Environ. Sci. Technol. 2000, 34, 2756-2760
Intermittent Exposure of Fish to Estradiol G R A C E H . P A N T E R , * ,†,‡ ROY S. THOMPSON,† AND JOHN P. SUMPTER‡ Brixham Environmental Laboratory, AstraZeneca Limited, Freshwater Quarry, Brixham, Devon, TQ5 8BA, United Kingdom, and Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH, United Kingdom
The majority of testing for the effects of chemicals on fish has, historically, been designed to provide continuous exposure to constant concentrations. However, in the environment, fish are typically exposed intermittently or to fluctuating levels of contaminants, due to their mobility between polluted and clean waters and to variations in effluent discharge concentrations. The effects of such intermittent exposure of fish to estrogenic substances are unknown. Therefore, male fathead minnows (Pimephales promelas) were exposed to nominal concentrations of 17-β estradiol (an endogenous and environmental estrogen) at 30, 60, or 120 ng L-1 dosed continuously, or 120 ng estradiol L-1 dosed intermittently, for 21 or 42 days. The estrogenic effects of these dosing regimes were evaluated by determination of plasma vitellogenin concentrations and changes in gonad weight. Intermittent exposure resulted in plasma vitellogenin concentrations approximately equal to those in response to continuous exposure to the same concentration and significantly higher than continuous exposure to the equivalent time-weighted average concentration. Thus, the response to the intermittent regime was greater than would be expected from a simple integration of concentration and time. Changes in gonadosomatic index were less sensitive to the dosing regimes. Exposed fish were also allowed to depurate to determine the rate at which the estrogenic response disappeared after removal of the stimulus. After a 21 day depuration, the elevated plasma vitellogenin concentrations remained high, suggesting that this estrogenic effect would be sustained in the fish for long periods after transient estrogenic exposure.
Introduction Conventional ecotoxicity testing with fish and aquatic invertebrates has predominantly employed systems designed to allow continuous exposure of the organisms to constant concentrations of a chemical, in order to define its potential to cause adverse effects and define the concentrationresponse relationship (1, 2). The laboratory testing of chemicals for their estrogenic effects on fish has, to date, followed the same principles (3, 4). However, these continuous exposures do not simulate the situation experienced * Corresponding author phone: 00 44 1803 882882; fax: 00 44 1803 882974; e-mail:
[email protected]. † AstraZeneca Limited. ‡ Brunel University. 2756
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 13, 2000
typically by fish populations in the real environment. Contaminant inputs to the aquatic environment may be episodic, due to accidental releases or batch processes, and concentrations in the environment may fluctuate due to changes in emission levels and varying dilution in the receiving water (5). Furthermore, fish in rivers display significant upstream and downstream movements (6, 7), and the recent development of methods for tagging fish have revealed regular daily movements in relation to feeding as well as seasonal and breeding migrations (8-10). Such daily movements of individuals may remove them periodically from the discharge plume of effluents. In addition, fish may show avoidance movements in response to particular contaminants (11) or to the lowered dissolved oxygen concentrations which may result from the oxygen demand of sewage or organic effluents, especially at times of low river flow (12, 13). Thus, such movements will result in intermittent or fluctuating degrees of exposure which may induce effects that are not predictable from criteria based on experiments employing continuous exposure. The aim of the present study was to determine whether the estrogenic response observed in fish exposed intermittently to a known estrogen was proportional to the timeweighted average concentration (the simple integration of concentration and duration) or was a function of the peak concentration. Estradiol was selected as the test compound in order to mimic the scenario of fish living near the discharge zone of a sewage treatment works, because this endogenous steroid in fish has been identified as one of the key causal agents for estrogenicity in such effluents (14). Tentative links have also been made between the estrogenicity of sewage effluent and the unusually high incidence of hermaphrodite fish found in British rivers (15). Thus, in this study, male fathead minnows (Pimephales promelas) were exposed continuously and intermittently to the environmental estrogen, estradiol. Changes in plasma vitellogenin levels and gonadosomatic index were used as estrogenic endpoints. Recovery from estrogenic stimulation was also assessed by monitoring these endpoints after allowing some of the exposed fish to depurate in clean water.
Experimental Section Animals. The fish used in this study were male fathead minnows, bred at the Brixham Environmental Laboratory. Individuals approaching sexual maturity (when males could be identified reliably by their external appearance) were employed. Each fish was weighed prior to the start, and the mean body weight was recorded: experiment 1, mean body weight 5.55 g (range: 3.19-9.63 g); experiment 2, mean body weight 3.90 g (range: 2.12-5.85 g); experiment 3, mean body weight 4.43 g (range: 1.79-6.44 g). These weights were used to set the daily feeding ration of 2% of body weight of Promin (Promin Ltd., Hampshire, U.K.) employed during the exposure period. All fish were acclimated to the test conditions for a period of 24 h before the start of exposure. At the start of experiments 1 and 2, seven fish were randomly allocated to each treatment and to an initial (day 0) sample group. At the start of experiment 3, 14 fish were randomly allocated to each treatment and seven to an initial (day 0) sample group. Test Conditions. The dilution water was dechlorinated mains water maintained at a temperature of 22 ( 1 °C. The hardness of the test water was 50 ( 5 mg L-1 (as CaCO3) and pH was 7.5 ( 0.5. The dissolved oxygen concentration remained >70% of the air saturation value throughout the 10.1021/es991117u CCC: $19.00
2000 American Chemical Society Published on Web 05/31/2000
experiments. A photoperiod of 16 h light and 8 h darkness, with 20 min transition periods, was provided. Test Substance and Dosing System. Stock solutions of estradiol (obtained from Sigma Chemical Co., Dorset, U.K.) were prepared in absolute ethanol. A continuous flow exposure system was employed. Each stock solution was dosed by syringe pump, at a rate of 1.157 µL min-1, to mix with the flow of dilution water (400 mL min-1) in a mixing chamber with magnetic stirring, prior to delivery to the test vessels. The test vessels were glass aquaria with a working volume of 13 L in experiments 1 and 2, and 25 L in experiment 3. The control vessel also received the same rate of addition of ethanol, such that all treatments contained 0.0029 mL ethanol L-1. Concentrations of the test compounds were not confirmed in the test vessels by chemical analysis. However, the dilution water and toxicant flow rates were checked at least four times per week to ensure that the test chemical was entering the tanks at the appropriate rate to obtain the desired concentrations. The flow rate was relatively high, providing 1.8 tank volumes per hour and a true 99% replacement time of
