PROFENOFOS TOXICITY TO THE EASTERN RAINBOW FISH (MELANOTAENIA DUBOULAYI)

Transcription

1 Environmental Toxicology and Chemistry, Vol. 17, No. 9, pp , SETAC Printed in the USA /98 $ PROFENOFOS TOXICITY TO THE EASTERN RAINBOW FISH (MELANOTAENIA DUBOULAYI) A. KUMAR and JOHN C. CHAPMAN* Centre for Ecotoxicology, Environment Protection Authority and University of Technology, Sydney, Westbourne Street, Gore Hill, New South Wales 2065, Australia (Received 11 April 1997; Accepted 9 January 1998) Abstract The 96-h median lethal concentration for profenofos in the Australian eastern rainbow fish (Melanotaenia duboulayi) was 0.9 mg/l, corresponding with an 83% reduction in acetylcholinesterase (AChE) activity in fish head. This is an order of magnitude less sensitive than the figure reported for crucian carp (Carassius carassius). Higher lethal body burdens were found in rainbow fish exposed to high concentrations of profenofos but the inhibition of AChE activity in these fish was less than in those killed by longer, normally sublethal, exposures. During sublethal exposure to 10 g/l for 10 d, the fish accumulated profenofos residues of 5.3 mg/kg associated with a 70% reduction in AChE activity in fish head. Decreases in food intake, food conversion efficiency, and growth were observed at 10 d and significant loss of weight was noted at 21 d. These were associated with markedly increased swimming activity and response to light. A clear relationship existed between the depression of AChE activity and observed behavioral responses, such as swimming activity and food intake. Keywords Profenofos Rainbow fish Residues Acetylcholinesterase inhibition Behavior INTRODUCTION Profenofos, a phosphorothiolate organophosphorus (OP) compound, is the most widely used OP pesticide in the cottongrowing areas of northern New South Wales (NSW) and Queensland (Australia) and can be applied up to two or three times to a field in a season, separated by at least 7 d. Profenofos is very toxic to fish and published 96-h median lethal concentration (LC50) values to three species varied from 80 to 300 g/l [1]. Toxicity of OP pesticides can vary widely in different species of fish [2] and obtaining comparative acute data on an Australian fish was considered to be important for interpretation of published acute lethality data and local sublethal information. Furthermore, profenofos has been implicated in at least one fish kill in NSW [3] but usually occurs in the presence of of other pesticides, so that its contribution to fish kills remains uncertain. Because this pesticide is not persistent in water, analysis of profenofos in fish would provide a better indication of lethal profenofos exposure than analysis in water [4]. However, Kumar et al. [5] have reported residues of profenofos in live fish collected from impoundments and streams in the cotton-growing areas of NSW and hence the presence of profenofos residues does not necessarily indicate that it caused a particular fish kill. Field data can be better interpreted when used in conjunction with residue and effect data from controlled laboratory experiments with an Australian fish under various exposures. Data on lethal body burdens (LBBs), measured as the concentration of the chemical inside the fish at the time of death, have been recommended as a good measure of the intrinsic toxicity of a chemical [6] and such data could be used to interpret residue data after fish kills [4]. Lack of information on the toxicity of profenofos to fish, particularly Australian fish species, indicates that further work * To whom correspondence may be addressed (chapmanj@epa.nsw.gov.au). is needed to accurately assess the impact of profenofos on Australian freshwater fauna. The mode of toxic action of OP pesticides is by the inhibition of the enzyme acetylcholinesterase (AChE), and the level of inhibition of AChE activity is used as a biochemical indicator of exposure to OP compounds [7]. Profenofos induced significant inhibitory effects on the AChE activity of the native shrimp Paratya australiensis during lethal and sublethal exposures [8]. The effects of profenofos on AChE activity in fish has not been reported and if AChE activity is to be of value as a field biomarker, it must be first evaluated under a range of controlled laboratory exposures and changes in activity must be related to observed effects [9]. A significant reduction in the AChE activity of an organism may be generated by a sublethal exposure to an OP but such an exposure may result in behavioral changes, which at the community level may be as profound as a lethal exposure. This project studied the interactions between concentrations of profenofos in water, residues of profenofos in a native fish, changes in AChE activity, and ecologically significant behavioral effects in the laboratory, to improve predictions of the impacts of profenofos on aquatic ecosystems. MATERIALS AND METHODS Exposure of eastern rainbow fish (Melanotaenia duboulayi) to profenofos Experiment 1 consisted of 96-h acute exposures of profenofos to the fish, to calculate 96-h LC50 values and to investigate dose response relationships in terms of AChE inhibition and profenofos accumulation under acute exposure. Experiment 2 consisted of acute lethal (4 mg/l) and sublethal (0.1 mg/l) exposures, to investigate LBBs in rainbow fish. Experiment 3 consisted of 21-d sublethal exposure (10 g/ 1799

