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This Guideline is designed 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 substance 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. All data are analysed either by a regression model to estimate the concentration that would cause X% reduction in the relevant endpoint, or by using hypothesis testing to determine a No Observed Effect Concentration (NOEC).
This Test Guideline describes an 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.
This Test Guideline describes an acute immobilisation assay on chronomids and is designed to complement existing Test Guidelines for chironomid chronic toxicity assays (TG 218, 219 and 233). The test method is based on TG 202: Daphnia sp. Acute Immobilisation Test. First instar Chironomus sp. larvae are exposed to a range of concentrations of the test substance in water-only vessels for a period of 48 hours. C. riparius is the preferred species but C. dilutus or C. yoshimatsui may also be used for the test. At least 20 larvae, preferably divided into four groups of five larvae each, should be used for each test concentration and for controls. In the definitive test, at least five test concentrations should be used, with a dilution water control and solvent control (if appropriate). Immobilisation is recorded at 24 and 48 hours, and if data allow, the EC50 is calculated at 24 and 48 hours. A limit test with a single concentration may also be performed at 100 mg/L of test substance or up to the practical limit of solubility (whichever is lowest) in order to demonstrate that the EC50 is greater than this concentration.
The test method described in this Test Guidelineis inteneded to determine the acute or letal toxicity of chemicals on embryonic stages of fish (Danio rerio).
Newly fertilised zebrafish eggs are exposed to the test chemical for a period of 96 hrs. Every 24 hrs. Twenty embryos (one embryo per well) are exposed to the chemical tested at each concentration level. The test includes five increasing concentrations of the chemical tested and a control. Every 24 hours, four apical observations are recorded as indicators of lethality: (i) coagulation of fertilised eggs, (ii) lack of somite formation, (iii) lack of detachment of the tail-bud from the yolk sac, and (iv) lack of heartbeat. At the end of the exposure period, acute toxicity is determined based on a positive outcome in any of the four apical observations recorded, and the LC50 is calculated. The test report also includes a number of other important information elements related to the conduct of the test, in particular: the concentration of dissolved oxygen, pH, total hardness, temperature et conductivity of solutions, measured concentrations of the chemical tested, and whether the validity criteria of the test were met.
This Test Guideline (TG) describes a honey bee brood acute toxicity test under laboratory conditions. The method aims at the determination of the lethal dose (72-h LD50) following single exposure of larvae to a chemical.
On day 1 (D1) of the study, first instar synchronised larvae are taken from the comb of three colonies (thirty-six larvae in total per group) and individually placed into 48 well-plates where they are fed a standardized amount of artificial diet. On day 4 (D4) of the test, a single dose of the test chemical is administered to the larvae with the diet. Each group of thirty-six larvae is administered a given dose in a range of five increasing doses. Mortalities are recorded on D5, D6, and D7 of the test. The 72-hr LD50 is calculated for larvae (cumulative mortality at D7). The study report also includes a number of important elements, in particular regarding test conditions (e.g., temperature and humidity).
This Test Guideline is designed to assess the toxicity of chemicals on the growth of submerged aquatic plants Myriophyllum spicatum growing in a sediment-free test system.
In a modified Andrews’ medium Myriophyllum spicatum plant cultures are exposed to at least five different concentrations of the test chemical over a period of 14 days. A minimum of 10 replicates for the controls and 5 replicates for each test concentration should be used. The measured variables include growth of shoot length, of lateral branches and roots, development of fresh and dry weight, increase of whorls. Both average specific growth rate (r) and yield (y) are determined and then used to expressed ErCx and EyCx respectively (x can be eg. 10,20,50)
In addition, the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) may be statistically determined.
This Test Guideline is designed to assess the toxicity of chemicals on the growth of the rooted aquatic plants (Myriophyllum spicatum) growing in water-sediment system.
