TG 229 is an OECD validated in vivo screening assay for fish reproduction 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). The short term reproduction assay was validated in the fathead minnow (Pimephales promelas) and this is the recommended species. The assay is run with three test chemical concentrations and the necessary controls, including a carrier control if necessary. For the fathead minnow, four replicate test vessels are used for each treatment level and control(s). During the conduct of the assay, the egg production is measured quantitatively daily in each test vessel. At termination of the 21-day exposure period, two biomarker endpoints are measured in males and females separately, as indicators of endocrine activity of the test chemical; these endpoints are vitellogenin and secondary sexual characteristics. Gonads of both sexes are also preserved and histopathology may be evaluated to assess the reproductive fitness of the test animals and to add to the weight of evidence of other endpoints.

TG 230 is an OECD validated in vivo screening assay for certain endocrine active substances 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). This assay covers the screening of oestrogenic and androgenic activity, and aromatase inhibition. The assay was validated on the fathead minnow (Pimephales promelas), the Japanese medaka (Oryzias latipes) and the zebrafish (Danio rerio); however zebrafish does not allow the detection of androgenic activity. 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.

TG 231 is an OECD validated assay intended to screen substances which may interfere with the normal functioning of the hypothalamo-pituitary-thyroid axis. The assay was validated with the species Xenopus laevis, which is recommended for use in the Guideline. The assay uses three test chemical concentrations and the necessary controls, including a carrier control if necessary. The assay starts with tadpoles at the development stage 51 on the Nieuwkoop and Faber scale and is extended for a duration of 21 days. Four replicate test vessels are used for each treatment level and control(s). After 7 days of exposure, a sub-set of tadpoles from each treatment level is sampled for the measurement of the length of the hind-limb. At termination of 21-day exposure period, developmental stage, snout-to-vent length and hind limb length are measured on all remaining tadpoles. A sub-set of tadpoles from each treatment level is fixed (whole-body or dissected) for histopathology of the thyroid gland.

TG 242 in an OECD validated test designed to assess potential effects of prolonged exposure to chemicals on reproduction and survival of parthenogenetic lineages of the freshwater mudsnail Potamopyrgus antipodarum. Adult female P. antipodarum are exposed to a concentration range of the test chemical. The test chemical is dispersed into the reconstituted dilution water, added to test beakers, and adult snails are subsequently introduced into the test beakers. When testing “difficult chemicals” (i.e. volatile, unstable, readily biodegradable and adsorbing chemicals) the test can be conducted under flow-through conditions as an alternative to the semi-static design with fixed renewal periods of the medium. P. antipodarum survival over the 28 days exposure period and reproduction at the end of the test after 28 days are examined. Reproduction is evaluated by counting the number of embryo in the brood pouch (without distinction of developmental stages) at the end of 28 days exposure.

TG 243 is an OECD validated test designed to assess effects of prolonged exposure to chemicals on the reproduction and survival of the hermaphrodite freshwater snail Lymnaea stagnalis (the Great Pond Snail). Reproducing adults of L. stagnalis are exposed to a concentration range of the test chemical and monitored for 28 days for their survival and reproduction (number of egg clutches). As additional information, the number of eggs per clutch may also be determined. Adult shell length increase may also be measured. The toxic effect of the test chemical on the cumulated number of clutches produced per individual-day.

TG 219 is an OECD validated test to assess the effects of prolonged exposure of chemicals to the sediment-dwelling larvae of the freshwater dipteran Chironomus sp. First instar chironomid larvae are exposed to at least five concentrations of the test chemical in sediment-water systems. The test starts by placing first instar larvae into the test beakers containing the sediment-water system and subsequently spiking the test substance into the water. Chironomid emergence and development rate is measured at the end of the test. The maximum exposure duration is 28 days for C. riparius, C. yoshimatsui, and 65 days for C. tentans. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate). The study report should include the development time and the total number of fully emerged midges (sex and number are recorded daily), the observation of any abnormal behaviour, the number of visible pupae that have failed to emerge and any egg masses deposition.