2 1800 Environ. Toxicol. Chem. 17, 1998 A. Kumar and J. C. Chapman L), to investigate the relationship between profenofos accumulation, AChE inhibition, and behavioral change. Test procedures Technical-grade profenofos (O-4-bromo-2-chlorophenyl O- ethyl S-propyl phosphorothioate) of 97% purity was supplied by Ciba-Geigy Australia (Pendle Hill, NSW, Australia). A stock solution was prepared by dissolving technical-grade profenofos in pesticide-grade acetone as a solvent carrier to make further test solutions with the desired concentrations. Rainbow fish were bred and maintained in the Centre for Ecotoxicology laboratory at Gore Hill (NSW, Australia) according to the method described by Sunderam et al. [10]. Glass test tanks were arranged at random, and fish of both sexes were assigned to each tank using random numbers. Fish were acclimated in the dilution water and test tanks at the test temperature for at least 2 weeks before running the experiments. The water flow was maintained so that the dissolved oxygen in water always exceeded 60% saturation. The sides of all the tanks were covered with foam sheets to avoid stressing the fish. The test tanks were covered with glass lids and were not aerated during experimentation to reduce toxicant evaporation. In experiments 1 and 2, fish were exposed to profenofos under continuous flow-through systems as described by Sunderam et al. [10]. The flow rate through the 60-L glass tanks was maintained at 150 ml/min, resulting in 90% replacement in 13 h. In experiment 3, rainbow fish were exposed to profenofos under semistatic conditions, that is, solutions were renewed every 24 h. The detailed test procedures for each experiment are given below. Experiment 1 (96-h acute lethality to rainbow fish). For definitive determination of flow-through acute dose response relationships, a control, a solvent control (0.01 ml/l), and seven concentrations of profenofos were used, ranging from 0.27 to 3.0 mg/l, and determined from the range-finding tests. Tests were conducted under flow-through conditions according to Sunderam et al. [10] and mortality data were analyzed by the trimmed Spearman Karber method of Hamilton et al. [11]. All surviving fish from the test and the control tanks were collected, rinsed in deionized water, and kept at 20 C for further analysis. Experiment 2 (LBB in rainbow fish). Rainbow fish weighing between 2.1 and 3.6 g were exposed to 4 and 0.1 mg/l of profenofos (10 fish at each concentration) in 60-L tanks under continuous flow-through system. The controls were run concurrently. Fish were expected to die within 1 d at 4 mg/l, whereas at 0.1 mg/l they were expected to die within 12 to 14 d. Moribund fish were collected from the respective test tanks, rinsed in deionized water, and kept at 20 C for further analysis. Controls were also sampled at regular intervals. Experiment 3 (sublethal exposure of rainbow fish to profenofos [10 g/l] for 21 d). Eighty rainbow fish weighing between 2.5 and 4.8 g were randomly allocated to eight test and eight control tanks, resulting in five fish per 30-L glass aquarium. After 7 d of acclimation to the experimental conditions, fish were individually marked on the region below the dorsal or caudal fins using a freeze-branding technique as described by Crossland [12]. Fish were acclimated to branding for 2 d before exposure to profenofos. Fish were fed during acclimation and experimentation with a weighed ration equivalent to 1% of their mean initial wet body weight, constituting an amount that the fish would consume within the following 15 min. Fish in the eight test tanks were exposed to 10 g/l profenofos and each test aquarium received a renewal of test solution every 24 h. Concurrently, the water in the control tanks was also renewed. Uneaten food was also removed at this time and was weighed after drying in an oven at 60 C to provide an accurate measure of the amount of food eaten by the fish. The fish were weighed initially (to 0.01 g) 2 d after branding (before exposure) and on days 10 and 21 of exposure. This involved weighing individual live fish in a preweighed 1.5-L beaker filled with water to minimize stress to the fish. Ten test and control fish were sacrificed at days 10 and 21 for analyses of AChE activity and profenofos residues. Twenty fish, 10 each from control and test tanks, at days 10 and 21 were analyzed individually for spontaneous swimming activity, by the method of Little et al. [13]. Exposed fish were transferred to a tank containing 5Lofwater at a profenofos concentration of 10 g/l. Control fish were tested concurrently in the same manner, except in clean water. After acclimation for 5 min, fish were observed by an overhead video camera for exactly 2 min and the amount of time the fish were in motion was timed with a stopwatch. After the swimming activity test, the response of these fish was observed by video camera for 2 min to a sudden external stimulus with a high flux of light (photokinesis) for 1 min. Physicochemical analysis Water temperature, ph, conductivity, and dissolved oxygen were measured at regular intervals in all the experiments using the instruments described by Sunderam et al. [10]. The concentration of profenofos in water from each test tank was measured at the beginning of each experiment and then at regular intervals of 24 h. In the semistatic experiment 3, profenofos was analyzed just before the renewal of test solution. All glassware used for extraction was rinsed with acetone and hexane before use. The profenofos concentration in water samples was analyzed according to the method described by Abdullah et al. [8]. Profenofos residues were also analyzed in all the test fish sampled during all the experiments. The whole-body profenofos burden was analyzed by the following procedure. Approximately 3goffishtissue was homogenized in 20 ml of hexane:acetone (60:40 v/v) solvent mixture and transferred into a glass-stoppered conical flask. The volume in the conical flask was then made up to 100 ml with the same solvent mixture and approximately 3gofanhydrous sodium sulfate was added to the contents in the flask to remove traces of water. The conical flasks were then stoppered and continuously agitated by mechanical shaker for 4 h followed by filtration through a Buchner funnel using a vacuum pump. The collected solvent filtrate was concentrated using a Turbovap II concentration workstation (Zymark, Hopkinton, MA, USA). Concentrated whole fish extract was dissolved in 10 ml hexane and was passed through the glass column packed with florisil, which was first activated at 450 C and then treated in hexane containing 4% water. The columns were eluted with 100 ml of 10% diethylether in hexane, then 5% acetone in hexane. Eluate was then concentrated by the Turbovap. The concentrates were removed from the water bath and allowed to cool. The final sample was made up to 5 ml with nanograde hexane and the samples were then transferred to clean hexane-rinsed glass vials for analysis.

