Ideally, the fish are exposed to the test chemical for 96 hours. Mortalities are noted at 24, 48, 72, and 96 hours, and where feasible, the concentrations that kill 50% of the fish (LC50) are identified.

It is up to the testing laboratory to decide whether to use one or more species. For both the controls and each test concentration, a minimum of seven fish must be employed.

At least five concentrations of the test material should be given in a geometric series with a factor of 2.2 or less. A single dose level of 100 mg/L is equivalent to the limit test. Fish observations at least 24 hours, 48 hours, 72 hours, and 96 hours are included in this study. On a logarithmic probability paper, the cumulative percentage mortality for every exposure period is plotted against concentration

On April 2, 2014, Test Guideline 204, "Fish, Prolonged Toxicity Test: 14-Day Study," was removed in response to the OECD Council's ruling.

Determining the fatal and sub-lethal effects of chemicals on the early life stages of the tested species is the goal of the test procedure outlined in this Test Guideline.

Five concentrations of the test material dissolved in water are given to fish in their early life stages, ideally in flow-through circumstances or, where suitable, semi-static conditions. Fertilised eggs (at least 80 per concentration level) are first placed in the test chambers (four at the very least), and the test continues until all of the control fish are eating themselves. To find the lowest observed effect concentration, the no observed effect concentration, or the effect concentration that causes an x% change in organisms for the observed effect, fatal and sub-lethal effects are evaluated and compared with control values.

Measurements of the test substance's concentrations in water at regular intervals (five at least), dissolved oxygen, temperature, pH, total hardness, and salinity, fish weight and length, observations of abnormal behaviour, appearance, hatching, and survival, as well as the effect concentration that causes an x% change in the organisms for the effect observed, should all be included in the study report.

The life phases from the freshly fertilised egg to the end of the sac-fry stage are exposed in this short-term toxicity test on fish embryo and sac-fry stages.

Five different quantities of the test material dissolved in water are given to fish in the embryonic and sac-fry phases. A semi-static or flow-through approach may be used, depending on the type of material being tested. At least 30 fertilised eggs are first evenly distributed among at least three replicate test chambers per concentration. The test ends just before the yolk sac of any larvae in any of the test chambers has been fully absorbed or before starvation-related deaths in controls begin.

To find the lowest observed effect concentration and, consequently, the no observed effect concentration, lethal and sub-lethal effects are evaluated and contrasted with control values. To estimate the concentration that would result in a specific percentage effect, they might also be evaluated using a regression model. The weekly measurement of oxygen concentration, temperature, and pH values; the daily counting of the offspring; the daily recording of the parent mortality; and the calculation of test substance concentrations should all be included in the study report. Observations of aberrant conduct, appearance, hatching, and survival should also be included.

The purpose of this test guideline is to evaluate how long-term chemical exposure affects young fish growth.

After being weighed, juvenile fish in the exponential growth period are often placed in test chambers where they are exposed to five sublethal concentrations of the test drug dissolved in water, ideally under flowthrough circumstances, but if that is not feasible, proper semi-static (static-renewal) conditions are used. The test lasts for twenty-eight days. After 14 days, the daily feeding ration may be revised based on the initial fish weights. Fish weights at the conclusion of the test, the measurement of the test substance's concentration, and the observation of outward abnormalities and abnormal behaviour should all be included in the study report.

To determine the concentration that would result in an x% fluctuation in growth rate, or ECx, effects on growth rates are examined using a regression model. The lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) can also be found by comparing the results with control values.

The in vivo screening assay for fish reproduction described in this Test Guideline involves holding sexually mature male and spawning female fish together while exposing them to a chemical for a restricted period of time (21 days) in their life cycle. The fathead minnow (Pimephales promelas), which is the suggested species, was used to validate the short-term reproduction experiment. Three test chemical concentrations and the required controls— including, if required, a carrier control—are used in the assay. Four duplicate test vessels are employed for each treatment dose and control for the fathead minnow. Every day of the assay, each test vessel's egg production is quantitatively assessed.

Two biomarker endpoints—vitellogenin and secondary sexual characteristics—are evaluated in males and females independently at the end of the 21-day exposure period as markers of the test chemical's endocrine action. Both sexes' gonads are likewise kept, and histopathology can be to gauge the test animals' reproductive fitness and support other objectives.

