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)

• (3)Fleming, R. et al. (1994), Sediment Toxicity Tests for Poorly Water-Soluble Substances, Final Report to the European Commission, Report No: EC 3738. August 1994. WRc, UK.

• (4)SETAC (1993), Guidance Document on Sediment toxicity Tests and Bioassays for Freshwater and Marine Environments, From the WOSTA Workshop held in the Netherlands.

• (5)ASTM International (2009), E1706-05E01: Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates, In: Annual Book of ASTM Standards, Volume 11.06, Biological Effects and Environmental Fate; Biotechnology. ASTM International, West Conshohocken, PA.

• (6)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.

• (7)US-EPA (2000), Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates, Second edition, EPA 600/R-99/064, March 2000, Revision to the first edition dated June 1994.

• (8)US-EPA/OPPTS 850.1735 (1996), Whole Sediment Acute Toxicity Invertebrates.

• (9)US-EPA/OPPTS 850.1790 (1996), Chironomid Sediment toxicity Test.

• (10)Milani, D., Day, K.E., McLeay, D.J. and R.S. Kirby (1996), Recent intra- and inter-laboratory studies related to the development and standardisation of Environment Canada's biological test methods for measuring sediment toxicity using freshwater amphipods (Hyalella azteca) and midge larvae (Chironomus riparius), Technical Report, Environment Canada, National Water Research Institute, Burlington, Ontario, Canada.

• (11)Norberg-King, T.J., Sibley, P.K., Burton, G.A., Ingersoll, C.G., Kemble, N.E., Ireland, S., Mount, D.R. and C.D. Rowland (2006), Interlaboratory evaluation of Hyalella azteca and Chironomus tentans short-term and long-term sediment toxicity tests, Environ. Toxicol. Chem., 25: 2662-2674.

• (12)Taenzler, V., Bruns, E., Dorgerloh, M., Pfeifle, V. and L. Weltje (2007), Chironomids: suitable test organisms for risk assessment investigations on the potential endocrine-disrupting properties of pesticides, Ecotoxicology, 16: 221-230.

• (13)Sugaya, Y. (1997), Intra-specific variations of the susceptibility of insecticides in Chironomus yoshimatsui, Jp. J. Sanit. Zool., 48: 345-350.

• (14)Kawai, K. (1986), Fundamental studies on chironomid allergy, I. Culture methods of some Japanese chironomids (Chironomidae, Diptera), Jp. J. Sanit. Zool., 37: 47-57.

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

• (16)OECD (2000), Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, Environment, Health and Safety Publications, Series on Testing and Assessment No. 23, ENV/JM/MONO(2000)6, OECD, Paris.

• (17)Weltje, L., Rufli, H., Heimbach, F., Wheeler, J., Vervliet-Scheebaum, M. and M. Hamer (2010), The chironomid acute toxicity test: development of a new test system, Integr. Environ. Assess. Management.

• (18)Environment Canada. (1995), Guidance Document on Measurement of Toxicity Test Precision Using Control Sediments Spiked with a Reference Toxicant, Report EPS 1/RM/30, September 1995.

• (19)Oetken, M, Nentwig, G., Löffler, D, Ternes, T. and J. Oehlmann (2005), Effects of pharmaceuticals on aquatic invertebrates, Part I, The antiepileptic drug carbamazepine, Arch. Environ. Contam. Toxicol., 49: 353-361.

• (20)Suedel, B.C. and J.H. Rodgers (1994), Development of formulated reference sediments for freshwater and estuarine sediment testing, Environ. Toxicol. Chem., 13: 1163-1175.

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

• (22)Dunnett, C.W. (1964), A multiple comparisons procedure for comparing several treatments with a control. J. Amer. Statis. Assoc., 50: 1096-1121.

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

• (24)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.

• (25)Williams, D.A. (1972), The comparison of several dose levels with a zero dose control. Biometrics, 28: 510-531.

• (26)Jungmann, D., Bandow, C., Gildemeister, T., Nagel, R., Preuss, T.G., Ratte, H.T., Shinn, C., Weltje, L. and H.M. Maes (2009), Chronic toxicity of fenoxycarb to the midge Chironomus riparius after exposure in sediments of different composition. J Soils Sediments, 9: 94-102.

• (27)Rao, J.N.K. and A.J. Scott (1992), A simple method for the analysis of clustered binary data. Biometrics, 48: 577-585.

• (28)Christensen, E.R. (1984), Dose-response functions in aquatic toxicity testing and the Weibull model, Water Res., 18: 213-221.

• (29)Bruce, R.D. and D.J. Versteeg (1992), A statistical procedure for modelling continuous toxicity data, Environ. Toxicol. Chem., 11: 1485-1494.

• (30)Slob, W. (2002), Dose-response modelling of continuous endpoints. Toxicol. Sci., 66: 298-312.

• (31)OECD (2006), Current Approaches in the Statistical Analysis of Ecotoxicity Data: a Guidance to Application, OECD Series on Testing and Assessment No. 54, 146 pp., ENV/JM/MONO(2006)18, OECD, Paris.

• (32)Benoit, D.A., Sibley, P.K., Juenemann, J.L. and G.T. Ankley (1997), Chironomus tentans life-cycle test: design and evaluation for use in assessing toxicity of contaminated sediments, Environ. Toxicol. Chem., 16: 1165-1176.

• (33)Vogt, C., Belz, D., Galluba, S., Nowak, C., Oetken, M. and J. Oehlmann (2007), Effects of cadmium and tributyltin on development and reproduction of the non-biting midge Chironomus riparius (Diptera) — baseline experiments for future multi-generation studies, J. Environ. Sci. Health Part A, 42: 1-9.

• (34)OECD (2010), Validation report of the Chironomid full life-cycle toxicity test, Forthcoming publication in the Series on Testing and Assessment, OECD, Paris.

Appendix 1

Definitions

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

Chemical is a substance or a mixture.

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

Overlying water is the water placed over sediment in the test vessel.

Interstitial water or pore water is the water occupying space between sediment and soil particles.

Spiked water is the test water to which test chemical has been added.

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

Appendix 2

Recommendations for culture of Chironomus riparius

• 1. Chironomus larvae may be reared in crystallising dishes or larger containers. Fine quartz sand is spread in a thin layer of about 5 to 10 mm deep over the bottom of the container. Kieselgur (e.g. Merck, Art 8117) has also been shown to be a suitable substrate (a thinner layer of up to a very few mm is sufficient). Suitable water is then added to a depth of several cm. Water levels should be topped up as necessary to replace evaporative loss, and prevent desiccation. Water can be replaced if necessary. Gentle aeration should be provided. The larval rearing vessels should be held in a suitable cage which will prevent escape of the emerging adults. The cage should be sufficiently large to allow swarming of emerged adults, otherwise copulation may not occur (minimum is ca. 30 × 30 × 30 cm).

• 2.Cages should be held at room temperature or in a constant environment room at 20 ± 2 °C with a photo period of 16 hour light (intensity ca. 1 000 lux), 8 hours dark. It has been reported that air humidity of less than 60 % RH can impede reproduction.

Dilution water

• 3.Any suitable natural or synthetic water may be used. Well water, dechlorinated tap water and artificial media (e.g. Elendt ‘M4’ or ‘M7’ medium, see below) are commonly used. The water should be aerated before use. If necessary, the culture water may be renewed by pouring or siphoning the used water from culture vessels carefully without destroying the tubes of larvae.

