate a fish assay capable of detecting certain endocrine active chemicals originates from the concerns that environmental levels of chemicals may cause adverse effects in both humans and wildlife due to the interaction of these chemicals with the endocrine system. In 1998, the OECD initiated a high-priority activity to revise existing guidelines and to develop new guidelines for the screening and testing of potential endocrine disrupters. One element of the activity was to develop a Test Guideline for the screening of chemicals active on the endocrine system of fish species. The 21-day Fish Endocrine Screening Assay underwent an extensive validation programme consisting of inter-laboratory studies with selected chemicals to demonstrate the relevance and reliability of the assay for the detection of oestrogenic and aromatase inhibiting chemicals (1, 2, 3, 4, 5) in the three fish species investigated (the fathead minnow, the Japanese medaka and the zebrafish); the detection of androgenic activity is possible in the fathead minnow and the medaka, but not in the zebrafish. This test method does not allow the detection of anti-androgenic chemicals. The validation work has been peer-reviewed by a panel of experts nominated by the National Coordinators of the Test Guideline Programme (6). The assay is not designed to identify specific mechanisms of hormonal disruption because the test animals possess an intact hypothalamic-pituitary-gonadal (HPG) axis, which may respond to chemicals that impact on the HPG axis at different levels. The Fish Short Term Reproduction assay (OECD TG 229) includes fecundity and, as appropriate, gonadal histopathology for the fathead minnow, as well as all endpoints included in this test method. OECD TG 229 provides a screening of chemicals which affect reproduction through various mechanisms including endocrine modalities. This should be considered prior to selecting the most appropriate test method.

• 2.This test method describes an in vivo screening assay where sexually mature male and spawning female fish are held together and exposed to a chemical during a limited part of their life-cycle (21 days). At termination of the 21-day exposure period, depending on the species used, one or two biomarker endpoint(s) are measured in males and females as indicators of oestrogenic, aromatase inhibition or androgenic activity of the test chemical; these endpoints are vitellogenin and secondary sexual characteristics. Vitellogenin is measured in fathead minnow, Japanese medaka and zebrafish, whereas secondary sex characteristics are measured in fathead minnow and Japanese medaka only.

• 3.This bioassay serves as an in vivo screening assay for certain endocrine modes of action and its application should be seen in the context of the ‘OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals’ (28).

INITIAL CONSIDERATIONS AND LIMITATIONS

• 4.Vitellogenin is normally produced by the liver of female oviparous vertebrates in response to circulating endogenous oestrogen. It is a precursor of egg yolk proteins and, once produced in the liver, travels in the bloodstream to the ovary, where it is taken up and modified by developing eggs. Vitellogenin is almost undetectable in the plasma of immature female and male fish because they lack sufficient circulating oestrogen; however, the liver is capable of synthesizing and secreting vitellogenin in response to exogenous oestrogen stimulation.

• 5.The measurement of vitellogenin serves for the detection of chemicals with various oestrogenic modes of action. The detection of oestrogenic chemicals is possible via the measurement of vitellogenin induction in male fish, and it has been abundantly documented in the scientific peer-reviewed literature (e.g. (7)). Vitellogenin induction has also been demonstrated following exposure to aromatizable androgens (8, 9). A reduction in the circulating level of oestrogen in females, for instance through the inhibition of the aromatase converting the endogenous androgen to the natural oestrogen 17β-estradiol, causes a decrease in the vitellogenin level, which is used to detect chemicals having aromatase inhibiting properties (10, 11). The biological relevance of the vitellogenin response following oestrogenic/aromatase inhibition is established and has been broadly documented. However, it is possible that production of VTG in females can also be affected by general toxicity and non-endocrine toxic modes of action, e.g. hepatotoxicity.

• 6.Several measurement methods have been successfully developed and standardised for routine use. This is the case of species-specific Enzyme-Linked Immunosorbent Assay (ELISA) methods using immunochemistry for the quantification of vitellogenin produced in small blood or liver samples collected from individual fish (12, 13, 14, 15, 16, 17, 18). Fathead minnow blood, zebrafish blood or head/tail homogenate, and medaka liver are sampled for VTG measurement. In medaka, there is a good correlation between VTG measured from blood and from liver (19). Appendix 6 provides the recommended procedures for sample collection for vitellogenin analysis. Kits for the measurement of vitellogenin are widely available; such kits should be based on a validated species-specific ELISA method.