3 Profenofos toxicity to the rainbow fish Environ. Toxicol. Chem. 17, Gas chromatographic analysis The concentrated extract was analyzed for profenofos using a Shimadzu CG 8A GC (Shimadzu, Kyoto, Japan) equipped with a 0.53 mm 30 m J&W DB5 megabore column (J&W Scientific, Folsom, CA, USA), a 63 Ni electron-capture detector (ECD), and a recorder. The optimized operating conditions for the Shimadzu gas chromatograph were as follows: temperature (injector/detector), 280 C; oven temperature, 220 C; carrier gas, nitrogen at 20 ml/min; injection volume, 2 l; attenuation, 3; range of the detector, Ap/mV. The identity of the profenofos residues in the water and tissue samples was confirmed by spiking extracts with a profenofos standard and reanalyzing the samples. Recoveries for the samples of profenofos in water varied from 98 to 105% and for tissue samples were greater than 89%. The method has a detection limit of mg/kg. Fish tissues were also analyzed for lipid content following the procedure outlined by Nowak and Julli [14]. Approximately 3gofwhole fish was homogenized in 10 ml hexane with3goftrisodium citrate, 3gofdisodium hydrogen orthophosphate, and 15 g anhydrous sodium sulfate. After 1 h, the mixture was emptied into glass columns and eluted with 60 ml of a 1:1 mixture of hexane and chloroform into beakers that had been dried to constant weight. The solvent was evaporated in a water bath and beakers dried to a constant weight to give the lipid content. For measurement of the AChE activity, fish heads were severed behind the operculum and homogenized in cold 0.1 M phosphate buffer (ph 8) containing 0.5% Triton X-100 (Ajax Chemicals, Auburn, NSW, Australia) and centrifuged at 3,000 rpm for 10 min. Acetylcholinesterase activity was measured in the supernatant by the method of Ellman et al. [15]. Protein content was determined by the method of Lowry et al. [16] using bovine serum albumen as the standard. Acetylcholinesterase activity was expressed as micromoles per minute per gram protein ( mol/min/g). Statistical analysis Data were analyzed by analysis of variance (ANOVA) [17] and results were considered to be statistically significant if p If significantly different, the data were compared using Tukey s honestly significant difference (HSD) multiple comparisons test [18]. Before ANOVA was performed, homogenity of variances was tested using Bartlett s test. If the variances were heterogenous, the Kruskal Wallis nonparametric rank test was used [18]. Detailed statistical analysis for each experiment is given below. In experiment 1 and 2 data were analyzed by one-way ANOVA. In experiment 1, correlation and regression analyses were also performed to determine the relationship among mortality, profenofos accumulation, and AChE inhibition in rainbowfish during 96-h acute exposures to different concentrations of profenofos. In experiment 3 data were analyzed by two-way ANOVA to determine if profenofos exposure and time after exposure had a significant effect on the percentage lipid content, AChE activity, food intake, growth rate, and food conversion efficiency in the rainbow fish. RESULTS Experiment 1 (96-h acute lethality to rainbow fish) The concentrations of profenofos in 96-h acute exposures remained consistent during the 96-h exposure, and generally Fig. 1. Median lethal concentration (LC50) values at different time intervals during rainbow fish exposure to profenofos. Error bars represent 95% upper and lower confidence intervals. good agreement was found between nominal and measured profenofos concentrations. The dissolved oxygen measured was always greater than 60% saturation and ph ranged from 6.9 to 7.3. The 96-h LC50 for these rainbow fish was calculated to be 0.91 mg/l but incipient toxicity was not reached at 96 h (Fig. 1). Mortality was directly proportional to profenofos concentration and time of exposure (slope 0.018, r ). The 96-h LC50 concentration in rainbow fish resulted in 83% reduction in AChE activity of surviving fish. A logarithmic relationship existed between increasing profenofos concentration and decreasing AChE activity (slope 11.5, r ; Fig. 2). Control fish had significantly greater AChE activities compared to those of profenofos-exposed fish (p ). Higher residues of profenofos accumulated in the rainbow fish at higher acute exposures (Fig. 2) and a logarithmic relationship existed between profenofos accumulation in rainbow fish and associated AChE inhibition (slope 15.1, r ; Fig. 3). Experiment 2 (LBB in rainbow fish) Measured profenofos concentrations during the 24-h lethal exposure (4 mg/l) varied from 3.5 to 3.7 mg/l (mean SE, Fig. 2. Relationship of profenofos concentrations in water to the profenofos accumulation and percent acetylcholinesterase (AChE) inhibition in rainbow fish during 96-h exposures.

