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Sublethal effects of azamethiphos on shelter use by juvenile lobsters (Homarus americanus)
Aquaculture 181 Ž2000. 1–10 www.elsevier.nlrlocateraqua-online
Sublethal effects of azamethiphos on shelter use by juvenile lobsters žHomarus americanus / P. Abgrall b, R.W. Rangeley a , L.E. Burridge b
a,)
, P. Lawton
a
a Department of Fisheries and Oceans, Biological Station, St. Andrews, New Brunswick, Canada E0G 2X0 McGill UniÕersity, Department of Biology, 1205 Docteur Penfield AÕenue, Montreal, QC, Canada H3A 1B1
Accepted 13 May 1999
Abstract The use of pesticides to treat sea lice infestations in aquaculture may have negative impacts on non-target organisms such as the American lobster Ž Homarus americanus .. Juvenile lobsters spend most of their time in shelter to avoid predation. This study examined: Ž1. whether the organophosphate pesticide azamethiphos affected shelter use by juvenile lobsters; Ž2. whether leaving shelter was a form of azamethiphos avoidance; and Ž3. whether azamethiphos affected shelter re-entry. The experiments were performed on juvenile lobsters Ž6.5–8 mm carapace length. in individual aquaria with an artificial shelter placed on a sand substrate. Azamethiphos concentrations of 0, 100, 500 and 1000 mg ly1 were used. Ten-minute short-term pulsed exposures to azamethiphos mimicking field conditions resulted in no shelter exits or lobster deaths. Under continuous exposure to azamethiphos, all lobsters left their shelters and the time to shelter exit and death decreased with increasing azamethiphos concentration. Survival of lobsters placed in fresh seawater following shelter exit was 100% for the 100 mg ly1 treatment, 50% for the 500 mg ly1 treatment and 33% for the 1000 mg ly1 treatment. Time to re-enter the shelter following exposure to azamethiphos was significantly shorter than the control for lobsters exposed to 100 mg ly1 and significantly longer than the control for lobsters exposed to 1000 mg ly1. Shelter exit appears to be a form of avoidance behavior to high concentrations of azamethiphos. At concentrations used by the aquaculture industry Ž100 mg ly1 and short exposure times., azamethiphos would not affect lobster shelter use. However, if the concentration or exposure time increased, mortality could occur directly due to this pesticide or indirectly as a consequence of leaving shelter. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Homarus americanus; Azamethiphos; Aquaculture
)
Corresponding author. E-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 2 4 - 0
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P. Abgrall et al.r Aquaculture 181 (2000) 1–10
1. Introduction Salmon aquaculture has recently grown into a leading industry in Eastern Canada. In 1996, preliminary statistics from the Department of Fisheries and Oceans established the value of salmon aquaculture to be $290 million. In New Brunswick, the aquaculture industry is valued at ) $110 million as compared to $31 million for commercial landings of pelagic fish, groundfish and shellfish. Aquaculture is often presented as a solution to the decline of wild fish stock ŽBeveridge, 1987.. However, problems arising from salmon aquaculture have resulted in a reassessment of its true value. Outbreaks of pests such as sea lice ŽCopepoda:Caligidae. represent a major problem facing the industry due to the potentially devastating economic losses they cause. Various treatments including formaldehyde, organophosphates Žmalathion, trichlorfon, dichlorvos and azamethiphos., ivermectin, pyrethrum, hydrogen peroxide and garlic and onions have been suggested in an attempt to control these infestations ŽRoth et al., 1993.. These treatments are tested for both their effectiveness and their effects on the infected fish ŽSievers et al., 1995; Roth et al., 1996.. There is, however, a growing concern about the effects of pesticides and insecticides on non-target organisms ŽFlannagan, 1973; Lockhart et al., 1973; Eidt, 1975; Takimoto and Miyamoto, 1976; Kikuchi et al., 1984a; Kleiner et al., 1984; Takimoto et al., 1984; Egidius and Moster, 1987; Cusack and Johnson, 1990; Burridge and Haya, 1993.. Most studies look at direct mortality caused by chemicals, but there is increasing interest in sublethal effects Že.g., Kikuchi et al., 1984b; Sampath et al., 1990; Baticados and Tendencia, 1991; Sarkar and Konar, 1993; Kent and Caux, 1995; Taylor et al., 1995.. Sublethal effects can have important consequences such as increased risk of predation or reduced reproduction. Azamethiphos targets arthropods by inhibiting cholinesterase thus causing the accumulation of acetylcholine and preventing the restoration of the sensitivity of the synapse ŽO’Brien, 1967.. The lethal effects of azamethiphos on lobsters have recently been studied ŽBurridge et al., 1999. since their harvest represents an important economic activity. It has recently been introduced for sea lice treatment in salmon aquaculture