Feeding larvae

• 1. Chironomus larvae should be fed with a fish flake food (Tetra Min®, Tetra Phyll® or other similar brand of proprietary fish food), at approximately 250 mg per vessel per day. This can be given as a dry ground powder or as a suspension in water: 1,0 g of flake food is added to 20 ml of dilution water and blended to give a homogenous mix. This preparation may be fed at a rate of about 5 ml per vessel per day. (shake before use.) Older larvae may receive more.

• 5.Feeding is adjusted according to the water quality. If the culture medium becomes ‘cloudy’, the feeding should be reduced. Food additions should be carefully monitored. Too little food will cause emigration of the larvae towards the water column, and too much food will cause increased microbial activity and reduced oxygen concentrations. Both conditions can result in reduced growth rates.

• 6.Some green algae (e.g. Scenedesmus subspicatus, Chlorella vulgaris) cells may also be added when new culture vessels are set up.

Feeding emerged adults

• 7.Some experimenters have suggested that a cotton wool pad soaked in a saturated sucrose solution may serve as a food for emerged adults.

Emergence

• 8.At 20 ± 2 °C adults will begin to emerge from the larval rearing vessels after approximately 13 - 15 days. Males are easily distinguished by having plumose antennae and thin body.

Egg masses

• 9.Once adults are present within the breeding cage, all larval rearing vessels should be checked three times weekly for deposition of the gelatinous egg masses. If present, the egg masses should be carefully removed. They should be transferred to a small dish containing a sample of the breeding water. Egg masses are used to start a new culture vessel (e.g. 2 - 4 egg masses/vessel) or are used for toxicity tests.

• 10.First instar larvae should hatch after 2 - 3 days.

Set-up of new culture vessels

• 11.Once cultures are established it should be possible to set up a fresh larval culture vessel weekly or less frequently depending on testing requirements, removing the older vessels after adult midges have emerged. Using this system a regular supply of adults will be produced with a minimum of management.

Preparation of test solutions ‘M4’ and ‘M7’

• 12.Elendt (1990) has described the ‘M4’ medium. The ‘M7’ medium is prepared as the ‘M4’ medium except for the substances indicated in Table 1, for which concentrations are four times lower in ‘M7’ than in ‘M4’. The test solution should not be prepared according to Elendt and Bias (1990) for the concentrations of NaSiO3 · 5H2O, NaNO3, KH2PO4 and K2HPO4 given for the preparation of the stock solutions are not adequate.

Preparation of the ‘M7’-medium

• 13.Each stock solution (I) is prepared individually and a combined stock solution (II) is prepared from these stock solutions (I) (see Table 1). Fifty ml from the combined stock solution (II) and the amounts of each macro nutrient stock solution which are given in Table 2 are made up to 1 litre of deionised water to prepare the ‘M7’ medium. A vitamin stock solution is prepared by adding three vitamins to deionised water as indicated in Table 3, and 0,1 ml of the combined vitamin stock solution are added to the final ‘M7’ medium shortly before use. The vitamin stock solution is stored frozen in small aliquots. The medium is aerated and stabilised.

Table 1

Stock solutions of trace elements for medium M4 and M7

Stock solutions (I)	Amount (mg) made up to 1 litre of deionised water	To prepare the combined stock solution (II): mix the following amounts (ml) of stock solutions (I) and make up to 1 litre of deionised water	To prepare the combined stock solution (II): mix the following amounts (ml) of stock solutions (I) and make up to 1 litre of deionised water	Final concentrations in test solutions (mg/l)	Final concentrations in test solutions (mg/l)
Stock solutions (I)	Amount (mg) made up to 1 litre of deionised water	M4	M7	M4	M7
H3BO3 [^E0203]	57 190	1,0	0,25	2,86	0,715
MnCl2·4H2O [^E0203]	7 210	1,0	0,25	0,361	0,090
LiCl [^E0203]	6 120	1,0	0,25	0,306	0,077
RbCl [^E0203]	1 420	1,0	0,25	0,071	0,018
SrCl2·6H2O [^E0203]	3 040	1,0	0,25	0,152	0,038
NaBr [^E0203]	320	1,0	0,25	0,016	0,004
Na2MoO4·2H2O [^E0203]	1 260	1,0	0,25	0,063	0,016
CuCl2·2H2O [^E0203]	335	1,0	0,25	0,017	0,004
ZnCl2	260	1,0	1,0	0,013	0,013
CaCl2·6H2O	200	1,0	1,0	0,010	0,010
KI	65	1,0	1,0	0,0033	0,0033
Na2SeO3	43,8	1,0	1,0	0,0022	0,0022
NH4VO3	11,5	1,0	1,0	0,00058	0,00058
Na2EDTA·2H2O [^E0203] [^E0204]	5 000	20,0	5,0	2,5	0,625
FeSO4·7H2O [^E0203] [^E0204]	1 991	20,0	5,0	1,0	0,249

These substances differ in M4 and M7, as indicated above.

These solutions are prepared individually, then poured together and autoclaved immediately.

Table 2

Macro nutrient stock solutions for medium M4 and M7

	Amount made up to 1 litre of deionised water (mg)	Amount of macro nutrient stock solutions added to prepare medium M4 and M7 (ml/l)	Final concentrations in test solutions M4 and M7 (mg/l)
CaCl2 · 2H2O	293 800	1,0	293,8
MgSO4 · 7H2O	246 600	0,5	123,3
KCl	58 000	0,1	5,8
NaHCO3	64 800	1,0	64,8
NaSiO3 · 9H2O	50 000	0,2	10,0
NaNO3	2 740	0,1	0,274
KH2PO4	1 430	0,1	0,143
K2HPO4	1 840	0,1	0,184

Table 3

Vitamin stock solution for medium M4 and M7

All three vitamin solutions are combined to make a single vitamin stock solution.

	Amount made up to 1 litre of deionised water (mg)	Amount of vitamin stock solution added to prepare medium M4 and M7 (ml/l)	Final concentrations in test solutions M4 and M7 (mg/l)
Thiamine hydrochloride	750	0,1	0,075
Cyanocobalamin (B12)	10	0,1	0,0010
Biotine	7,5	0,1	0,00075

REFERENCES

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.

Elendt, B.P. (1990), Selenium deficiency in Crustacea, Protoplasma, 154: 25-33.

Elendt, B.P. and W.-R. Bias (1990), Trace nutrient deficiency in Daphnia magna cultured in standard medium for toxicity testing, Effects on the optimisation of culture conditions on life history parameters of D. magna, Water Research, 24: 1157-1167.

Appendix 3

Preparation of formulated sediment

SEDIMENT COMPOSITION

The composition of the formulated sediment should be as follows:

Constituent	Characteristics	% of sediment dry weight
Peat	Sphagnum moss peat, as close to pH 5,5-6,0 as possible, no visible plant remains, finely ground (particle size ≤ 1 mm) and air dried	4 - 5
Quartz sand	Grain size: > 50 % of the particles should be in the range of 50-200 μm	75 - 76
Kaolinite clay	Kaolinite content ≥ 30 %	20
Organic carbon	Adjusted by addition of peat and sand	2 (± 0,5)
Calcium carbonate	CaCO3, pulverised, chemically pure	0,05 - 0,1
Water	Conductivity ≤ 10 μS/cm	30 - 50

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 once again and is adjusted to 6,5 to 7,5 with CaCO3 if necessary. Samples of the sediment are taken to determine the dry weight and the organic carbon content. Then, before it is used in the chironomid toxicity test, it is recommended that the formulated sediment be conditioned for seven days under the same conditions which prevail in the subsequent test.