4 1802 Environ. Toxicol. Chem. 17, 1998 A. Kumar and J. C. Chapman Table 2. Acetylcholinesterase (AChE) activity ( mol/min/g protein) and profenofos (PF) residues in rainbow fish during sublethal exposure to 10 g/l for 21 d Treatment Time (d) PF residues in fish a (mg/kg) AChE activity a % AChE inhibition b Control Exposed ND d ND a Each value represents mean standard error. b Compared with control. c ND not detected. Fig. 3. Relationship between profenofos accumulation in rainbow fish and acetylcholinesterase (AChE) inhibition during 96-h profenofos exposures mg/l) and in the 14-d sublethal exposure (0.1 mg/ L), profenofos concentrations varied from 0.07 to 0.09 mg/l (mean SE, mg/l). No significant differences were found in the profenofos concentrations of test tanks during sublethal exposure (p 0.9). The ph varied from 7.1 to 7.5, oxygen content varied from 66 to 78% saturation, and the temperature varied between 21.2 and 21.8 C. The LBB in fish that were exposed to 4 mg/l of profenofos was significantly higher (97 3 mg/kg wet weight) than that of fish that were killed by longer exposure for 14 d at 0.1 mg/ L (19 3 mg/kg wet weight) (df 16, p ; Table 1). Lethal body burdens based on lipid weight basis were uniformly 12 times greater than those expressed on a wet weight basis. The fish that survived the exposure to 0.1 mg/l profenofos exhibited a body burden of mg/kg, which is significantly lower than the mean LBB of fish that did not survive the exposure (df 8, p 0.01). The inhibition of AChE activity in moribund rainbow fish exposed to the lower concentration (0.1 mg/l) for 14 d was greater than 90%, in comparison to 72% inhibition in the fish exposed to 4 mg/l for less than 24 h (Table 1). The inhibition of AChE activity was also greater than for those rainbow fish that survived the sublethal exposure for 14 d (up to 70% inhibition; Table 1). Table 1. Lethal body burden (LBB; mg/kg) in rainbow fish at high (3.6 mg/l) and low (80 g/l) exposures to profenofos Measured aqueous a concn. (mg/l) c LBB a (mg/kg wet wt.) LBB a (mg/kg lipid wt.) 1, a Each value represents mean standard error. b Compared with control. c Data from two live fish. % Acetylcholinesterase inhibition b Experiment 3 (sublethal exposure of rainbow fish to profenofos [10 g/l] for 21 d) The profenofos concentration varied from 6.2 to 7.0 g/l as measured on days 1, 5, 10, 15, and 21 of exposure. The mean value was 6.6 g/l. Water quality parameters varied as follows: ph 7.1 to 7.7, temperature 21.2 C to 22.3 C, and dissolved oxygen 64 to 78% saturation. Profenofos residues were significantly higher in exposed fish on day 21, reaching mg/kg, compared to mg/kg at day 10 (df 10, p ; Table 2). The percentage lipid content in the profenofos-exposed fish decreased significantly from % at day 10 to % at day 21 (p 0.005). In contrast, no significant difference was found in the percentage lipid content of the control fish on day 10 ( %) or day 21 ( %) of the exposure regime (p 0.6). A significant decrease was found in AChE activity of exposed fish on both day 10 and day 21, compared to that in control fish (df 28, p ). In contrast, the control fish did not exhibit any significant differences in AChE activity between day 10 and day 21 (Table 2). After 10 d of exposure, the AChE activity of the exposed rainbow fish was inhibited by 70% and was reduced to 81% inhibition after 21 d of exposure. The data for food intake, growth rate, and food conversion efficiency of rainbow fish exposed to a sublethal concentration of profenofos for 21 d are presented in Table 3. No significant differences were found in the initial weights of fish in controls and exposed fish (df 78, p 0.7). The feeding rate, growth rate, and food conversion efficiency of control rainbow fish did not vary significantly on days 10 and 21. When exposed to 10 g/l profenofos, the growth rate decreased significantly to mg/g live fish/ d on day 10 of exposure. By day 21 the fish were found to have lost weight, and a negative growth rate of mg/g live fish/d occurred (df 12, p 0.01; Table 3). However, the feeding rate and food conversion efficiency in the exposed fish were not significantly different from those of the controls on day 10, but they were significantly lower on day 21 (df 12, p 0.01). The feeding rate, growth rate, and food conversion efficiency of the profenofos-exposed fish on day 10 were significantly different from those on day 21 (df 12, p 0.01). The cumulative time spent in swimming, a measure of spontaneous swimming activity, increased with an increase in duration of the sublethal exposure (Table 3) and exposed test fish were in motion throughout the observation period. Exposed fish also demonstrated pronounced reactions to the light stim-