Shoot apices of healthy and non-flowering plant are potted in standardised, artificial sediment supplemented with additional nutrient and exposed to at least five concentrations of the chemical over a period of 14 days. A minimum of 6 replicates for the untreated control and 4 replicates for each test concentration should be used. The measured quantitative variables include growth of shoot length and development of fresh and dry weight, and the measured qualitative variables include presence or not of chlorosis and necrosis or growth deformities. Both average specific growth rate (r) and yield (y) are determined and then used to expressed ErCx and EyCx respectively (x can be e.g. 10, 20, 50.)
In addition, the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) may be statistically determined.
This Test Guideline describes the Medaka Extended One Generation Test (MEOGRT), 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.
The test guideline of the Larval Amphibian Growth and Development Assay (LAGDA) describes a toxicity 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.
The Potamopyrgus antopodarumon reproduction test is 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 (see paragraph 29). 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. The toxic effect of the test chemical on embryo numbers is expressed as ECX by fitting an appropriate regression model in order to estimate the concentration that would cause x % reduction in embryo numbers or alternatively as the No Observed Effect Concentration and Lowest Observed Effect Concentration (NOEC/LOEC) value (2).
This Test Guideline is 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 is expressed as ECx by fitting an appropriate regression model to the data in order to estimate the concentration that would cause x% reduction in the reproductive output. Alternatively, the toxic effect of the test chemical can be expressed as the No Observed Effect Concentration and Lowest Observed Effect Concentration (NOEC/LOEC) values. Both ECx and NOEC/LOEC can be determined from a single study.
This Test Guideline describes a method to assess effects of a test chemical on the phagocytotic activity of activated sludge containing protozoan organisms under defined conditions in the presence of different concentrations of the test chemical. The principle of biological sewage-treatment plants (STP) is to transform the organic matter of incoming waste-water in microbial biomass, which in turn is separated from the liquid yielding a purified effluent. The purpose of the test is to provide a means to record effects of test chemicals on ciliated protozoa in sewage treatment plants, which due to their grazing on bacteria considerably contribute to the functioning of STPs.
This Test Guideline describes a chronic oral toxicity test on adult worker honey bees under laboratory conditions over an exposure period of 10 days.
Young bees (max. 2 days old) are exposed to 50 % (w/v) aqueous sucrose solution containing the test chemical by continuous and ad libitum feeding over a period of 10 days. Mortality and behavioural abnormalities are observed and recorded daily during the 10 day test period. The chronic effects of the test chemical are evaluated by comparing the results of the test chemical treated group to those of the respective control group. The test is designed for the determination of the following endpoints LC50 (median Lethal Concentration) and the LDD50 (median Lethal Dietary Dose) values after 10 days of exposure, and NOEC (No Observed Effect Concentration) and NOEDD (No Observed Effect Dietary Dose).
This test guideline is a laboratory test method, designed to assess the acute contact toxicity of pesticides and other chemicals to adult worker bumblebees.
Adult worker bumblebees are exposed to the test chemical dissolved in an appropriate carrier, by direct application to the dorsal thorax (droplet). The test duration is at least 48 h. Mortality is recorded daily and compared with control values. Results are analysed in order to calculate the LD50 and NOED, if possible, at 24 h & 48 h and furthermore at 72 h & 96 h in case the study is prolonged.
This test guideline is a laboratory test method, designed to assess the acute oral toxicity of pesticides and other chemicals to adult worker bumblebees.
Adult worker bumblebees are exposed to 50 % (w/v) aqueous sucrose solution containing the test chemical. The test duration is at least 48 h. Mortality is recorded daily and compared with control values. Results are analysed in order to calculate the LD50 and NOED, if possible, at 24 h & 48 h and furthermore at 72 h & 96 h in case the study is prolonged.