and has been registered for use by Health Canada. The juvenile phase of the American lobster is divided in three stages: shelter-restricted, emergent Žmostly shelter confined. and vagile ŽLawton and Lavalli, 1995.. During the shelter-restricted juvenile stage, the lobsters have been found to be nocturnally active ŽPottle and Elner, 1982; Lawton, 1987. and prefer to shelter in more complex habitats ŽPottle and Elner, 1982; Johns and Mann, 1987.. These behaviors have been suggested to be adaptations to minimize risk of predation. Juvenile American lobsters are known to use more than one shelter ŽPottle and Elner, 1982. and thus, do not have an exclusive bond to a particular shelter. The introduction of a chemical could change the lobster’s normal behavior and result in the lobster leaving its shelter and becoming subject to increased predation risk. If azamethiphos additions inhibited the lobster’s physical ability to then return to shelter, this would also increase the risk of predation. We tested the hypothesis that the shelter use of shelter-restricted juvenile lobsters would be modified if exposed to short-term pulsed exposures ŽExperiment 1. or continuous exposure ŽExperiment 2. to azamethiphos. We predicted that Ž1. lobsters
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would leave their shelters, Ž2. the latency to exit shelter would decrease, and Ž3. the number of exits would increase with increasing azamethiphos concentration. Latency to death and between shelter exit and death were also expected to decrease with increasing concentration. We then tested whether leaving the shelter was an avoidance behavior ŽExperiment 3.. If it was an avoidance behavior, the lobster should survive if transferred to fresh seawater following shelter exit. In addition, we tested whether the exposure to azamethiphos affected the lobster’s ability to re-enter the shelter ŽExperiment 4.. We predicted that the time spent before the lobster re-entered the shelter would increase as the concentration of azamethiphos increased.
2. Materials and methods Artificial shelters Žsections of PVC pipe 50 mm long = 10 mm in diameter. were placed on the sand covered bottom of glass aquaria Ž24 cm = 19 cm = 20.5 cm deep.. Seawater was pumped directly from the Bay of Fundy surrounding the Biological Station in St. Andrews, New Brunswick Žtemperature 9.0–10.98C, salinity ; 31‰.. In Experiments 1 and 4, a through flow of seawater insured aeration and constant water temperature. Experiments 2 and 3 were performed in static seawater where air stones insured proper aeration and the aquaria were placed in a 10-cm high flowing seawater bath to insure constant water temperature. Water depth in aquaria was 20.5 cm. The light level reaching the shelters averaged 1.36 LUX. Lobster culture facilities in use at the Biological Station included individual housing of juvenile lobsters Žfollowing mass rearing. in small cells within plexiglass holding trays. These cells Ž; 5 cm = 6.5 cm = 6 cm deep with a seawater flow. contained neither substrate, nor shelter materials. Randomly picked, non-moulting laboratory reared shelter-restricted juvenile lobsters Ž6.5–8 mm carapace length. were transferred from individual holding cells to aquaria the evening before the experiment and left overnight to acclimatize. There was one lobster per aquarium. All lobster transfers in every experiment were done using a net. In Experiments 1, 2 and 3, the observer was standing on an elevated platform approximately 1 m away from the closest aquarium and avoided any disturbance to the lobsters. In Experiment 4, the observer was again standing 1 m away, but on the same level as the aquaria to facilitate the numerous lobster transfers required. The shelters were always placed perpendicularly in relation to the observer’s vision to allow simultaneous observation of both shelter openings. The latency measurements were taken to the nearest second using a stopwatch. 2.1. Experiment 1 The purpose of this experiment was to test whether short pulses of azamethiphos would modify the shelter use of juvenile lobsters. Three pulses of azamethiphos lasting 10 min each were added to a continuous ambient seawater flow Žflow rate s 0.4 lrmin.. at 1 h intervals. The seawater inflow was placed on the bottom of the aquarium 4 cm from the shelter and directed toward it. The azamethiphos was added by drops using marriott bottles to produce the desired concentration. Four concentrations were tested: 0,