5 Profenofos toxicity to the rainbow fish Environ. Toxicol. Chem. 17, Table 3. Feeding rate, growth rate, food conversion efficiency, and behavioral responses in rainbow fish during sublethal exposure to profenofos (10 g/l) Treatment Time (d) Feeding rate a (mg/g dry food/d) Growth rate a (mg/g live fish/d) Food conversion a efficiency % Swimming activity a (s) Light response a (s) Control Control Exposed Exposed a Each value represents mean standard error. ulus and they swam in circles and knocked on the edges of the tank. In contrast, the control fish were hyperactive only during the initial observation period and did not show any such hyperactive swimming behavior and response to stimulus. The Kruskal Wallis rank test (nonparametric test) showed that the swimming activity and the response to light stimulus in the control and profenofos-exposed fish on days 10 and 21 were significantly different (H 33.3, P 0.05). No significant difference was found between these behaviors in either the control on days 10 and 21 or the exposed groups on days 10 or 21. DISCUSSION This investigation compared three toxicologic endpoints for acute lethality of profenofos to rainbow fish: the 96-h LC50, the LBB and inhibition of the enzyme AChE. The residues in the exposed fish were directly proportional to the concentration of profenofos in water. Mount and Boyle [19] exposed the brown bullhead (Ictalurus nebulosus) to various concentrations of technical-grade parathion for time periods up to 30 d and observed a close relationship between concentration of parathion in the water and that in the blood. Bender [20] found that the uptake of malathion in flesh of common carp (Cyprinus carpio) was related to its concentration in water; 96-h exposure to 1, 2.5, 5.0, and 7.5 mg/l malathion resulted in residues up to 0.9, 7.9, 28.4, and 41.6 mg/kg, respectively. In the present acute exposures to rainbow fish, no significant difference was found in residue accumulation at the lower concentrations below 52 g/l. The concentration that caused 50% mortality (96-h LC50: 0.91 mg/l) was associated with up to 59 3 mg/kg profenofos body burden in fish and 83% inhibition in AChE activity. In general, LC50 values have been associated with 70 to 90% reduction in AChE activity for OP compounds [21]. Coppage and Matthews [22] claimed that a mean reduction in AChE activity of about 80% is critical in short-term (24-h) poisoning of the estuarine fish tested. However, extended exposure (21 d) of rainbow fish to profenofos (10 g/l) in the present study also resulted in a very low AChE levels (84% inhibition). Rainbow fish are more resistant to acute profenofos exposure than other species; the 96-h LC50 reported for the crucian carp (Carassius carassius) was 0.09 mg/l, for rainbow trout (Oncorhynchus mykiss) the 96-h LC50 was 0.08 mg/l, and for bluegill (Lepomis machrochirus) it was 0.3 mg/l [1]. Vittozzi and Angelis [2] also reported great variation in the acute toxicity of OP pesticides to various fish species. According to that report, the main biological factor that determines the acute lethal effect might be the cholinergic system, particularly AChE inhibition. This, together with residues, may allow determination of relative sensitivity of various species to profenofos, without the need for full LC50 determinations. The AChE inhibition in rainbow fish was greater at higher concentrations of profenofos and a strong correlation existed between the amount of residues present in the fish and associated percentage AChE inhibition (r ). This accords with other studies with fish [23 25]. The rainbow fish exposed to 27 g/l for 96 h had only 43% reduction in AChE activity, associated with a body burden of mg/kg profenofos. In contrast, the fish exposed to 570 g/l had 74% reduction in AChE activity, associated with a body burden of 45 2 mg/kg. A major shortcoming of acute concentration response data is that they do not give information on the concentration of the chemical in the target tissues. Several important aspects such as kinetic behavior, bioavailability, and biotransformation can influence the results of LC50 tests. Especially in shortterm experiments with relatively hydrophobic chemicals, steady state is not achieved and toxicity may be severely underestimated [26]. It is apparent from Figure 1 that a steady state (incipient level) had not been reached with profenofos after 96 h. De Bruijn et al. [6] and Sijm et al. [27] proposed that the LBB gave a better description of the intrinsic toxicity of chemicals to fish than the LC50, because the LBB gives the concentration of the chemical inside the fish at the time of death. These relationships are being developed further for chemicals with specific modes of action but more work is required before body residues can be readily applied to assessing the aquatic ecological risk of chemicals with specific modes of action such as OP compounds [28]. The different LBB values that were observed upon exposure to high and low concentrations of profenofos could be related to the fact that for greater inhibition of AChE, profenofos must be first oxidized to the oxon analog by the mixed-function oxidase system. Subsequently, the oxon analogs must reach their site of action and phosphorylate AChE. For the higher concentration used in this experiment, it is possible that the internal distribution and subsequent biotransformation into toxic metabolites was relatively slow compared to the uptake of the parent compound; therefore, the direct toxic effects of the parent compound may be predominating. For the low concentration of profenofos, it is postulated that the offered concentration was not high enough to reach harmful effect levels inside the fish and the toxic effects were dominated by inhibition of AChE. de Bruijn et al. [6] reported that at low exposure levels of chlorothion and methidathion, much lower LBB values were observed (0.41 mol/g and 0.75 mg/kg fish, respectively), probably due to a more specific mode of action at lower concentrations. However, fish that died within 24 h after exposure to high concentrations of these compounds had LBB values that were 6 to 10 times higher. This dependence of LBBs on aqueous levels of administered chemicals was considered by de Bruijn et al. [6] to be due to effects on the biotransformation processes. This suggests that LBB experi-