STORAGE

The dry constituents for preparation of the artificial sediment may be stored in a dry and cool place at room temperature. The formulated (wet) sediment should not be stored prior to its use in the test. It should be used immediately after the 7 days conditioning period that ends its preparation.

REFERENCES

OECD (1984), Earthworm, Acute Toxicity Test, Test Guideline No. 207, Guidelines for the Testing of Chemicals, OECD, Paris.

Meller, M., Egeler, P., Roembke, J., Schallnass, H., Nagel, R. and B. Streit (1998), Short-term toxicity of lindane, hexachlorobenzene and copper sulfate on tubificid sludgeworms (Oligochaeta) in artificial media, Ecotox. Environ. Safety, 39: 10-20.

Appendix 4

Chemical Characteristics of an Acceptable Dilution water

CONSTITUENT	CONCENTRATIONS
Particulate matter	< 20 mg/l
Total organic carbon	< 2 mg/l
Unionised ammonia	< 1 μg/l
Hardness as CaCO3	< 400 mg/l [^E0205]
Residual chlorine	< 10 μg/l
Total organophosphorus pesticides	< 50 ng/l
Total organochlorine pesticides plus polychlorinated biphenyls	< 50 ng/l
Total organic chlorine	< 25 ng/l

However, it should be noted that if there is an interaction suspected between hardness ions and the test chemical, lower hardness water should be used (and thus, Elendt Medium M4 should not be used in this situation).

Appendix 5

Guidance for test performance

Example of a breeding cage:

Image omitted; see official source

A	:	gauze on the top and at least one side of the cage (mesh size ca. 1 mm)
---	---	---
B	:	aperture for placing the emerged adults inside the breeding cage and to remove the laid egg ropes from the crystallisation dishes (not shown in this graphic)
---	---	---
C	:	breeding cage size minimum 30 cm length, 30 cm height and 30 cm width

Example of a test vessel:

Image omitted; see official source

A	:	pasteur pipette for air supply of the overlying water
---	---	---
B	:	glass lid to prevent emerged midges from escaping
---	---	---
C	:	water surface layer
---	---	---
D	:	test vessel (glass beaker minimum 600 ml)
---	---	---
E	:	sediment layer

Example of an exhauster for capturing adult midges (arrows indicate air flow direction):

Image omitted; see official source

A	:	glass tube (inner diameter ca. 5 mm) connected to a self-priming pump
---	---	---
B	:	cork of vulcanised rubber, perforated with glass tube (A). On the inside, the opening of glass tube (A) is covered with some cotton and a gauze (mesh size ca. 1 mm) to prevent damaging the midges when they are sucked into the exhauster
---	---	---
C	:	transparent container (plastic or glass, length ca. 15 cm) for captured midges
---	---	---
D	:	cork of vulcanised rubber, perforated with tube (E). To release midges into the breeding cage, cork D is released from container C
---	---	---
E	:	tube (plastic or glass, inner diameter ca. 8 mm) to collect adult midges from vessel

Schematic presentation of a life-cycle test:

Image omitted; see official source

A	:	1st generation — test vessels containing a sediment-water system, eight replicates, 20 first instar larvae per vessel
---	---	---
B	:	four test vessels for each breeding cage, A and B
---	---	---
C	:	breeding cages (A and B) for swarming, mating and oviposition
---	---	---
D	:	crystallising dishes for deposition of egg ropes
---	---	---
E	:	micro plates, one well for each egg rope
---	---	---
F	:	2nd generation — test vessels containing a sediment-water system, eight replicates, 20 first instar larvae per vessel.

C.41.   FISH SEXUAL DEVELOPMENT TEST

INTRODUCTION

• 1.This test method is equivalent to OECD test guideline (TG) 234 (2011). It is based on a decision from 1998 to develop new or update existing test methods for the screening and testing of potential endocrine disrupters. The Fish Sexual Development Test (FSDT) was identified as a promising test method covering a sensitive fish life stage responsive to both oestrogen and androgen-like chemicals. The test method went through an inter-laboratory validation exercise from 2006 to 2010, where Japanese medaka (Oryzias latipes), zebrafish (Danio rerio) and three spined stickleback (Gasterosteus aculeatus) were validated and fathead minnow (Pimephales promelas) was partially validated (41) (42) (43). This protocol includes Japanese medaka, the three-spined stickleback and zebrafish. The protocol is in principle an enhancement of OECD TG 210 Fish, Early Life Stage Toxicity Test (1), where the exposure is continued until the fish are sexually differentiated, i.e. about 60 days post-hatch (dph) for Japanese medaka, the three-spined stickleback and zebrafish (the exposure period can be shorter or longer for other species that are validated in the future), and endocrine-sensitive endpoints are added. The FSDT assesses early life-stage effects and potential adverse consequences of putative endocrine disrupting chemicals (e.g. oestrogens, androgens and steroidogenesis inhibitors) on sexual development. The combination of the two core endocrine endpoints, vitellogenin (VTG) concentration and phenotypic sex ratio enable the test to indicate the mode of action of the test chemical. Due to the population-relevant change in phenotypic sex ratio, the FSDT can be used for hazard and risk assessment. However, if the test is used for hazard or risk assessment, the stickleback should not be used because the validation data available so far showed that in this species the alterations of phenotypic sex ratio by the test chemicals were uncommon.

• 2.The protocol is based on fish exposed via water to chemicals during the sex labile period in which the fish is expected to be most sensitive to the effects of endocrine disrupting chemicals that interfere with sexual development. Two core endpoints are measured as indicators of endocrine-associated developmental aberrations, the VTG concentrations and sex ratios (proportions of sex) determined via gonad histology. Gonadal histopathology (evaluation and staging of oocytes and spermatogenetic cells) is optional. Additionally, the genetic sex is determined whenever possible (e.g. in Japanese medaka and the three spined stickleback). The presence of a genetic sex marker is a considerable advantage as it increases the power of the sex ratio statistics and enables the detection of individual phenotypic sex reversal. Other apical endpoints that should be measured include hatching rate, survival, length and body weight. The test method might be adaptable to other species than those mentioned above provided that the other species undergo a validation equal to the one accomplished for Japanese medaka, the three-spined stickleback and zebrafish, that the control fish are sexually differentiated at the end of the test, that VTG levels are sufficiently high to detect significant chemical-related variations, and that sensitivity of the test system is established using endocrine active reference chemicals ((anti)-oestrogens, (anti)-androgens, aromatase inhibitors etc). In addition, any validation report(s) referring to FSDT data using other species should be reviewed by the OECD, and the validation outcome should be considered as satisfactory.

Initial considerations and limitations

• 3.VTG is normally produced by the liver of female oviparous vertebrates in response to circulating endogenous oestrogen (2). 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. The VTG synthesis is very limited, though detectable, in immature fish and adult male fish because they lack sufficient circulating oestrogen. However, the liver is capable of synthesising and secreting VTG in response to exogenous oestrogen stimulation (3) (4) (5).