• 7.Secondary sex characteristics in male fish of certain species are externally visible, quantifiable and responsive to circulating levels of endogenous androgens; this is the case for the fathead minnow and the medaka — but not for zebrafish, which does not possess quantifiable secondary sex characteristics. Females maintain the capacity to develop male secondary sex characteristics, when they are exposed to androgenic chemicals in water. Several studies are available in the scientific literature to document this type of response in fathead minnow (20) and medaka (21). A decrease in secondary sex characteristics in males should be interpreted with caution because of low statistical power, and should be based on expert judgement and weight of evidence. There are limitations to the use of zebrafish in this assay, due to the absence of quantifiable secondary sex characteristics responsive to androgenic acting chemicals.

• 8.In the fathead minnow, the main indicator of exogenous androgenic exposure is the number of nuptial tubercles located on the snout of the female fish. In the medaka, the number of papillary processes constitutes the main marker of exogenous exposure to androgenic chemicals in female fish. Appendix 5A and Appendix 5B indicate the recommended procedures to follow for the evaluation of sex characteristics in fathead minnow and in medaka, respectively.

• 9.Definitions used in this test method are given in Appendix 1.

PRINCIPLE OF THE TEST

• 10.In the assay, male and female fish in a reproductive status are exposed together in test vessels. Their adult and reproductive status enables a clear differentiation of each sex, and thus a sex-related analysis of each endpoint, and ensures their sensitivity towards exogenous chemicals. At test termination, sex is confirmed by macroscopic examination of the gonads following ventral opening of the abdomen with scissors. An overview of the relevant bioassay conditions is provided in Appendix 2. The assay is normally initiated with fish sampled from a population that is in spawning condition; senescent animals should not be used. Guidance on the age of fish and on the reproductive status is provided in the section on Selection of fish. The assay is conducted using three chemical exposure concentrations as well as a water control, and a solvent control if necessary. Two vessels or replicates per treatment are used (each vessel containing 5 males and 5 females) in medaka and zebrafish, whereas four vessels or replicates per treatment are used (each vessel containing 2 males and 4 females) in fathead minnow. This is to accommodate the territorial behaviour of male fathead minnow while maintaining sufficient power of the assay. The exposure is conducted for 21 days and sampling of fish is performed at day 21 of exposure.

• 11.On sampling at day 21, all animals are killed humanely. Secondary sex characteristics are measured in fathead minnow and medaka (see Appendix 5A and Appendix 5B); blood samples are collected for determination of vitellogenin in zebrafish and fathead minnow, alternatively head/tail can be collected for the determination of vitellogenin in zebrafish (Appendix 6); liver is collected for VTG analysis in medaka (Appendix 6).

TEST ACCEPTANCE CRITERIA

• 12.For the test results to be acceptable the following conditions apply:

• — the mortality in the water (or solvent) controls should not exceed 10 % at the end of the exposure period;

• — the dissolved oxygen concentration should be at least 60 % of the air saturation value (ASV) throughout the exposure period;

• — the water temperature should not differ by more than ± 1,5 °C between test vessels at any one time during the exposure period and be maintained within a range of 2 °C within the temperature ranges specified for the test species (Appendix 2);

• — evidence should be available to demonstrate that the concentrations of the test chemical in solution have been satisfactorily maintained within ± 20 % of the mean measured values.

DESCRIPTION OF THE METHOD

Apparatus

• 13.Normal laboratory equipment and especially the following:

• (a) oxygen and pH meters;

• (b) equipment for determination of water hardness and alkalinity;

• (c) adequate apparatus for temperature control and preferably continuous monitoring;

• (d) tanks made of chemically inert material and of a suitable capacity in relation to the recommended loading and stocking density (see Appendix 2);

• (e) spawning substrate for fathead minnow and zebrafish, Appendix 4 gives the necessary details;

• (f) suitably accurate balance (i.e. accurate to ± 0,5 mg).

