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This Test Guideline describes a method to assess bioaccumulation of sediment-associated chemicals in endobenthic oligochaetes worms. It applies to stable, neutral organic chemicals having log Kow values between 3.0 and 6.0, superlipophilic substances that show a log Kow of more than 6.0, or stable metallo-organic compounds which tend to associate with sediments.The test consists of two phases. During the uptake phase, worms are exposed to sediment spiked with the test substance, topped with reconstituted water and equilibrated as appropriate. Groups of control worms are held under identical conditions. The duration of the uptake phase is by default 28 days, unless a steady-state has been reached before. For the elimination phase, the worms are transferred to a sediment-water-system free of test substance. This second phase is terminated when either the 10% level of steady state concentration, or of the concentration measured in the worms on day 28 of the uptake phase, is reached, or after a maximum of 10 days. Change of the concentration of the test substance in/on the worms is monitored throughout both phases of the test. The uptake rate constant (ks), the elimination rate constant (ke) and the kinetic bioaccumulation factor (BAFK = ks/ ke) are calculated. Radiolabelled test substances may be used to determine whether metabolites identification and quantification should be made. The minimum number of treated replicates for kinetic measurements should be three per sampling point throughout uptake and elimination phase. To ensure the test validity (cumulative mortality of the worms < 20% of the initial number), toxicity tests should also be conducted at regular intervals. Besides, the worm lipid content, the sediment total organic carbon content and the residue level in worms at the end of the elimination phase are useful for the interpretation of the results.

This Test Guideline describes a method to assess the extent and kinetics of primary and ultimate biodegradation of organic chemicals whose route of entry into the environment begins with their discharge to wastewater. It consists of five simulation tests in a) sewer systems, b) activated sludge, c) anaerobic digester sludge, d) treated effluent in the mixing zone of surface water, and e) untreated wastewater that is directly discharged to surface water. These tests are appropriate for chemicals released continuously or episodically to wastewater.The five test methods described are open batch systems or closed gas flow-through batch systems. The principle objectives are to i) measure the rate of primary biodegradation, ii) measure the rate of mineralization and iii ) follow the formation and decay of major transformation products when appropriate.Typically, a test chemical, radiolabelled (14C) in an appropriate position, is incubated with a representative environmental sample. Abiotic and biotic treatments are prepared for each test chemical and condition. The level of parent and degradation products is determined using chromatographic separation and radio-analytical detection methods.

This Test Guideline describes a method to estimate the developmental toxicity of a test chemical to the dung dwelling life stages of dung-dependent dipteran species. Two test species can be used. The test chemical is mixed with bovine faeces, to which either 10 eggs of Scathophaga stercoraria or 10 larvae of Musca autumnalis are added. The test will be terminated 5 days after emergence of the last adult in the control (> 18 days for S. stercoraria, >13 days for M. autumnalis). Then the possible impacts of the test chemical on the following measurement endpoints are assessed under controlled conditions: sex and total number of emerged adult flies, retardation of emergence indicated by the developmental rate and morphological change. Depending on the experimental design, the No Observed Effect Concentration (NOEC) or the Effect concentration for x percent effect (ECx) can be determined. This Guideline can be used for water soluble or insoluble substances, but is not applicable to volatile substances. If the toxicity of the chemical is unknown, five nominal test concentrations should be conducted. A positive control should be tested periodically. The test is considered valid if in the controls hatching of larvae is superior or equal to 70% of the number of introduced eggs, emergence of adults is superior or equal to 70% and superior or equal to 50% of the respectively hatched and introduced larvae and if the emergence of adult flies starts after 18 +- 2 days (S. stercoraria) or after 13 +- 2 days(M. autumnalis).

This Test Guideline describes a method to assess the effects of chemical substances in soil on the reproductive output of the soil mite species Hypoaspis (Geolaelaps) aculeifer Canestrini (Acari: Laelapidae). It can be used for water soluble or insoluble substances, but not with volatile substances.Adult females of similar age are exposed to a range of concentrations of the test substance mixed into 20 g dry mass of artificial soil 28-35 days after the start of the egg laying period. Depending on the endpoint (ECx, NOEC or both), five to twelve concentrations should be tested. At least two to four replicates for each test concentrations and six to eight control replicates, of 10 animals each, are recommended. At 20¡ãC, the test lasts 14 days after introducing the females, which usually allows the control offspring to reach the deutonymph stage. The number of surviving females (mortality ¡Ü 20% for a valid test) and the number of juveniles per test vessel (at least 50 for a valid test) are determined. The fecundity of the mites exposed to the test substance is compared to that of controls in order to determine the ECx (e.g. EC10, EC50) or the No Observed Effect Concentration (NOEC). Any observed differences between the behaviour and the morphology of the mites in the control and the treated vessels should be recorded.

The test method described in this Test Guideline assesses the effect of chemicals on the reproductive output of Daphnia magna Straus. To this end, young female Daphnia are exposed to the test substance 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. Reproductive output of the parent animals and the total number of living offspring produced per parent alive at the end of the test should be reported. The study report should also include: 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, the sex ratio of the offspring may be recorded. The reproductive output of the animals exposed to the test substance is compared to that of the control in order to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC). In addition, and as far as possible, the data are analyzed using a regression model in order to estimate the concentration that would cause an x % reduction in reproductive output.

This method provides information on health hazard likely to arise from exposure to test substance via oral administration. The method is based on the repeated oral administration of the substance of interest during one limited period (one dose level daily during 28 days). This Guideline is intended primarily for use with rodents (rat preferably). At least 10 animals (5 female and 5 male) should be used for each dose level. Three tests groups, at least, should be used. The test compound is administered by gavage or via the diet or drinking water. A limit test may be performed if no effects would be expected at a dose of 1000 mg/kg bw/d. The report of this study will include results from clinical and functional observations, body weight and food/water consumption measurements, hematology and clinical biochemistry; as well as gross necropsy and histopathology.

The method permits estimation of an LD50 with a confidence interval and the results allow a substance to be classified for acute toxicity according to the Globally Harmonised System of classification and labelling of chemicals.It is easiest to apply to materials that produce death within couple of days. This Test Guideline is intended for use with rodents (rat female preferably). There are a limit test and a main test. The limit test can be used efficiently to identify chemicals that are likely to have low toxicity. The test substance is administered generally in a single dose by gavage to animals fasted prior to dosing. Single animals are dosed in sequence usually at 48h intervals. The first animal is dosed a step below the best preliminary estimates of the LD50. The second animal receives a lower dose (if the first animal dies) or a higher dose (if the first animal survives). Animals are observed with a special attention given during the first 4 hours and daily thereafter, for a total of 14 days generally. Weights Animals should be determined at least weekly. All the animals should be subjected to gross necropsy. Globally the LD50 is calculated using the maximum likelihood method. Following this, it may be possible to compute interval estimates for the LD50; most narrow is the interval and better is LD50 estimation.Software to be used with TG 425, 432, 455. Click here. Software not part of the Mutual Acceptance of Data.