• 4.The measurement of VTG serves for the detection of chemicals with oestrogenic, anti-oestrogenic, androgenic modes of action and chemicals that interfere with steroidogenesis as for example aromatase inhibitors. The detection of oestrogenic chemicals is possible via the measurement of VTG induction in male fish, and it has been abundantly documented in the scientific peer-reviewed literature. VTG induction has also been demonstrated following exposure to aromatisable androgens (6) (7). 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-oestradiol, causes a decrease in the VTG concentration, which is used to detect chemicals having aromatase inhibiting properties or steroidogenesis inhibitors more broadly (33). The biological relevance of the VTG response following oestrogenic/aromatase inhibition is established and has been broadly documented (8) (9). However, it is possible that production of VTG in females can also be affected by general toxicity and non-endocrine toxic modes of action.

• 5.Several measurement methods have been successfully developed and standardised for routine use to quantify VTG in blood, liver, whole body or head/tail homogenate samples collected from individual fish. This is the case for zebrafish, three-spined stickleback and Japanese medaka and also the partially validated species fathead minnow; species-specific Enzyme-Linked Immunosorbent Assay (ELISA) methods using immunochemistry for the quantification of VTG are available (5) (10) (11) (12) (13) (14) (15) (16). In Japanese medaka and zebrafish, there is a good correlation between VTG measured from blood plasma, liver and homogenate samples although homogenates tend to show slightly lower values than plasma (17) (18) (19). Appendix 5 provides the recommended procedures for sample collection for VTG analysis.

• 6.Change in the phenotypic sex ratio (proportions of sex) is an endpoint reflecting sex reversal. In principle, oestrogens, anti-oestrogens, androgens, anti-androgens and steroidogenesis inhibiting chemicals can affect the sex ratio of developing fish (20). It has been shown that this sex reversal is partly reversible in zebrafish (21) following oestrogen-like chemical exposure, whereas sex reversal following androgen-like chemical exposure is permanent (30). The sex is defined as female, male, intersex (both oocytes and spermatogenetic cells in one gonad) or undifferentiated, determined in individual fish via histological examination of the gonads. Guidance is given in Appendix 7 and in the OECD Guidance Document on the Diagnosis of Endocrine-Related Histopathology of Fish Gonads (22).

• 7.Genetic sex is examined via genetic markers when they exist in a given fish species. In Japanese medaka the female XX or male XY genes can be detected by Polymerase Chain-Reaction (PCR), or the Y-linked DM domain gene (DMY) can be analysed (DMY negative or positive) as described in (23) (24). In three-spined stickleback, there is an equivalent PCR method for genetic sex determination described in Appendix 10. Where the genetic sex can be individually linked to the phenotypic sex, the power of the test is improved and therefore genetic sex should be determined in species with documented genetic sex markers.

• 8.The two core endocrine endpoints, VTG and sex ratio, can in combination demonstrate the endocrine mode of action (MOA) of the chemical (Table 1). The sex ratio is a population relevant biomarker (25) (26) and for some well defined modes of action, the FSDT results may be used for hazard and risk assessment purposes when deemed appropriate by the regulatory agency. These modes of action are at present oestrogens, androgens and steroidogenesis inhibitors.

Table 1

Reaction of the endocrine endpoints to different modes of action of chemicals

↑ = increasing, ↓ = decreasing, — = not investigated

MOA	VTG ♂	VTG ♀	Sex ratio	References
Weak oestrogen agonist	↑	↑	↑♀ or ↑Undiff	(27) (40)
Strong oestrogen agonist	↑	↑	↑♀ or ↑Undiff, No ♂	(28) (40)
Oestrogen antagonist	—	—	↓♀, ↑Undiff.	(29)
Androgen agonist	↓ or —	↓ or —	↑♂, No ♀	(28) (30)
Androgen antagonist	—	—	↑♀ ↑Intersex	(31)
Aromatase inhibitor	↓	↓	↓♀	(33)

• 9.The FSDT does not cover the reproductive life stage of the fish and therefore chemicals that are suspected to affect reproduction at lower concentrations than sexual development should be examined in a test that covers reproduction.

• 10.Definitions for the purpose of this Test Method are given in Appendix 1.

• 11.The in vivo FSDT is intended to detect chemicals with androgenic and oestrogenic properties as well as anti-androgenic, anti-oestrogenic and steroidogenesis inhibiting properties. The FSDT validation phases (1 and 2) did cover oestrogenic, androgenic and steroidogenesis inhibiting chemicals. The effects in the FSDT of oestrogen- and androgen antagonists can be seen in Table 1 but these MOA are less documented at present time.

PRINCIPLE OF THE TEST

• 12.In the test, fish are exposed, from newly fertilised egg until the completion of sexual differentiation, to at least three concentrations of the test chemical dissolved in water. The test conditions should be flow-through unless not possible due to the availability or nature (e.g. limited solubility) of the test chemical. The test starts with the placing of newly fertilised eggs (before cleavage of the blastodisc) in the test chambers. The loading of the chambers is described for each species in paragraph 27. For the validated fish species, Japanese medaka, the three-spined stickleback and zebrafish, the test is terminated at 60 dph. At test termination, all fish are euthanised humanely. A biological sample (blood plasma, liver or head/tail homogenate) is collected for VTG analysis from each fish and the remaining part is fixed for histological evaluation of the gonads to determine the phenotypic sex; optionally, histopathology (e.g. staging of gonads, severity of intersex) can be performed. A biological sample (the anal- or the dorsal fin) for the determination of the genetic sex is taken in species possessing appropriate markers (Appendices 9 and 10).

• 13.An overview of relevant test conditions specific for validated species: Japanese medaka, the three-spined stickleback and zebrafish is provided in Appendix 2.

INFORMATION ON THE TEST CHEMICAL

• 14.Results from an acute toxicity test or other short-term toxicity assay [e.g. test method C.14 (34) and OECD TG 210 (1)], preferably performed with the species chosen for this test, should be available. This implies that the water solubility and the vapour pressure of the test chemical are known and a reliable analytical method for the quantification of the chemical in the test chambers, with known and reported accuracy and limit of detection, is available.

• 15.Other useful information includes the structural formula, purity of the chemical, stability in water and light, pKa, Pow and results of a test for ready biodegradability (Test Method C.4) (35).

Test acceptance criteria

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

• — The dissolved oxygen concentration should be at least 60 per cent of the air saturation value (ASV) throughout the test;

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

• — A validated method for analysis of the exposure chemical with a detection limit well below the lowest nominal concentration should be available and evidence should be gathered to demonstrate that the concentrations of the test chemical in solution have been satisfactorily maintained within ± 20 % of the mean measured values;

• — Overall survival of fertilised eggs in the controls and, where relevant, in the solvent controls, should be greater than or equal to the limits defined in Appendix 2;

• — Acceptance criteria related to growth and proportions of sex at termination of the test are based on data from the control groups (pooled solvent and water control unless they are significantly different, then solvent only): Japanese medaka Zebrafish Three-spined stickleback Growth Fish wet weight, blotted dry

  150 mg 75 mg 120 mg Length (standard length) 20 mm 14 mm 20 mm Sex ratio (% males or females) 30-70 % 30-70 % 30-70 %

• — When a solvent is used it should have no statistical significant effect on survival and should not produce any endocrine disrupting effects or other adverse effects on the early-life stages as revealed by a solvent control.