Water

• 14.Any water in which the test species shows suitable long-term survival and growth may be used as test water. It should be of constant quality during the period of the test. The pH of the water should be within the range 6,5 to 8,5, but during a given test it should be within a range of ± 0,5 pH units. In order to ensure that the dilution water will not unduly influence the test result (for example by complexion of test chemical), samples should be taken at intervals for analysis. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, and Ni), major anions and cations (e.g. Ca²⁺, Mg²⁺, Na⁺, K⁺, Cl^-, and SO4 ^2-), pesticides (e.g. total organophosphorus and total organochlorine pesticides), total organic carbon and suspended solids should be made, for example, every three months where dilution water is known to be relatively constant in quality. If water quality has been demonstrated to be constant over at least one year, determinations can be less frequent and intervals extended (e.g. every six months). Some chemical characteristics of acceptable dilution water are listed in Appendix 3.

Test solutions

• 15.Test solutions of the chosen concentrations are prepared by dilution of a stock solution. The stock solution should preferably be prepared by simply mixing or agitating the test chemical in dilution water by using mechanical means (e.g. stirring or ultrasonication). Saturation columns (solubility columns) can be used for achieving a suitable concentrated stock solution. The use of a solvent carrier is not recommended. However, in case a solvent is necessary, a solvent control should be run in parallel, at the same solvent concentration as the chemical treatments. For difficult test chemicals, a solvent may be technically the best solution; the OECD Guidance Document on aquatic toxicity testing of difficult substances and mixtures should be consulted (22). The choice of solvent will be determined by the chemical properties of the chemical. The OECD Guidance Document recommends a maximum of 100 μl/l, which should be observed. However a recent review (23) highlighted additional concerns when using solvents for endocrine activity testing. Therefore it is recommended that the solvent concentration, if necessary, is minimised wherever technically feasible (dependent on the physical-chemical properties of the test chemical).

• 16.A flow-through test system will be used. Such a system continually dispenses and dilutes a stock solution of the test chemical (e.g. metering pump, proportional diluter, saturator system) in order to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals, preferably daily, during the test and should not vary by more than 10 % throughout the test. Care should be taken to avoid the use of low-grade plastic tubing or other materials that may contain biologically active chemicals. When selecting the material for the flow-through system, possible adsorption of the test chemical to this material should be considered.

Holding of fish

• 17.Test fish should be selected from a laboratory population, preferably from a single stock, which has been acclimated for at least two weeks prior to the test under conditions of water quality and illumination similar to those used in the test. It is important that the loading rate and stocking density (for definitions, see Appendix 1) be appropriate for the test species used (see Appendix 2).

• 18.Following a 48-hour settling-in period, mortalities are recorded and the following criteria applied:

• — mortalities of greater than 10 % of population in seven days: reject the entire batch;

• — mortalities of between 5 % and 10 % of population: acclimation for seven additional days; if more than 5 % mortality during second seven days, reject the entire batch;

• — mortalities of less than 5 % of population in seven days: accept the batch

• 19.Fish should not receive treatment for disease during the acclimation period, in the pre-exposure period, or during the exposure period.

Pre-exposure and selection of fish

• 20.A one-week pre-exposure period is recommended, with animals placed in vessels similar to the actual test. Fish should be fed ad libitum throughout the holding period and during the exposure phase. The exposure phase is started with sexually dimorphic adult fish from a laboratory supply of reproductively mature animals (e.g. with clear secondary sexual characteristics visible as far as fathead minnow and medaka are concerned), and actively spawning. For general guidance only (and not to be considered in isolation from observing the actual reproductive status of a given batch of fish), fathead minnows should be approximately 20 (± 2) weeks of age, assuming they have been cultured at 25 ± 2 °C throughout their lifespan. Japanese medaka should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 25 ± 2 °C throughout their lifespan. Zebrafish should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 26 ± 2 °C throughout their lifespan.

TEST DESIGN

• 21.Three concentrations of the test chemical, one control (water) and, if needed, one solvent control are used. The data may be analysed in order to determine statistically significant differences between treatment and control responses. These analyses will inform whether further longer term testing for adverse effects (namely, survival, development, growth and reproduction) is required for the chemical, rather than for use in risk assessment (24).