6 1804 Environ. Toxicol. Chem. 17, 1998 A. Kumar and J. C. Chapman ments should be undertaken at lower water concentrations to allow sufficient time for the chemical to be distributed inside the organism and to reach either the site of toxic action or the sites of metabolism. The lower level of AChE inhibition in the dead fish exposed to the higher level of profenofos (4 mg/l) is consistent with a lower rate of biotransformation. The greater AChE inhibition in the rainbow fish exposed to 0.1 mg/l profenofos is consistent with its biotransformation into the much more potent oxon AChE inhibitor during the longer exposure time. The sublethal exposure of rainbow fish to 10 g/l (nominal) of profenofos resulted in significant decreases in growth rates, food consumption rates, and food conversion efficiency, and after 21 d of exposure the rainbow fish exhibited loss in weight. For comparison, concentrations of profenofos between 1 and 5 g/l were measured in water from creeks and lagoons in northern NSW during the spraying season [5]. Crossland [29] reported that after 28 d of exposure to 3,4-dichloroaniline at concentrations of 39, 71, 120, and 210 g/l, growth rates in juvenile rainbow trout were reduced by 16, 19, 52, and 61%, respectively. Webb and Brett [30] suggested that toxicant-induced reduction in growth could be caused by a reduction in feeding rate or reduced food conversion efficiency. According to Mathers et al. [31], the reduction in growth rates due to exposure to toxicant could be caused by physiologic stress on food metabolism (reflected by reduced growth efficiency) and/ or a reduction in the food consumption rate (a reflection of reduction in motivation). Previous studies have shown that vertebrates, including salmon, will reject food contaminated with OP compounds [32]. During this study, sublethal exposure of rainbow fish to profenofos seemed to influence both the motivation to feed and the ability to grow. Profenofos-related reduction in the food conversion efficiency could be a result of change in the amount of food energy available for growth after metabolic demands were met. For instance, the hyperventilation observed in profenofos-exposed fish could result in less energy being available for growth and also in additional caloric requirements. Bostrom and Johansson [33] found that pentachlorophenol alters energy metabolism of fish by uncoupling oxidative phosphorylation and suppressing the activity of pyruvate kinase, thereby reducing glycolysis and stimulating the activity of some enzymes of the citric acid cycle and hexose monophosphate shunt. A consequence of this activity is an increase in respiration and increase in the rate at which lipid is used, presumably as substrate for the citric acid cycle [33]. In the present study, the lipid content in the whole body of rainbow fish exposed to profenofos was reduced by 25%, whereas in the control fish the lipid content increased. Hanes et al. [34] showed that, even when adequately fed, young coho salmon (Oncorhynchus kisutch), exposed over 14 d to 100 g/l pentachlorophenol, depleted their whole body lipid stores by 27%, whereas untreated fish increased their lipid reserves by 0.2%. This does not necessarily indicate similar mechanisms with profenofos exposure because pentachlorophenol is a respiratory uncoupler and has been used as a method of weight reduction in humans to specifically enhance metabolism and utilization of body lipid stores. The responses seen in profenofos-exposed fish are most probably related to reduced feeding and possible increased redirection of energy to maintaining the normal functions of the body. Increased caloric requirements caused by hyperactivity in the fish may have been responsible, at least partially, for the decrease in growth rate. Two measures of behavior, spontaneous activity and photokinesis, were assessed as indicators of sublethal toxicity in rainbow fish. Swimming activity is one of the most common and easily measured behavioral responses observed during toxicity studies and can be evaluated by observing orientation, posture, frequency, duration, and speed of swimming movements. Normal response to light stimulus may be modified by sublethal exposure to the chemical. Parameters measured in photobehavior may include changes in direction of orientation to a light stimulus (phototaxis), the intensity of the phototactic response, or the swimming rate (photokinesis). Deviation in spontaneous swimming activity may influence the ability of fish to capture prey by limiting the area of food search or may increase their conspicuousness to predators [35]. In the present study, spontaneous swimming activity increased in the fish exposed to sublethal profenofos; the rate of speed and time spent in swimming increased with