• If a deviation from the test acceptance criteria is observed, the consequences should be considered in relation to the reliability of the test data and these considerations should be included in the reporting.

DESCRIPTION OF THE TEST METHOD

Test chambers

• 17.Any glass, stainless steel or other chemically inert chambers can be used. The dimensions of the chambers should be large enough to allow compliance with loading rate criteria given below. It is desirable that test chambers be randomly positioned in the test area. A randomised block design with each concentration being present in each block is preferable to a completely randomised design. The test chambers should be shielded from unwanted disturbance.

Selection of species

• 18.Recommended fish species are given in Appendix 2. The procedures for inclusion of new species are given in paragraph 2.

Holding of parental fish

• 19.Details on holding the parental fish under satisfactory conditions may be found in OECD TG 210(1). Parental fish should be fed once or twice a day with appropriate food.

Handling of embryos and larvae

• 20.Initially, embryos and larvae may be exposed within a main chamber in smaller glass or stainless steel chambers, fitted with mesh sides or ends to permit a flow of test chemical through the chamber. Non-turbulent flow through these small chambers may be induced by suspending them from an arm arranged to move the chamber up and down but always keeping the organisms submerged.

• 21.Where egg containers, grids or meshes have been used to hold eggs within the main test chamber, these restraints should be removed after the larvae hatch, except that meshes should be retained to prevent the escape of the fish. If there is a need to transfer the larvae, they should not be exposed to the air and nets should not be used to release fish from egg containers. The timing of this transfer varies with the species and transfer may not always be necessary.

Water

• 22.Any water in which the test species shows control survival at least as good as in water described in Appendix 3 is suitable as test water. It should be of constant quality during the period of the test. In order to ensure that the dilution water will not unduly influence the test result (for example by reacting with the test chemical) or adversely affect the performance of the brood stock, samples should be taken at intervals for analysis. Total organic carbon, conductivity, pH and suspended solids should be measured, for example every three months where dilution water is known to be relatively constant in quality. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca²⁺, Mg²⁺, Na⁺, K⁺, Cl^–, SO4 ^2–) and pesticides should be done, if water quality is questionable. Details about chemical analysis and water collection can be found in paragraph 34.

Test solutions

• 23.Flow-through system should be used if practically possible. For flow-through tests, a system that continually dispenses and dilutes a stock solution of the test chemical (e.g. metering pump, proportional diluter, and saturator system) is necessary to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals during the test and should not vary by more than 10 % throughout the test. A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (1). Care should be taken to avoid the use of plastic tubing or other materials, some of which may contain biologically active chemicals or may adsorb the test chemical.

• 24.The stock solution should preferably be prepared without the use of solvents by simply mixing or agitating the test chemical in the dilution water by using mechanical means (e.g. stirring or ultrasonication). If the test chemical is difficult to dissolve in water, procedures described in the OECD Guidance Document on aquatic toxicity testing of difficult substances and mixtures should be followed (36). The use of solvents should be avoided but may be necessary in some cases in order to produce a suitably concentrated stock solution. Examples of suitable solvents are given in (36).

• 25.Semi-static test conditions should be avoided unless justification is provided on compelling reasons associated with the test chemical (e.g. stability, limited availability, high cost or hazard). For the semi-static technique, two different renewal procedures may be followed. Either new test solutions are prepared in clean chambers and surviving eggs and larvae gently transferred into the new chambers, or the test organisms are retained in the test chambers whilst a proportion (at least two thirds) of the test water is changed daily.

PROCEDURE

Conditions of Exposure

Collection of eggs and duration

• 26.To avoid genetic bias, eggs are collected from a minimum of three breeding pairs or groups, mixed and randomly selected to initiate the test. For the three-spined stickleback, see the description of artificial fertilisation in Appendix 11. The test should start as soon as possible after the eggs have been fertilised, the embryos preferably being immersed in the test solutions before cleavage of the blastodisc commences, or as close as possible after this stage and no later than 12 h post fertilisation. The test should continue until sexual differentiation in the control group is completed (60 dph for Japanese medaka, the three-spined stickleback and zebrafish).

Loading

• 27.The number of fertilised eggs at the start of the test should be at least 120 per concentration divided between a minimum of 4 replicates (square root allocation to control is accepted). The eggs should be randomly distributed (by using statistical tables for randomisation) among treatments. The loading rate (for definition, see Appendix 1) should be low enough in order that a dissolved oxygen concentration of at least 60 % of the ASV can be maintained without direct aeration of the chambers. For flow-through tests, a loading rate not exceeding 0,5 g/l per 24 hours, and not exceeding 5 g/l of solution at any time is recommended. No later than 28 days post fertilisation the number of fish per replicate should be redistributed, so that each replicate contains as equal a number of fish as possible. If exposure related mortality occurs, the number of replicates should be reduced appropriately so that fish density between treatment levels is kept as equal as possible.

Light and temperature

• 28.The photoperiod and water temperature should be appropriate for the test species (see Appendix 2 for experimental conditions for the FSDT).

Feeding

• 29.Food and feeding are critical, and it is essential that the correct food for each stage is supplied at appropriate time intervals and at a level sufficient to support normal growth. Feeding should be ad libitum whilst minimising the surplus. To obtain a sufficient growth rate, fish should be fed at least twice daily (accepting once daily on weekends), separated by at least three hours between each feed. Surplus food and faeces should be removed, as necessary, to avoid accumulation of waste. As experience is gained, food and feeding regimes are continuously being refined to improve survival and optimise growth. Effort should therefore be made to confirm the proposed regime with acknowledged experts. Feeding should be withheld 24 hours before ending the test. Examples of appropriate food items are listed in Appendix 2 (see also the OECD Fish Testing Framework (39).

Test concentrations

• 30.Test chemicals should be spaced as described in Appendix 4. A minimum of three test concentrations in at least four replicates should be used. The curve relating LC50 to period of exposure in the acute studies available should be considered when selecting the range of test concentrations. Five test concentrations are recommended if the data are to be used for risk assessment.

• 31.Concentrations of the chemical higher than 10 % of the acute adult LC50 or 10 mg/l, whichever is the lower, need not be tested. The maximum test concentration should be 10 % of the LC50 on the larval/juvenile life-stage.

Controls

• 32.A dilution water control (≥ 4 replicates) and, if relevant, a solvent control (≥ 4 replicates) should be run in addition to the test concentrations. Only solvents that have been investigated not to have any statistical significant influence on the test endpoints should be used in the test.

• 33.Where a solvent is used, its final concentration should not be greater than 0,1 ml/l (36) and it should be the same concentration in all test chambers, except the dilution water control. However, every effort should be made to avoid the use of such solvent or keep solvent's concentrations to a minimum.

Frequency of Analytical Determinations and Measurements

• 34.Chemical analysis of the test chemical concentration should be performed before initiation of the test to check compliance with the acceptance criteria. All replicates should be analysed individually at the beginning and termination of the test. One replicate per test concentration should be analysed at least once per week during the test, changing systematically between replicates (1,2,3,4,1,2…). If samples are stored to be analysed at a later time, the storage method of the samples should be previously validated. Samples should be filtered (e.g. using a 0,45 μm pore size) or centrifuged to ensure that the determinations are made on the chemical in true solution.

