Metier (modular ecotoxicity tests incorporating ecological relevance) for difficult substances: Aedes aegypti (diptera: culicidae) initial module test development using 3,4‐dichloroaniline

Abstract
There is an urgent need to develop regulatory guidelines for compounds classed as “difficult substances” ‐ poorly water‐soluble, volatile, readily biodegradable or photodegradable, or hydrolytically unstable compounds. The project METIER (modular ecotoxicity tests incorporating ecological relevance) aims to develop a series of ecologically relevant toxicity tests allowing the full breadth of response of aquatic organisms to chemical contaminants to be assessed under realistic exposure scenarios. This involves the development of a standard flow‐through exposure system and a series of modular toxicity tests linked to a mechanistic physiological model of contaminant effects. This paper addresses the initial stages of the development and test design of the module with Aedes aegypti larvae (Diptera: Culicidae), using 3,4–dichloroaniline (3,4–DCA), a poorly water‐soluble compound included in the European Community Priority 1 List of Chemicals. A viable culture system, suitable test medium, and the experimental design for loading effects and test duration are investigated. Moreover, the dissolution process and stability of the 3,4–DCA solutions are presented. Up to 50 mg L−1 3,4‐DCA was dissolved without the use of a carrier. There was no loss of chemical during 4 d after dissolution. Use of one larva per vessel in 96‐h acute toxicity tests is recommended. Also, 24‐, 48‐, 72‐, and 96‐h acute toxicity tests were run in low and high volumes of medium. Results of these tests reveal that A. aegypti larvae are more sensitive in higher volumes of test medium and belong to a moderately tolerant group of organisms regarding 3,4‐DCA acute toxicity.