TG 211 in an OECD validated test that assesses the effect of chemicals on the reproductive output of Daphnia magna Straus. To this end, young female Daphnia are exposed to the test chemical added to water at a range of concentrations (at least five). For semi-static tests, at least 10 animals at each test concentration and for flow-through tests, 40 animals divided into four groups of 10 animals at each test concentration are used. The test duration is 21 days. The total number of living offspring produced per parent animal and the number of living offspring produced per surviving parent animal at the end of the test are reported. The study report also includes: the daily counting of the offspring, the daily recording of the parent mortality, the weekly measurement of oxygen concentration, temperature, hardness and pH values and the determination of the concentrations of test substance. Optionally other effects can be reported, including the sex ratio of the offspring.

TG 210 is an OECD validated test intended to define the lethal and sub-lethal effects of chemicals on the early life stages of the species tested. The early-life stages of fish are exposed to five concentrations of the test substance dissolved in water, preferably under flow-through conditions, or where appropriate, semi-static conditions. The test starts with placing fertilised eggs (at least 80 per concentration level) in the test chambers (four at the minimum) and continues at least until all the control fishes are free-feeding. Lethal and sub-lethal effects are assessed and compared with control values to either determine the lowest observed effect concentration and the no observed effect concentration, or the effect concentration leading to x% change on organisms for the effect observed. The study report should include measurement of the concentrations of the test substance in water at regular intervals (five at least), the dissolved oxygen, the temperature, pH, total hardness and salinity, fish weight and length, observations of abnormal appearance, abnormal behaviour, hatching and survival, as well as the no-observed effect level or the effect concentration leading to x% change in the organisms for the effect observed.

TG 234 is an OECD validated assay that assesses early life-stage effects and potential adverse consequences of putative endocrine disrupting chemicals (e.g. oestrogens, androgens and steroidogenesis inhibitors) on fish sexual development. In the test, fish are exposed, from newly fertilized egg until the completion of sexual differentiation at about 60 days post hatch, to at least three concentrations of the test substance dissolved in water. In each treatment level and control(s) group(s), a minimum of four replicates is recommended. At termination of the test, two core endpoints are measured in each fish: vitellogenin concentration from head and tail or from blood sampling, and proportion of males, females, intersex and undifferentiated fish through gonadal histology. In fish species possessing a genetic sex marker, the genetic sex is identified to determine sex reversal in individual fish. The combination of the two core endocrine endpoints, vitellogenin concentration and phenotypic (and possibly genotypic) sex ratio, enable the test to indicate the mode of action of the test chemical.

TG 241 is an OECD validated test with an amphibian species (African clawed frog (Xenopus laevis)) that considers growth and development from fertilization through the early juvenile period. It is an assay (typically 16 weeks) that assesses early development, metamorphosis, survival, growth, and partial reproductive maturation. It also enables measurement of a suite of other endpoints that allows for diagnostic evaluation of suspected endocrine disrupting chemicals (EDCs) or other types of developmental and reproductive toxicants. The LAGDA serves as a higher tier test with an amphibian for collecting more comprehensive concentration-response information on adverse effects suitable for use in hazard identification and characterization, and in ecological risk assessment. The general experimental design entails exposing X. laevis embryos at Nieuwkoop and Faber (NF) stage 8-10 (3) to a minimum of four different concentrations of test chemical and control(s) until 10 weeks after the median time to NF stage 62. There are four replicates in each test concentration with eight replicates for the control. Endpoints evaluated during the course of the exposure (at the interim sub-sample and final sample at completion of the test) include those indicative of generalized toxicity: mortality, abnormal behaviour, and growth determinations (length and weight), as well as endpoints designed to characterize specific endocrine toxicity modes of action targeting oestrogen, androgen or thyroid-mediated physiological processes.

