Physiological stress in fish during toxicological procedures: a potentially confounding factor

An increasing body of evidence suggests that stress may modulate the response of an animal to toxicological challenge. The degree of stress inherent in the procedures comprising a fish LC50 study (OECD Guidelines for the testing of chemicals "Fish, acute toxicity test" No. 203, adopted 12 May 1981) was assessed by measuring plasma cortisol levels (rainbow trout) and whole-body levels of immunoreactive corticosteroids (zebra fish). Aspects of the 96 h LC50 protocol which were found to evoke corticosteroidogenic activity in the test fish included transfer from holding tanks to test tanks, tank transfer during semi-static procedures, and disturbance associated with water quality measurement. There was evidence for acclimation of the fish to repetitive stimuli such as tank transfer but, overall, the data suggest that the 96h test protocols evoke a state of chronic stress in the test population. The removal of fish from test vessels during longer-term studies was also found to evoke a stress response in fish remaining within the vessel, suggesting that these conditions may expose test fish to a regime of intermittent periods of acute stress. The possibility that physiological stress may interfere with the accurate estimation of toxicological endpoints was examined. In three independent LC50 tests the toxicity of the arylmethane dye, malachite green, to rainbow trout was found to be markedly and consistently reduced when the degree of disturbance and handling during the test period was minimised.