Method of determining the effect of a test substance on a clone of a Centroptilum triangulifer mayfly

A method of testing for the presence of any test substance in an environment to which any life stage of at least one clone of a Centroptilum triangulifer mayfly is exposed and which has an effect on the mayfly comprising the steps:

• (A) identifying and selecting at least one clone of Centroptilum triangulifer from an isolate as a test clone to be subjected to the test substance and a corresponding control clone from a different aliquot of the test clone against which the test clone will be compared;
• (B) exposing each test clone, but not the corresponding control 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 and the corresponding control clone for a lethal or sublethal effect to determine whether the test clone has a lethal or sublethal effect in comparison to the corresponding control clone;
• and
• (ii) sacrificing the test clone and the corresponding control clone, and analyzing the sacrificed test clone and corresponding control clone for at least one of
• (a) the presence in the test clone compared to the corresponding control clone of at least one of the test substance and any of its metabolites,
• (b) the quantity in the test clone compared to the corresponding control clone 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 the test clone compared to the corresponding control clone.