the increase in duration of exposure. Similar behavioral responses were observed in the catfish Mystus vittatus [36] and Channa punctatus [37] when exposed to various concentrations of insecticides. Henry and Atchinson [38] observed hyperactivity in bluegills exposed to methyl parathion. The hyperactivity was defined as an almost continuous swimming combined with numerous jerks, partial jerks, and flickering of fins. Photokinesis behavior in the rainbow fish exposed to 10 g/l of profenofos increased significantly compared to the control fish, but no significant differences were found in the behavior observed on days 10 and 21 of exposure. The AChE activity was found to be inhibited by 81%, associated with profenofos residues up to mg/kg in the rainbow fish exposed for 21 d. Even after 10 d of exposure, AChE levels were inhibited by 70% and residues were mg/kg. Thus, profenofos not only puts a physiologic stress on fish but it also induces changes in behavior. An apparent correlation existed among the concentration of profenofos in fish, AChE inhibition, and behavioral changes monitored in rainbow fish during sublethal exposure. Fish exposed to higher concentrations of profenofos (1.2, 2.0, and 3.0 mg/l) showed abnormal behavioral patterns similar to those described in the literature, and these are consistent with increased inhibition of the AChE activity. McKim et al. [39] indicated that fish poisoned with cholinesterase inhibitors lose equilibrium, swim in a spiral or cork screw pattern, overreact to stimuli, have increased amplitude of respiration, and may have terminal tetany and convulsions. Sheepshead minnow (Cyprinodon variegatus) poisoned with diazinon had abnormal forward extension of the pectoral fins and were excessively reactive when startled [40]. Similar signs were observed in the Asiatic catfish Heteropneustes fossilis poisoned with malathion [41]. Some of these patterns were also observed in fish continuously exposed to only 0.1 mg/l but not until after 7 d of exposure. Drummond and Russom [42] classified chemicals according to general mode of action using behavioral and morphologic signs of stress from acute poisoning. The data from these tests favor the physical deformity syndrome described by Drummond and Russom [42], which is the specific mode of action for anticholinesterase agents. Thus, behavioral studies of test organisms conducted in conjunction with measurement of AChE inhibition and bioconcentration of toxicant residues may provide data to clarify the toxic effects caused by sublethal chemical concentrations of OP compounds. Once these relationships are established, the behavioral studies alone could

7 Profenofos toxicity to the rainbow fish Environ. Toxicol. Chem. 17, yield more comprehensive and cost-effective assessment of toxicity than provided by other biochemical and physiologic parameters. CONCLUSIONS The following conclusions can be drawn on the basis of the laboratory investigations of rainbowfish under acute and sublethal profenofos exposures. The eastern rainbow fish is considerably less sensitive to profenofos than are other reported species. The inhibition of AChE and the body residues of profenofos in the rainbow fish were found to be concentration-dependent during 96-h lethal and sublethal exposures. Profenofos exposures resulted in increased AChE inhibition, which could be related to greater accumulation of profenofos residues in the fish. Sublethal exposure of rainbow fish for longer duration resulted in lower body burdens in rainbow fish, and it is speculated that this is associated with biotransformation of profenofos. Sublethal exposures to profenofos affected food intake, growth rates, feeding rates, and food conversion efficiency in rainbow fish, which could be related to significant AChE inhibition. Significant behavioral changes such as spontaneous swimming activity and photokinesis during sublethal exposure of fish to profenofos could be related to significant AChE inhibition due to accumulation of profenofos residues in the bodies of the fish. Acknowledgement A. Kumar gratefully acknowledges the support of Environment Protection Authority, New South Wales, Sydney, Australia, and scholarship support from Ciba-Geigy Australia Limited. REFERENCES 1. Worthing CR, Walker SB The Pesticide Manual A World Compendium, 8th ed. British Crop Protection Council, Thornton Heath, UK. 2. Vittozzi L, Angelis GD A critical review of comparative acute toxicity data on freshwater fish. Aquat Toxicol 19: Land and Environment Court Unreported, Stein J, No 50003/92, , NSW vs Barlow. Environment Protection Authority, New South Wales, Sydney, Australia. 4. Nowak B, Goodsell A, Julli M Residues of endosulfan in carp as an indicator of exposure conditions. Ecotoxicology 4: Kumar A, Chapman JC, Ivantsoff W Profenofos residues in wild fish for cotton growing areas of NSW. Abstracts, 2nd World Congress, Society of Environmental Toxicology and Chemistry, Vancouver, BC, Canada, November 5 9, p de Bruijn J, Yedema E, Seinen W, Hermens J Lethal body burdens of four organophosphorous pesticides in guppy (Poecilia reticulata). Aquat Toxicol 20: Mineau P Chemicals in Agriculture: Cholinesterase Inhibiting Insecticides Their Impact on Wildlife and the Environment, Vol 2. Elsevier, London, United Kingdom. 