• 35.During the test, dissolved oxygen, pH, total hardness, conductivity, salinity (if relevant), and temperature should be measured in all test chambers. As a minimum dissolved oxygen, salinity (if relevant), and temperature should be measured weekly, and pH, conductivity and hardness at the beginning and at the end of the test. Temperature should preferably be monitored continuously in at least one test chamber.

• 36.Results should be based on measured concentrations. However, if the 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.

Observations and measurements

Stage of embryonic development

• 37.The exposure should begin as soon as possible after fertilisation and before cleavage of the blastodisc commences and no later than 12 h post fertilisation to ensure exposure during early embryonic development.

Hatching and survival

• 38.Observations on hatching and survival should be made at least once daily and numbers recorded. Dead embryos, larvae and juvenile fish should be removed as soon as observed since they can decompose rapidly and may be broken up by the actions of the other fish. Extreme care should be taken when removing dead individuals not to knock or physically damage adjacent eggs/larvae, these being extremely delicate and sensitive. Criteria for death vary according to life stage:

• — for eggs: particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance;

• — for larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of central nervous system and/or lack of reaction to mechanical stimulus.

Abnormal appearance

• 39.The number of larvae or fish showing abnormality of body form should be recorded, and the appearance and the nature of the abnormality described. It should be noted that abnormal embryos and larvae occur naturally and can be of the order of several per cent in the control(s) in some species. Abnormal animals should only be removed from the test chambers on death. However, in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, if abnormalities result in pain, suffering and distress or lasting harm, and death can be reliably predicted, animals should be anaesthetised and euthanised according to the description in paragraph 44 and treated as mortality for data analysis..

Abnormal behaviour

• 40.Abnormalities, e.g. hyperventilation, uncoordinated swimming, atypical quiescence and atypical feeding behaviour should be recorded at appearance.

Weight

• 41.At the end of the test all surviving fish should be euthanised (anaesthetised if blood samples should be taken), and individual wet weight (blotted dry) should be measured.

Length

• 42.At the end of the test, individual lengths (standard length) should be measured.

• 43.These observations will result in some or all of the following data being available for reporting:

• — cumulative mortality;

• — numbers of healthy fish at end of test;

• — time to start of hatching and end of hatching;

• — length and weight of surviving animals;

• — numbers of deformed larvae;

• — numbers of fish exhibiting abnormal behaviour.

Sampling of fish

• 44.Fish sampling is performed at termination of the test. Sampled fish should be euthanised with e.g. MS-222 (100-500 mg per l buffered with 200 mg NaHCO3 per l) or FA-100 (4-allyl-2-methoxyphenol: eugenol) and individually measured and weighed as wet weight (blotted dry) or anaesthetised if a blood sample should be taken (see paragraph 49).

Sampling for VTG analysis and sex determination via histological evaluation

• 45.All fish should be sampled and prepared for analysis of sex and VTG. All fish should be analysed histologically to determine sex. For the VTG measurements, a sub-sampling of at least 16 fish from each replicate is accepted. More fish should be analysed for VTG if the results of the sub-sampling turn out to be unclear.

• 46.The sampling procedure for VTG and sex determination is dependent on the VTG analysis method:

Head/tail homogenate method for VTG analysis

• 47.The fish is euthanised. Head and tail of each fish are separated from the body of the fish by cuts made right behind the pectoral fins, and right behind the dorsal fin, using a scalpel (See Figure 1). The head and tail part from each fish are pooled, weighed and individually numbered, frozen in liquid nitrogen and stored at – 70° or less for VTG analysis. The body part of the fish is numbered and fixed in an appropriate fixative for histological evaluation (22). By use of this method VTG and histopathology are evaluated on each individual and a possible change in the VTG level can thus be related to the phenotypic sex of the fish or genetic sex (Japanese medaka and the three-spined stickleback) of the fish. For further information see guidance for homogenisation (Appendix 5) and guidance for VTG quantification (Appendix 6).

Liver homogenate method for VTG analysis

• 48.The fish is euthanised. The liver is dissected out and stored at – 70 °C or below. Recommended procedures for liver excision and pre-treatment are available in OECD TG 229 (37) or Chapter C.37 of this Annex (38). Livers are then individually homogenised as described in OECD TG 229 or Chapter C.37 of this Annex. The supernatant collected is used for measuring VTG with a homologous ELISA technique (see Appendix 6 for an example of quantification in zebrafish or OECD TG 229 (37) for Japanese medaka). Following this approach, it is also possible to have individual fish data on both VTG and gonad histology.

Blood plasma method for VTG analysis

• 49.Blood is collected from the anaesthetised fish by cardiac puncture, caudal vein or tail cutting, and centrifuged at 4 °C for plasma collection. The plasma is stored at – 70 °C or below until use. The whole fish is euthanised and fixed for histology. Both plasma samples and fish are numbered individually to relate VTG levels to the sex of the fish.

• Figure 1

How to cut a fish for measurement of VTG in head/tail homogenate and histological evaluation of the mid section Image omitted; see official source

Genetic sex determination

• 50.A biological sample for the determination of the genetic sex is taken from individual fish in species possessing appropriate markers. For Japanese medaka, the anal fin or dorsal fin is collected. A detailed description is given in Appendix 9 including tissue sampling and sex determination by a PCR-method. Equally, for the three spined stickleback, a description of tissue sampling and a sex determining PCR-method is given in Appendix 10.

VTG measurement

• 51.The measurement of VTG should be based upon a quantitative and analytically validated method. Information should be available upon the intra-assay and inter-assay variability of the method used in a given laboratory. The source of inter- and intra-laboratory variability is (most likely) based on the different developing stages of the fish population. Considering the variability of VTG measurement, NOECs based on this endpoint alone should be treated with great care. Different methods are available to assess VTG production in the fish species considered in this assay. A measurement technique that is both relatively sensitive and specific is the determination of protein concentrations via enzyme-linked immunosorbent assay (ELISA). Homologous antibodies (raised against VTG of the same species) and most important homologous standards should be used.

Sex determination

• 52.Dependent on the VTG sampling procedure, whole fish or the remaining mid-section of each fish is placed in a pre-labelled processing cassette and fixed in an appropriate fixative for histological determination of sex (optionally also for evaluation of gonadal staging). Guidance on fixation and embedding is provided in Appendix 7 as well as in the OECD Guidance Document on the Diagnosis of Endocrine-Related Histopathology of Fish Gonads (22). After processing, the fish are embedded in paraffin blocks. The individuals should be placed longitudinally in the paraffin block. At least six longitudinal sections (3-5 μm in thickness) in a frontal plane including gonadal tissue from both gonads are taken from each individual. The interval between these sections should be approximately 50 μm for males and 250 μm for females. However, since each block will often contain males and females (if more than one individual are embedded in each block), the interval between sections from these blocks should be approximately 50 μm until at least six sections of the gonads from each male are obtained. Thereafter, the interval between sections can be increased to approximately 250 μm for the females. Sections are stained with haematoxylin and eosin and examined by light-microscopy with focus on sex (male, female, intersex or undifferentiated). Intersex is defined as presence of more than one oocyte in testis per six sections analysed or spermatogenic cells (yes/no) in ovaries. Histopathology and staging of ovaries and testis is optional but if investigated, the results should be statistically analyzed and reported. It should be noted that some fish species naturally lack a fully developed pair of gonads and only one gonad may be present (e.g. Japanese medaka and occasionally zebrafish). All such observations should be recorded.

