A method of determining the effect of a test substance on any life stage of at least one clone of a Centroptilum triangulifer mayfly comprising (A) identifying and selecting at least one clone of Centroptilum triangulifer as a test clone to be subjected to the test substance; (B) exposing each test clone to the test substance for a predetermined time; and (C) determining the effect of the test substance on each test clone by at least one of (i) observing the test clone for a lethal or sublethal effect and (ii) Sacrificing the test clone, and analyzing the sacrificed test clone for at least one of (a) the presence of at least one of the test substance and any of its metabolites, (b) the quantity of at least one of the test substance and any of its metabolites, and (c) a lethal or sublethal effect of at least one of the test substance and any of its metabolites. In a preferred embodiment for testing for a substance and any of its metabolites, the substance and any metabolites being tested are extracted and separated from the sacrificed mayflies in a solvent mixture, and the substance and any metabolites are identified and quantified using appropriate equipment and techniques.
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