Novel Reproduction Toxicity Test Method for Hyalella azteca Using Sexually Mature Amphipods Provides More Robust Data Than Standard Methods in Exposures to Imidacloprid

Hyalella azteca is an epibenthic crustacean used in ecotoxicology, but there are challenges associated with standard methods using reproduction as an endpoint. A novel, 28‐day reproduction toxicity test method for H. azteca was created to address these issues by initiating tests with sexually mature amphipods to eliminate the confounding effects of growth, using a sex ratio of seven females to three males to reduce reproductive variability, and conducting tests in water‐only conditions to make recovery of juveniles easier and expand testing capabilities to water‐soluble compounds. In the present study, we evaluated the 28‐day novel method by comparing it with the 42‐day standard test method in duplicate and parallel water‐only, static‐renewal exposures to sublethal concentrations of imidacloprid (0.5–8 µg/L). Both methods showed similar effects on survival, with survival approaching 50% in the highest test concentration (8 µg/L). However, the 42‐day median effect concentrations (EC50s) for growth were more sensitive in the standard method (1.5–3.2 µg/L) compared with the 28‐day EC50s generated by the novel method (>8 µg/L). Reproduction endpoints (juveniles/female) produced similar EC50s between methods, but the data were less variable in novel tests (smaller coefficients of variation); therefore, fewer replicates would be required to detect effects on reproduction compared with the standard method. In addition, novel tests generated 28 days of reproduction data compared with 14 days using standard tests and allowed survival and growth of sexes to be assessed independently. Thus, the novel method shows promise to improve the use of reproduction as an endpoint in water‐only toxicity tests with H. azteca . Environ Toxicol Chem 2024;43:723–735.