8. Abdullah AR, Kumar A, Chapman JC Inhibition of acetylcholinesterase in the freshwater shrimp (Paratya australiensis) by profenofos. Environ Toxicol Contam 13: Chapman JC The role of ecotoxicity testing in assessing water quality. J Ecol 20: Sunderam RIM, Cheng DMH, Thompson GB Toxicity of endosulfan to native and introduced fish in Australia. Environ Toxicol Chem 11: Hamilton MA, Russo RC, Thurston RV Trimmed Spearman Karber methods for estimating median lethal concentration in toxicity bioassays. Environ Sci Technol 11: Correction. 12:417 (1978). 12. Crossland NO A method to evaluate effects of toxic chemicals on fish growth. Chemosphere 14: Little EE, Archeski RD, Flerov BA, Kozlovskaya VI Behavioural indicators of sublethal toxicity in rainbow trout. Arch Environ Contam Toxicol 19: Nowak B, Julli M Residues of endosulfan in wild fish from cotton growing areas in New South Wales, Australia. Toxicol Environ Chem 33: Ellman GL, Courtney KD, Anders V, Featherstone RM A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 3: Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ Protein measurement with the Folin phenol reagent. J Biol Chem 193: Underwood AJ Techniques of analysis of variance in experimental biology and ecology. Oceanogr Mar Biol Annu Rev 19: Zar JH Biostatistical Analysis. Prentice-Hall, Upper Saddle River, NJ, USA. 19. Mount DI, Boyle HW Parathion Use of blood concentration to diagnose mortality of fish. Environ Sci Technol 3: Bender ME Uptake and retention of malathion by carp. Prog Fish Cult 31: Zinkl JG, Lockhart WL, Kenny SA, Ward FJ The effects of cholinesterase inhibiting insecticides on fish. In Mineau P, ed, Chemicals in Agriculture: Cholinesterase Inhibiting Insecticides Their Impact on Wildlife and the Environment, Vol 2. Elsevier, London, UK, pp Coppage DL, Matthews E Short-term effects of organophosphate pesticides on cholinesterases of estuarine fishes and pink shrimp. Bull Environ Contam Toxicol 11: Post G, Leasure RA Sublethal effect of malathion to three salmonid species. Bull Environ Contam Toxicol 12: Verma SR, Tyagi AK, Bhatnagar MC, Dalela RC Organophosphate poisoning to some freshwater teleosts acetylcholinesterase inhibition. Bull Environ Contam Toxicol 21: Klaverkamp JF, Holden BR Brain acetylcholinesterase inhibition and hepatic activation of acephate and fenitrothion in rainbow trout (Salmo gairdneri). Can J Fish Aquat Sci 37: van Hoogen GJ, Opperhuizen A Toxicokinetics of chlorobenzenes in fish. Environ Toxicol Chem 7: Sijm DTHM, Schipper M, Opperhuizen A Toxicokinetics of halogenated benzenes in fish: Lethal body burden as a toxicological endpoint. Environ Toxicol Chem 12: Shephard BK Tissue screening concentrations for use in assessing ecological risks of chemical residues in aquatic biota. Abstracts, 2nd World Congress, Society of Environmental Toxicology and Chemistry, Vancouver, BC, Canada, November 5 9, p Crossland NO A method for evaluating effects of toxic chemicals on fish growth rates. In Adams WJ, Chapman GA, Landis WG, eds, Aquatic Toxicology and Hazard Assessment, Vol 10. STP 971. American Society for Testing and Materials, Philadelphia, PA, pp Webb PW, Brett JR Effects of sublethal concentrations of sodium pentachlorophenate on growth rate and conversion efficiency, and swimming performance in underyearling sockeye salmon (Oncorhynchus nerka). J Fish Res Board Can 30: Mathers RA, Brown JA, Johansen PH The growth and feeding behaviour response of largemouth bass (Micropterus salmoides) exposed to pentachlorophenol. Aquat Toxicol 6: Symons PEK Behaviour of young Atlantic salmon (Salmo salar) exposed to or force-fed fenitrothion, an organophosphate insecticide. J Fish Res Board Can 30: Bostrom SL, Johansson RG Effects of pentachlorophenol on enzymes involved in energy metabolism in the liver of the eel. Comp Biochem Physiol B 41: Hanes D, Kreuger H, Tinsley I, Bond C Influence of pentachlorophenol on fatty acids of coho salmon (Oncorhynchus kisutch). Proc West Pharmacol Soc 11: Laurence GC Comparative swimming abilities of fed and starved larval largemouth bass (Micropterus salmoides). J Fish Biol 4: Arunachalam S, Jeyalakshmi K, Aboobucker S Toxic and sublethal effects of carbaryl on a freshwater catfish, Mystus vittatus (Bloch). Arch Environ Contam Toxicol 9: Anees MA Acute toxicity of four organophosphorous insecticides to a freshwater teleost fish, Channa punctatus. Pak J Zool 7:

8 1806 Environ. Toxicol. Chem. 17, 1998 A. Kumar and J. C. Chapman 38. Henry MG, Atchinson GJ Behavioural effects of methyl parathion on social groups of bluegill (Lepomis macrochirus). Environ Toxicol Chem 3: McKim JM, Bradbury SP, Niemi GJ Fish acute toxicity syndromes and their use in the QSAR approach to hazard assessment. Environ Health Perspect 71: Goodman LR, Hansen DL, Coppage DL, Moore JC, Matthews E Diazinon: Chronic toxicity to, and brain acetylcholinesterase inhibition in, the sheepshead minnow (Cyprinodon variegatus). Trans Am Fish Soc 108: Singh VP, Gupta S, Saxena PK Evaluation of acute toxicity of carbaryl and malathion to freshwater teleosts Channa punctatus and Heteropneustes fossilis. Toxicol Lett 20: Drummond RA, Russom CL Behavioural toxicity syndromes: A promising tool for assessing toxicity mechanisms in juvenile fathead minnows. Environ Toxicol Chem 9:37 46.