TG 206 is an OECD validated test to determine the effects on the reproduction of a chemical administered to birds. Birds are fed a diet containing the test chemical in various concentrations for a duration of not less than 20 weeks. At least three dietary concentrations of the test chemical are required. The maximum recommended test concentration is 1000 ppm. Birds may be kept in pens as pairs (at least 12 pens per test group) or as groups of one male and two or three females (at least 8-12 pens per group). Birds are induced, by photoperiod manipulation, to lay eggs. Eggs are collected over a ten-week period, artificially incubated and hatched, and the young maintained for 14 days. Suitable facilities for rearing birds, preferably indoors, are necessary. Mortality of adults, egg production, cracked eggs, egg shell thickness (at least two eggs from each pen), viability, hatchability and effects on young birds are observed during the study.

TG 233 is an OECD validate test to assess the effects of prolonged exposure of chemicals to the life-cycle of the sediment-dwelling freshwater dipteran Chironomus sp. First instar chironomid larvae are exposed to five concentrations of the test chemical in sediment-water systems. The test chemical is spiked into the water or alternatively the sediment, and first instar larvae are subsequently introduced into test beakers in which the sediment and water concentrations have been stabilised. Chironomid emergence, time to emergence, and sex ratio of the fully emerged and alive midges are assessed. Emerged adults are transferred to breeding cages, to facilitate swarming, mating and oviposition. The number of egg ropes produced and their fertility are assessed. From these egg ropes, first instar larvae of the 2nd generation are obtained. These larvae are placed into freshly prepared test beakers (spiking procedure as for the 1st generation) to determine the viability of the 2nd generation through an assessment of their emergence, time to emergence and the sex ratio of the fully emerged and alive midges.

TG 240 is an OECD validated test which exposes fish over multiple generations to give data relevant to ecological hazard and risk assessment of chemicals, including suspected endocrine disrupting chemicals (EDCs). Exposure in the MEOGRT starts with spawning fish (P or F0 generation) and continues until hatching (until two weeks post fertilization, wpf) in the second (F2) generation. This Test Guideline measures several biological endpoints. Primary emphasis is given to potential adverse effects on population relevant parameters including survival, gross development, growth and reproduction (fecundity). Secondarily, in order to provide mechanistic information and provide linkage between results from other kinds of field and laboratory studies, where there is a posteriori evidence for a chemical having potential endocrine disrupter activity (e.g. androgenic or oestrogenic activity in other tests and assays) then other useful information is obtained by measuring vitellogenin (vtg) mRNA (or vitellogenin protein, VTG), phenotypic secondary sex characteristics (SSC) as related to genetic sex, and evaluating histopathology.

Specific guidance is provided for in vivo screens and tests using non-mammalian vertebrates and invertebrates for evaluating endocrine disrupting chemicals. The assays in this section have not been fully validated as OECD test guidelines, but have undergone partial validation (Androgenised Female Stickleback Screen (GD 148), Harpacticoid Copepod Development and Reproduction Test with Amphiascus (GD 201)); have been validated at the national level ( Fish Life Cycle Toxicity Test (FLCTT), Avian Two-Generation Toxicity Test in the Japanese Quail (ATGT)); are draft test guidelines in the OECD validation process (Short-Term Juvenile Hormone Activity Screening Assay using Daphnia magna, EASZY assay for detection of substances acting through estrogen receptors using zebrafish embryos, Juvenile Medaka Anti-Androgen Screening Assay, Xenopus Embryonic Thyroid Signalling Assay (XETA), Zebrafish Extended One-Generation Reproduction Test (ZEOGRT), RADAR: Rapid Androgen Disruption Adverse Outcome Reporter Assay); or are assays proposed as OECD test guidelines (Daphnia Multigeneration Test for Assessment of Endocrine-Active Chemicals (DMGT)) at the time of publication of this document.