Bioconcentration factors (BCFs) are determined by fish flow-through tests performed according to Organisation for Economic Co-operation and Development test guideline 305. These are time-consuming and expensive and use a large number of animals. An alternative test design using the freshwater amphipod Hyalella azteca for bioconcentration studies has been recently developed and demonstrated a high potential. For bioconcentration studies using H. azteca, male amphipods are preferred compared with female organisms. Manual sexing of male adult amphipods is, however, timeconsuming and requires care and skill. A new fully automatic sorting and dispensing machine for H. azteca based on image analysis has recently been developed by the company Life Science Methods. Nevertheless, an anesthesia step is necessary prior to the automatic selection. In the present study, we show that a single-pulse of 90 min of tricaine at the concentration of 1 g/L can be used and is recommended to select H. azteca males manually or automatically using the sorting machine. In the second part, we demonstrate that the machine has the ability to select, sort, and disperse the males of a culture batch of H. azteca as efficiently as manual procedures. In the last part of the study, BCFs of two organic substances were evaluated using the H. azteca bioconcentration test (HYBIT) protocol, with an anesthetizing step and robotic selection compared with manual selection without an anesthetizing step. The different BCF values obtained were in accordance with those indicated in the literature and showed that an anesthetizing step had no effect on the BCF values. Therefore, these data validated the interest in this sorting machine for selecting males to perform bioconcentrations studies with H. azteca. Environ Toxicol Chem 2023;42:1075-1084.

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