The purpose of the test is to measure the capacity of a chemical to activate or inhibit the transcription of a genetic construct (THb/ZIP-GFP X. laevis eleutheroembryos) either by binding to the thyroid hormone receptor or by modifying the amount of thyroid hormone available for transcription. The test is based on the use of a transgenic frog (possessing the genetic construction THb/ZIP-GFP) of the species X. laevis. GFP is a fluorescent protein that can be used to affirm the presence of the substance and to follow its action. This test is based on the quantification of fluorescence, and thus of the GFP protein, throughout the eleutheroembryo exposed to a test chemical. Indeed, if the fluorescence is > 12% by including the highest concentration of the test, the test is considered positive. Organisms are exposed in chemically inert cell culture plates (6 wells). Each well contains ten organisms in solution. In a run, 20 organisms are exposed to each test concentration. Tadpoles are exposed to the test chemical at NF stage 45 for a duration of 72 hours. A positive result of the XETA test indicates in vivo that a substance induces damage to the receptor and metabolization of the thyroid hormone. A negative result indicates that, under test conditions, the test substance does not induce detectable damage to the thyroid and its functioning.
The RTgill-W1 cell line assay describes a 24-well plate format fish cell line acute toxicity test using the permanent cell line from rainbow trout (Oncorhynchus mykiss) gill, RTgill-W1. After 24 h of exposure to the test chemical, cell viability is assessed based on three fluorescent cell viability indicator dyes, measured on the same set of cells. Resazurin enters the cells in its non-fluorescent form and is converted to the fluorescent product, resorufin, by mitochondrial, microsomal or cytoplasmic oxidoreductases. A reduction in the fluorescence of resorufin indicates a decline in cellular metabolic activity, including disruption of mitochondrial membranes. The data are expressed as the percent cell viability of unexposed control values versus the test chemical concentration. The resulting concentration-response curves serve to determine the effective concentrations causing 50% loss in cell viability, i.e. the EC50 value.
The test is designed to (i) predict fish acute toxicity in product testing; (ii) range-finding and pre-screening before conducting a full fish acute or other fish-based toxicity test; (iii) generation of toxicity information to be used for hazard assessment in combination with other lines of evidences (e.g., Quantitative Structure Activity Relationships (QSAR), weight of evidence (WoE)) within Integrated Testing Strategy (ITS)/Integrated Approach to Testing and Assessment (IATA).
The EASZY assay is a mechanism-based in vivo screening assay designed to detect endocrine active chemicals acting as agonist through estrogen receptors (ERs), by inducing the expression of the green fluorescent protein (GFP) driven by the cyp19a1b promoter. The EASZY assay allows for the detection of estrogenic activity of chemicals on transgenic tg(cyp19a1b:GFP) Zebrafish embryos exposed for 96 hours during the embryonic stages of development. At the end of the experiment, the fluorescence of each newly hatched eleutheroembryo is measured using fluorescence microscope. Because the skull of early developmental stages of zebrafish is transparent, GFP is observed, imaged and quantified in vivo. The intensity of fluorescence is then quantified using image analysis software.
The Rapid Androgen Disruption Activity Reporter (RADAR) Test Guideline describes an aquatic assay that utilizes transgenic Oryzias latipes (O. latipes, Japanese medaka) eleutheroembryos at day post hatch zero, in a multi-well format to detect chemicals active on the androgen axis. The RADAR assay was designed as a screening tool to provide a short-term assay to measure the response of eleutheroembryos to chemicals potentially active on the androgen axis. Test organisms are exposed for 72 hours in six-well plates in the presence and absence of a co-treatment with 17-methyl testosterone. The assay comprises five concentrations of the test chemical. At the end of the exposure, fluorescence imaging is measured and transformed into a numerical format; the statistical analysis and data interpretation procdure enable to determine whether the test chemical is considered active or not active.
This Test Guideline describes a Rapid Estrogen ACTivity In Vivo (REACTIV) Assay in an aquatic assay that utilises transgenic Oryzias latipes (Japanese medaka) eleutheroembryos at day post hatch zero, in a multi-well plate format to identify chemicals active on the estrogen axis. The REACTIV assay was designed as a screening assay to provide a medium throughput and short-term assay to measure the response of eleutheroembryos to chemicals potentially active on the estrogen axis.