• 53.Genetic sex determination in individual Japanese medaka is based on the presence or absence of the medaka male-sex determining gene, DMY, which is located on the Y chromosome. The genotypic sex of medaka can be identified by sequencing the DMY gene from DNA extracted from for instance a piece of anal fin or dorsal fin. The presence of DMY indicates a XY (male) individual regardless of phenotype, while the absence of DMY indicates a XX (female) individual regardless of phenotype (23). Guidance for tissue preparation and PCR method is given in Appendix 9. The genetic sex determination in individual three-spined stickleback is also performed via a PCR method, described in Appendix 10.

• 54.The occurrence of intersex (for definition, see Appendix 1) should be reported.

Secondary sexual characteristics

• 55.Secondary sexual characteristics are under endocrine control in species like the Japanese medaka; therefore observations of physical appearance of the fish should if possible be made at the end of the exposure. In the Japanese medaka, the papillary formation on the posterior part of the anal fin in females is androgen sensitive. Chapter C.37 of this Annex (38) provides relevant photographs of male secondary sex characteristics and androgenised females.

DATA AND REPORTING

Treatment of results

• 56.It is important that the strongest valid statistical test determine the endpoint. The replicate is the experimental unit but intra-replicate variability should be included in the statistical testing. A decision flow-chart is available in Appendix 8 to help with the most appropriate statistical test to use based on the characteristic of the data obtained from the test. Statistical significance level is 0,05 for all endpoints included.

Proportions of sex and genetic sex

• 57.The proportions of sex should be analysed for significant effect (NOEC/LOEC approach) of exposure by Jonckheere-Terpstra (Trend test) if a monotone dose-response exists. If non-monotonicity is found then a pair wise test should be applied: Use Dunnett's test if normality and homogenous variance can be obtained. Use Tamhane-Dunnett if heterogeneous variance is present. Otherwise use exact Mann-Whitney test with Bonferroni-Holm adjustment. A flow chart describing the statistics of the proportions of sex is placed in Appendix 8. The proportions of sex should be presented in tables as concentration proportions ± SD of males, females, intersex and undifferentiated. Statistical significance should be highlighted. Examples are presented in the FSDT Phase 2 validation report (42). Genetic sex should be reported as percentage of phenotypic sex reversal of males, females, intersex and undifferentiated.

VTG concentrations

• 58.VTG concentrations should be analysed for significant effect (NOEC/LOEC approach) of exposure. The Dunnett test is preferable to the t-test with Bonferroni correction. Where a Bonferroni correction is used, the Bonferroni-Holm correction is preferable. Allowance should be made for log-transformation of VTG to achieve normality and variance homogeneity. Next, if the concentration-response is consistent with monotonicity, then the JonckheereTerpstra test is preferable to any of the above. If t-tests or Dunnett's test is used, there is no need for a ANOVA significance F-test in order to proceed. For details see the flow chart in Appendix 8. Results should be reported in tables as concentration means ± SD for males, females, intersex and undifferentiated separately. Statistical significance for phenotypic females and phenotypic males should be highlighted. Examples are presented in the FSDT Phase 2 validation report (42).

Test chemical actual concentrations

• 59.The actual chamber concentrations of the test chemical should be analysed in frequencies described in paragraph 34. Results should be reported in tables as mean concentration ± SD on replicate basis as well as on concentration basis with information on number of samples and with outliers from the mean treatment concentration ± 20 % highlighted. Examples can be found in the FSDT Phase 2 validation report (42).

Interpretation of results

• 60.The test results should be interpreted with caution where measured test chemical concentrations in test solutions occur at levels near the detection limit of the analytical method.

Test report

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

Test chemical

• — Relevant physical-chemical properties; chemical identification data including purity and analytical method for quantification of the test chemical.

Test conditions

• — Test procedure used (e.g. flow-through semi-static/renewal); test design including test concentrations, method of preparation of stock solutions (in an Annex), frequency of renewal (the solubilising agent and its concentration should be given, when used);

• — The nominal test concentrations, the means of the measured values and their standard deviations in the test chambers and the method by which these were attained (the analytical method used should be presented in an Annex);Evidence that the measurements refer to the concentrations of the test chemical in true solution;

• — Water quality within test chambers: pH, hardness, temperature and dissolved oxygen concentration;

• — Detailed information on feeding (e.g. type of food(s), source, amount given and frequency and analyses for contaminants (e.g. PCBs, PAHs and organochlorine pesticides) if relevant.

Results

• — Evidence that controls met the validity criteria: data on hatching rate should be presented in tables as percentage per replicate and per concentration. Outliers from the acceptance criteria (in controls) should be highlighted. Survival should be presented as percentage per replicate and per concentration. Outliers from the validity criteria (in controls) should be highlighted;

• — Clear indication of the results obtained on the different endpoints observed: embryo survival and hatching success; external abnormalities; length and weight; VTG measurements (ng/g homogenate, ng/ml plasma or ng/mg liver); gonadal histology, sex ratio, genetic sex data; incidence of any unusual reactions by the fish and any visible effects produced by the test chemical.

• 62.The results should be presented as mean values ± standard deviation (SD) or standard error (SE). Statistics should be reported as a minimum as NOEC and LOEC and confidence intervals. The statistical flow chart (Appendix 8) should be followed.

LITERATURE

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

• (2)Jobling, S., D. Sheahan, J.A. Osborne, P. Matthiessen, and J.P. Sumpter, 1996, ‘Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals’, Environmental Toxicology and Chemistry 15, pp. 194-202.

• (3)Sumpter, J.P. and S. Jobling, 1995, ‘Vitellogenesis As A Biomarker for Estrogenic Contamination of the Aquatic Environment’, Environmental Health Perspectives 103, pp. 173-178.

• (4)Tyler, C.R., R.van Aerle, T.H. Hutchinson, S. Maddix, and H. Trip (1999), ‘An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin’, Environmental Toxicology and Chemistry 18, pp. 337-347.

• (5)Holbech, H., L. Andersen, G.I. Petersen, B. Korsgaard, K.L. Pedersen, and P. Bjerregaard (2001a), ‘Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio)’, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 130, pp. 119-131.

• (6)Andersen, L., P. Bjerregaard, and B. Korsgaard (2003), ‘Vitellogenin induction and brain aromatase activity in adult male and female zebrafish exposed to endocrine disrupters’, Fish Physiology and Biochemistry 28, pp. 319-321.

• (7)Orn, S., H. Holbech, T.H. Madsen, L. Norrgren, and G.I. Petersen (2003), ‘Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone’, Aquatic Toxicology 65, pp. 397-411.

• (8)Panter, G.H., T.H. Hutchinson, R. Lange, C.M. Lye, J.P. Sumpter, M. Zerulla, and C.R. Tyler (2002), ‘Utility of a juvenile fathead minnow screening assay for detecting (anti-)estrogenic substances’, Environmental Toxicology and Chemistry 21, pp. 319-326.