• 22.For zebrafish and medaka, on day 21 of the experiment, males and females from each treatment level (5 males and 5 females in each of the two replicates) and from the control(s) are sampled for the measurement of vitellogenin and secondary sex characteristics, where applicable. For fathead minnow, on day 21 of exposure, males and females (2 males and 4 females in each of the four replicates) and from the control(s) are sampled for the measurement of vitellogenin and secondary sex characteristics.

Selection of test concentrations

• 23.For the purposes of this test, the highest test concentration should be set by the maximum tolerated concentration (MTC) determined from a range finder or from other toxicity data, or 10 mg/l, or the maximum solubility in water, whichever is lowest. The MTC is defined as the highest test concentration of the chemical which results in less than 10 % mortality. Using this approach assumes that there are existing empirical acute toxicity data or other toxicity data from which the MTC can be estimated. Estimating the MTC can be inexact and typically requires some professional judgment.

• 24.Three test concentrations, spaced by a constant factor not exceeding 10, and a dilution-water control (and solvent control if necessary) are required. A range of spacing factors between 3,2 and 10 is recommended.

PROCEDURE

Selection and weighing of test fish

• 25.It is important to minimise variation in weight of the fish at the beginning of the assay. Suitable size ranges for the different species recommended for use in this test are given in Appendix 2. For the whole batch of fish used in the test, the range in individual weights for male and female fish at the start of the test should be kept, if possible, within ± 20 % of the arithmetic mean weight of the same sex. It is recommended to weigh a subsample of the fish stock before the test in order to estimate the mean weight.

Conditions of exposure

Duration

• 26.The test duration is 21 days, following a pre-exposure period. The recommended pre-exposure period is one week.

Feeding

• 27.Fish should be fed ad libitum with an appropriate food (Appendix 2) at a sufficient rate to maintain body condition. Care should be taken to avoid microbial growth and water turbidity. As a general guidance, the daily ration may be divided into two or three equal portions for multiple feeds per day, separated by at least three hours between each feed. A single larger ration is acceptable particularly for weekends. Food should be withheld from the fish for 12 hours prior to sampling/necropsy.

• 28.Fish food should be evaluated for the presence of contaminants such as organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs). Food with an elevated level of phytoestrogens that would compromise the response of the assay to known oestrogen agonist (e.g. 17-beta estradiol) should be avoided.

• 29.Uneaten food and faecal material should be removed from the test vessels at least twice weekly, e.g. by carefully cleaning the bottom of each tank using a siphon.

Light and temperature

• 30.The photoperiod and water temperature should be appropriate for the test species (see Appendix 2).

Frequency of analytical determinations and measurements

• 31.Prior to initiation of the exposure period, proper function of the chemical delivery system should be ensured. All analytical methods needed should be established, including sufficient knowledge on the chemical stability in the test system. During the test, the concentrations of the test chemical are determined at regular intervals, as follows: the flow rates of diluent and toxicant stock solution should be checked preferably daily but as a minimum twice per week, and should not vary by more than 10 % throughout the test. It is recommended that the actual test chemical concentrations be measured in all vessels at the start of the test and at weekly intervals thereafter.

• 32.It is recommended that results be based on measured concentrations. However, if concentration of the test chemical in solution has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test, then the results can either be based on nominal or measured values.

• 33.Samples may need to be filtered (e.g., using a 0,45 μm pore size) or centrifuged. If needed, then centrifugation is the recommended procedure. However, if the test material does not adsorb to filters, filtration may also be acceptable.

• 34.During the test, dissolved oxygen, temperature, and pH should be measured in all test vessels at least once per week. Total hardness and alkalinity should be measured in the controls and one vessel at the highest concentration at least once per week. Temperature should preferably be monitored continuously in at least one test vessel.

Observations

• 35.A number of general (e.g. survival) and core biological responses (e.g. vitellogenin levels) are assessed over the course of the assay or at termination of the assay. Measurement and evaluation of these endpoints and their utility are described below.

Survival

• 36.Fish should be examined daily during the test period and any mortality should be recorded and the dead fish removed as soon as possible. Dead fish should not be replaced in either the control or treatment vessels. Sex of fish that die during the test should be determined by macroscopic evaluation of the gonads.

