Development of a gradual hypoxia chamber for assessing copper toxicity on air-breathing behavior in Lymnaea stagnalis.

Behavioral endpoints are of increasing interest in toxicology because of their sensitivity, but require clear guidance for experimental design. This study describes the design of a hypoxia chamber for use with pond snails, Lymnaea stagnalis. Studies assessing the switch from water- to air-breathing in hypoxic conditions have previously utilized methods that neglect intricacies of animal behavior such as handling stress and acclimation. The chamber provides a linear decline in dissolved oxygen, against which surfacing behavior for air-breathing can be precisely measured. The maximum biomass of snails suitable for use in the hypoxia chamber, such that the nitrogen-driven deoxygenation curve is not altered by the snails' own metabolism, was established to be greater than 10 adult snails. The capacity of most analysis softwares is below accurately tracking 10 individuals at once, indicating this is likely not a limitation. The size of snails determined the amount of time each episode of aerial respiration was, with smaller snails spending more time air-breathing. A proof-of-principle experiment using acute copper exposure (0 - 60 µg/L) yielded a concentration-response curve, with greater copper concentrations inhibiting air-breathing. The chamber described in the present study provides an improved framework for assessing hypoxic response and is presented in a manner allowing for further modification to meet unique research needs.