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100, 500 and 1000 mg ly1 . Six trials were performed at each concentration. The number of times that the lobsters completely exited the shelter, the latency to exit shelter and the latency to death were measured relative to the time of pesticide addition. Death was defined as the time at which no movement of any part of the lobster could be observed. 2.2. Experiment 2 The purpose of this experiment was to test whether continuous exposure to azamethiphos would modify the shelter use of juvenile lobsters. Lobsters were continuously exposed to a known concentration of azamethiphos. Azamethiphos Žor seawater for the control. was added to the sea water using a pipette and stirred to produce the desired azamethiphos concentration in the aquarium. Four concentrations were tested: 0, 100, 500 and 1000 mg ly1 . Six trials were performed at each concentration. The number of times that the lobsters completely exited the shelter, the latency to exit shelter and the latency to death were measured relative to the time of pesticide addition. 2.3. Experiment 3 The purpose of this experiment was to test whether the shelter exit was an avoidance behavior. The same methodology as in Experiment 2 was used with the exception that the lobsters were taken out of the aquaria immediately after their first shelter exit. They were then placed in the holding cells from which they were initially taken. These holding cells had seawater flowing through them at the same temperature and salinity as the water in the aquaria. Three concentrations were tested: 100, 500 and 1000 mg ly1 . Twelve trials were performed at each concentration. The survival of the lobsters was assessed 1 and 24 h following their removal from aquaria. 2.4. Experiment 4 The purpose of this experiment was to test whether the exposure to azamethiphos affected the lobster’s ability to return in shelter. Lobsters were taken from their aquaria and placed individually in a 25-ml beaker containing a fixed concentration of azamethiphos for a 10-min time period. After this time, the lobsters were removed, dipped in fresh seawater, and returned to the same aquaria which all had a fresh seawater input. Four concentrations were tested: 0, 100, 500 and 1000 mg ly1 . Six trials were done at each concentration. The latency to re-enter shelter was measured relative to the time at which they were put back in the aquaria. The latency to re-enter shelter was defined as the time it took for the entire lobster to re-enter the shelter. 2.5. Statistical analysis Wilcoxon–Mann–Whitney tests were performed to test the significance of the difference of latency to exit shelter, latency to death or latency between shelter exit and death between the different concentrations in Experiments 2 and 4. Fisher exact tests were used to test the difference in the number of deaths between various concentrations
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of azamethiphos and to compare lobster survival at different concentrations between 1 and 24 h in Experiment 3. Finally, a Kruskal–Wallis one-way analysis of variance by rank was used to test the relationship between the latencies to re-enter shelter in Experiment 4. All statistical tests were performed according to Siegel and Castellan Ž1988..
3. Results 3.1. Experiment 1 Three 10-min pulses of azamethiphos at 1 h intervals did not affect shelter use of juvenile lobsters. The lobsters did not leave their shelter during the first two 10-min
Fig. 1. ŽA. Latency of lobsters to exit shelter in relation to concentration of azamethiphos during constant exposure ŽA.. ŽB. Latency of lobsters to death in relation to concentration of azamethiphos during constant exposure ŽB.. For both A and B, six lobsters were exposed to each concentration and were numbered from 1 to 18.
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exposures at any of the four treatment concentrations. Only one exit was observed during the third exposure. No deaths were observed at any concentration. 3.2. Experiment 2 All the lobsters continuously exposed at 100, 500 and 1000 mg ly1 of azamethiphos left their shelters once and subsequently died. No shelter exits or deaths were observed in the controls. As azamethiphos concentration increased, the latency to exit shelter ŽFig. 1A. and to death ŽFig. 1B. decreased. The difference of latency to exit shelter and latency to death between all concentrations was significant ŽWilcoxon–Mann–Whitney tests, m s n s 6, C L F 22 and p - 0.005 for all.. There was a significant difference of latency between shelter exit and death between 100 and 500 mg ly1 and 100 and 1000 mg ly1 ŽWilcoxon–Mann–Whitney tests, m s n s 6, C L s 23 and p - 0.05 for both., but not between 500 and 1000 mg ly1 ŽWilcoxon–Mann–Whitney tests, m s n s 6, C L s 38 and p ) 0.05.. 3.3. Experiment 3 No deaths were observed in lobsters following their removal from the aquaria with a 100 mg ly1 solution of azamethiphos. However, 67% and 50% of the lobsters died within 1 h of shelter exit after having been exposed to a 500 and 1000 mg ly1 solution of azamethiphos, respectively ŽFig. 2.. One hour following transfer to fresh seawater, there was a difference in the proportion of deaths between 100 and 500 mg ly1 ŽFisher
Fig. 2. Survival of lobsters put in fresh seawater following shelter exit. Comparison of survival after 1 Žopen bar. and 24 h Žshaded bar. in fresh seawater. Sample sizes12 lobsters in each treatment.