Behaviour and appearance

• 37.Any abnormal behaviour (relative to controls) should be noted; this might include signs of general toxicity including hyperventilation, uncoordinated swimming, loss of equilibrium, and atypical quiescence or feeding. Additionally external abnormalities (such as haemorrhage, discoloration) should be noted. Such signs of toxicity should be considered carefully during data interpretation since they may indicate concentrations at which biomarkers of endocrine activity are not reliable. Such behavioural observations may also provide useful qualitative information to inform potential future fish testing requirements. For example, territorial aggressiveness in normal males or masculinised females has been observed in fathead minnows under androgenic exposure; in zebrafish, the characteristic mating and spawning behaviour after the dawn onset of light is reduced or hindered by oestrogenic or anti-androgenic exposure.

• 38.Because some aspects of appearance (primarily colour) can change quickly with handling, it is important that qualitative observations be made prior to removal of animals from the test system. Experience to date with fathead minnows suggests that some endocrine active chemicals may initially induce changes in the following external characteristics: body colour (light or dark), coloration patterns (presence of vertical bands), and body shape (head and pectoral region). Therefore observations of physical appearance of the fish should be made over the course of the test, and at conclusion of the study

Humane killing of fish

• 39.At day 21, i.e. at termination of the exposure, the fish should be euthanized with appropriate amounts of Tricaine (Tricaine methane sulfonate, Metacain, MS-222 (CAS 886-86-2), 100-500 mg/l buffered with 300 mg/l NaHCO3 (sodium bicarbonate, CAS 144-55-8) to reduce mucous membrane irritation; blood or tissue is then sampled for vitellogenin determination, as explained in the Vitellogenin section.

Observation of secondary sex characteristics

• 40.Some endocrine active chemicals may induce changes in specialised secondary sex characteristics (number of nuptial tubercles in male fathead minnow, papillary processes in male medaka). Notably, chemicals with certain modes of action may cause abnormal occurrence of secondary sex characteristic in animals of the opposite sex; for example, androgen receptor agonists, such as trenbolone, methyltestosterone and dihydrotestosterone, can cause female fathead minnows to develop pronounced nuptial tubercles or female medaka to develop papillary processes (11, 20, 21). It also has been reported that oestrogen receptor agonists can decrease nuptial tubercle numbers and size of the dorsal nape pad in adult males (25, 26). Such gross morphological observations may provide useful qualitative and quantitative information to inform potential future fish testing requirements. The number and size of nuptial tubercles in fathead minnow and papillary processes in medaka can be quantified directly or more practically in preserved specimens. Recommended procedures for the evaluation of secondary sex characteristics in fathead minnow and medaka are available from Appendix 5A and Appendix 5B, respectively.

Vitellogenin (VTG)

• 41.Blood is collected from the caudal artery/vein with a heparinised microhematocrit capillary tubule, or alternatively by cardiac puncture with a syringe. Depending upon the size of the fish, collectable blood volumes generally range from 5 to 60 μl per individual for fathead minnows and 5-15 μl per individual for zebrafish. Plasma is separated from the blood via centrifugation, and stored with protease inhibitors at – 80 °C, until analysed for vitellogenin. Alternatively, in medaka the liver will be used, and in zebrafish the head/tail homogenate can be used as tissue-source for vitellogenin determination (Appendix 6). The measurement of VTG should be based upon a validated homologous ELISA method, using homologous VTG standard and homologous antibodies. It is recommended to use a method capable to detect VTG levels as low as few ng/ml plasma (or ng/mg tissue), which is the background level in unexposed male fish.

• 42.Quality control of vitellogenin analysis will be accomplished through the use of standards, blanks and at least duplicate analyses. For each ELISA method, a test for matrix effect (effect of sample dilution) should be run to determine the minimum sample dilution factor. Each ELISA plate used for VTG assays should include the following quality control samples: at least 6 calibration standards covering the range of expected vitellogenin concentrations, and at least one non-specific binding assay blank (analysed in duplicate). Absorbance of these blanks should be less than 5 % of the maximum calibration standard absorbance. At least two aliquots (well-duplicates) of each sample dilution will be analysed. Well-duplicates that differ by more than 20 % should be re-analysed.