• (9)Sun, L.W., J.M. Zha, P.A. Spear, and Z.J. Wang (2007), ‘Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults’, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, pp. 533-541.

• (10)Parks, L.G., A.O. Cheek, N.D. Denslow, S.A. Heppell, J.A. McLachlan, G.A. LeBlanc, and C.V.Sullivan (1999), ‘Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds’, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 123, pp. 113-125.

• (11)Brion, F., B.M. Nilsen, J.K. Eidem, A. Goksoyr, and J.M. Porcher (2002), ‘Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio)’, Environmental Toxicology and Chemistry 21, pp. 1699-1708.

• (12)Nishi, K., M. Chikae, Y. Hatano, H. Mizukami, M. Yamashita, R. Sakakibara, and E. Tamiya (2002), ‘Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin’, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 132, pp. 161-169.

• (13)Hahlbeck, E., I. Katsiadaki, I. Mayer, M. Adolfsson-Erici, J. James, and B.E. Bengtsson (2004), ‘The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruption — II — kidney hypertrophy, vitellogenin and spiggin induction’, Aquatic Toxicology 70, pp. 311-326.

• (14)Tatarazako, N., M. Koshio, H. Hori, M. Morita, and T. Iguchi (2004), ‘Validation of an enzyme-linked immunosorbent assay method for vitellogenin in the medaka’, Journal of Health Science 50, pp. 301-308.

• (15)Eidem, J.K., H. Kleivdal, K. Kroll, N. Denslow, R. van Aerle, C. Tyler, G. Panter, T. Hutchinson, and A. Goksoyr (2006), ‘Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin. Aquatic Toxicology’, 78, pp. 202-206.

• (16)Jensen, K.M. and G.T. Ankley (2006), ‘Evaluation of a commercial kit for measuring vitellogenin in the fathead minnow (Pimephales promelas)’, Ecotoxicology and Environmental Safety 64, pp. 101-105.

• (17)Holbech, H., Petersen, G. I., Norman, A., Örn, S, Norrgren, L., and Bjerregaard, P (2001b), ‘Suitability of zebrafish as test organism for detection of endocrine disrupting chemicals. Comparison of vitellogenin in plasma and whole body homogenate from zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss)’, Nordic Council of Ministers, TemaNord 2001:597, pp. 48-51.

• (18)Nilsen, B.M., K. Berg, J.K. Eidem, S.I. Kristiansen, F. Brion, J.M. Porcher, and A. Goksoyr (2004), ‘Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening’, Analytical and Bioanalytical Chemistry 378, pp. 621-633.

• (19)Orn, S., S. Yamani, and L. Norrgren (2006), ‘Comparison of vitellogenin induction, sex ratio, and gonad morphology between zebrafish and Japanese medaka after exposure to 17 alpha-ethinylestradiol and 17 beta-trenbolone’, Archives of Environmental Contamination and Toxicology 51, pp. 237-243.

• (20)Scholz, S. and N. Kluver (2009), ‘Effects of Endocrine Disrupters on Sexual, Gonadal Development in Fish, Sexual Development 3’, pp. 136-151.

• (21)Fenske, M., G. Maack, C. Schafers, and H. Segner (2005), ‘An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio’, Environmental Toxicology and Chemistry 24, pp. 1088-1098.

• (22)OECD (2010), Guidance Document on the Diagnosis of Endocrine-related Histopathology in Fish Gonads, Series on Testing and Assessment No. 123, ENV/JM/MONO(2010)14, OECD, Paris.

• (23)Kobayashi, T., M. Matsuda, H. Kajiura-Kobayashi, A. Suzuki, N. Saito, M. Nakamoto, N. Shibata, and Y. Nagahama (2004), ‘Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes’, Developmental Dynamics 231, pp. 518-526.

• (24)Shinomiya, A., H. Otake, K. Togashi, S. Hamaguchi, and M. Sakaizumi (2004), ‘Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations’, Zoological Science 21, pp. 613-619.

• (25)Kidd, K.A., P.J. Blanchfield, K.H. Mills, V.P. Palace, R.E. Evans, J.M. Lazorchak, and R.W. Flick (2007), ‘Collapse of a fish population after exposure to a synthetic estrogen’, Proceedings of the National Academy of Sciences of the United States of America 104, pp. 8897-8901.

• (26)Palace,V.P., R.E. Evans, K.G. Wautier, K.H. Mills, P.J. Blanchfield, B.J. Park, C.L. Baron, and K.A. Kidd (2009), ‘Interspecies differences in biochemical, histopathological, and population responses in four wild fish species exposed to ethynylestradiol added to a whole lake’, Canadian Journal of Fisheries and Aquatic Sciences 66, pp. 1920-1935.

• (27)Panter, G.H., T.H. Hutchinson, K.S. Hurd, J. Bamforth, R.D. Stanley, S. Duffell, A. Hargreaves, S. Gimeno, and C.R. Tyler (2006), ‘Development of chronic tests for endocrine active chemicals — Part 1. An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas)’, Aquatic Toxicology 77, pp. 279-290.

• (28)Holbech, H., K. Kinnberg, G.I. Petersen, P. Jackson, K. Hylland, L. Norrgren, and P. Bjerregaard (2006), ‘Detection of endocrine disrupters: Evaluation of a Fish Sexual Development Test (FSDT)’, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 144, pp. 57-66.

• (29)Andersen, L., K. Kinnberg, H. Holbech, B. Korsgaard, and P. Bjerregaard (2004), ‘Evaluation of a 40 day assay for testing endocrine disrupters: Effects of an anti-estrogen and an aromatase inhibitor on sex ratio and vitellogenin concentrations in juvenile zebrafish (Danio rerio)’, Fish Physiology and Biochemistry 30, pp. 257-266.

• (30)Morthorst, J.E., H. Holbech, and P. Bjerregaard (2010), ‘Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations’, Aquatic Toxicology 98, pp. 336-343.

• (31)Kiparissis,Y., T.L. Metcalfe, G.C. Balch, and C.D. Metcalf (2003), ‘Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes)’, Aquatic Toxicology 63, pp. 391-403.

• (32)Panter, G.H., T.H. Hutchinson, K.S. Hurd, A. Sherren, R.D. Stanley, and C.R. Tyler (2004), ‘Successful detection of (anti-) androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development’, Aquatic Toxicology 70, pp. 11-21.

• (33)Kinnberg, K., H. Holbech, G.I. Petersen, and P. Bjerregaard (2007), ‘Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio)’, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, pp. 165-170.

• (34)Chapter C.14 of this Annex, Fish Juvenile Growth Test.

• (35)Chapter C.4 of this Annex, Ready Biodegradability.

• (36)OECD (2000), Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, Series on Testing and Assessment No. 23, OECD, Paris.

• (37)OECD (2009), Fish Short Term Reproduction Assay, Test Guideline No. 229, Guidelines for the Testing of Chemicals, OECD, Paris.

• (38)Chapter C.37 of this Annex, 21-Day Fish Assay: A Short Term Screening for Oestrogenic and Androgenic Activity, and Aromatase Inhibition.

• (39)OECD (2012), Fish Toxicity Testing Framework, Series on Testing and Assessment No. 171, OECD, Paris