The in vivo screening assay for specific endocrine active chemicals described in this Test Guideline involves holding sexually mature male and spawning female fish together while exposing them to a chemical for a restricted period of time (21 days) during their life cycle. Oestrogenic and androgenic activity screening as well as aromatase inhibition are covered by this assay. The zebrafish (Danio rerio), Japanese medaka (Oryzias latipes), and fathead minnow (Pimephales promelas) were used to confirm the assay; however, zebrafish do not permit the detection of androgenic activity

Depending on the species, one or two biomarker endpoints—vitellogenin and secondary sexual characteristics—are measured in males and females at the end of the 21-day exposure period as indicators of the test chemical's oestrogenic, aromatase-inhibiting, or androgenic activity. While secondary sex traits are only tested in Japanese medaka and fathead minnows, vitellogenin is measured in zebrafish, Japanese medaka, and fathead minnows.

An assay that evaluates the early life-stage effects and possible negative outcomes of suspected endocrine disrupting substances (such as oestrogens, androgens, and steroidogenesis inhibitors) on fish sexual development is described in this Test Guideline. The test involves exposing fish to at least three concentrations of the test material dissolved in water from the time of fertilisation until the conclusion of sexual differentiation, which occurs approximately 60 days after hatching. At least four replicates are advised for each treatment level and control group.

Two primary goals are assessed at the end of the test for each fish: the percentage of males, females, intersex, and undifferentiated fish based on gonadal histology, and the amount of vitellogenin from the head and tail or from blood samples. Sex reversal in individual fish is determined by identifying the genetic sex in fish species that have a genetic sex marker. The test can reveal the test chemical's route of action by combining the two primary endocrine endpoints, vitellogenin concentration and phenotypic (and potentially genotypic) sex ratio.

The purpose of the test procedure outlined in this Test Guideline is to ascertain whether substances are acutely or laterally harmful to fish (Danio rerio) embryos.

The test chemical is applied to freshly fertilised zebrafish eggs for 96 hours. each day. Twenty embryos are exposed to the chemical under test at each concentration level, with one embryo per well. A control and five escalating concentrations of the chemical under test are included in the test. As markers of lethality, four apical observations are noted every 24 hours: (i) fertilised eggs coagulating; (ii) no somite formation; (iii) the tail-bud not detaching from the yolk sac; and (iv) no heartbeat.

A positive result in any of the four apical observations made at the conclusion of the exposure period indicates acute toxicity, and the LC50 is computed. The concentration of dissolved oxygen, pH, total hardness, temperature, and conductivity of solutions, as well as the measured concentrations of the chemical tested and whether the test's validity requirements were fulfilled, are among the other significant informational components that are included in the test report.

The in vivo screening assay for fish reproduction described in this Test Guideline involves holding sexually mature male and spawning female fish together while exposing them to a chemical for a restricted period of time (21 days) in their life cycle. The fathead minnow (Pimephales promelas), which is the suggested species, was used to validate the short-term reproduction experiment. Three test chemical concentrations and the required controls— including, if required, a carrier control—are used in the assay. Four duplicate test vessels are employed for each treatment dose and control for the fathead minnow. Every day of the assay, each test vessel's egg production is quantitatively assessed.

The in vivo screening assay for fish reproduction described in this Test Guideline involves holding sexually mature male and spawning female fish together while exposing them to a chemical for a restricted period of time (21 days) in their life cycle. The fathead minnow (Pimephales promelas), which is the suggested species, was used to validate the short-term reproduction experiment. Three test chemical concentrations and the required controls— including, if required, a carrier control—are used in the assay. Four duplicate test vessels are employed for each treatment dose and control for the fathead minnow. Every day of the assay, each test vessel's egg production is quantitatively assessed.

Two biomarker endpoints—vitellogenin and secondary sexual characteristics—are evaluated in males and females independently at the end of the 21-day exposure period as markers of the test chemical's endocrine action. Both sexes' gonads are likewise kept, and histopathology can be examined to gauge the test animals' reproductive fitness and support other objectives.