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Fig. 3. Latency to re-enter shelter following a 10-min exposure to azamethiphos. Six lobsters were exposed to each concentration.
exact test, p - 0.001. and between 100 and 1000 mg ly1 ŽFisher exact test, p - 0.01., but not between 500 and 1000 mg ly1 ŽFisher exact test, p s 0.89.. There was no significant increase in the number of deaths between 1 and 24 h following transfer to fresh seawater ŽFisher exact test, p - 0.05 for all concentrations.. No additional deaths were observed in the following two weeks. 3.4. Experiment 4 There was an overall increase in the lobsters’ latency to re-enter shelter as the concentration of azamethiphos to which it had previously been exposed increased ŽKruskal–Wallis one-way analysis of variance by ranks, H s 10.27, df s 3, p - 0.05. ŽFig. 3.. The latency to re-enter shelter was significantly lower following exposure to 100 mg ly1 and higher following exposure to 1000 mg ly1 than in the control ŽWilcoxon–Mann–Whitney tests, m s n s 6, C L F 27 and p - 0.05 for both.. The difference between 500 mg ly1 and the control was not significant ŽWilcoxon–Mann– Whitney test, m s n s 6, C L s 34, p s 0.24.. 4. Discussion Experiment 1 which was designed to mimic field applications through pulsed exposures showed that azamethiphos had no effect on the shelter use of juvenile lobsters
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at the four treatment concentrations used. Water samples collected from inside the shelter during additional trials have, however, indicated that the concentration to which the lobsters were actually exposed to was lower than expected by an average of 27.4% Žrange: 19.5–35.2%, n s 2.. The concentration values reported in this experiment are the ones initially entering the aquaria. These concentrations were all well controlled. The observed dilution can be explained by the 4-cm distance between the outflow and the shelter opening. The fact that the concentrations of azamethiphos reaching the shelter were partly diluted is not of great importance in this case because we still obtain a maximum exposure concentration averaging 726 mg ly1 . This is more than seven times greater than the concentration used to treat the cages. Experiments using a dye to follow the dispersion of chemicals ŽDobson and Tack, 1991. have shown that the concentration of pesticide reaching the sea floor was much lower than the initial concentration released at the surface. Therefore, under natural conditions, it does not seem that azamethiphos would constitute a threat to juvenile lobster’s through increased predation risk from shelter exit. Additional tests also demonstrated a residue of 7.2% of the original concentration Žrange: 1.9–16.7%, n s 3. left in the aquaria following the 50-min period separating the azamethiphos pulses. This means that the lobsters were not actually exposed to independent pulses of azamethiphos and were in fact exposed to the pesticide continuously at lower doses with periods of high doses. This does not affect the conclusion since there were no effects observed in any concentration even with this prolonged exposure to azamethiphos. However, if the concentration reaching the shelters lasted long enough, it could represent a risk to the lobster as shown in Experiment 2 where all of the lobsters only exited their shelters once and died due to their continuous exposure to azamethiphos. None of these deaths occurred in shelter and, as predicted, the latency to exit shelter and latency to death both decreased with increasing azamethiphos concentration ŽFig. 1A and B.. From this experiment, it seems that the duration of exposure is the critical factor to control when applying azamethiphos in the field. Since the exact concentration reaching the lobsters in the field is not known, we can determine a maximum tolerance time to azamethiphos exposure using the treatment concentration Ž100 mg ly1 .. We can predict that exposures of less than 200 min should not affect the lobster’s shelter use ŽFig. 1B.. We then tested if this shelter exit affected post-exit survival and potentially allowed the lobster to escape the noxious area. If the lobsters always die following shelter exit, the shelter exit would not represent an important behavior in terms of increasing survival and death through predation could not really be considered as an indirect mortality due to the exposure to azamethiphos. A 100% survival was observed at the field treatment concentration Ž100 mg ly1 ., but only 33–50% of the lobsters survived at 500 and 1000 mg ly1 , respectively ŽFig. 2.. This shows that if lobsters exit and get to clean water they can survive, in the absence of predators. If the lobster exits the shelter, but is not physically able to move out of the range of the pesticide, the shelter exit will not increase its survival. The important factors needed in this case to further understand the risk facing the lobsters will be the juvenile lobster’s mobility, the concentration surrounding the lobster and the range over which the
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pesticide is found. These factors will allow the determination of the duration of exposure following shelter exit. Further studies are necessary to determine the interaction between the dispersal and action of the pesticide and the behavioral responses of the lobsters. Lobsters in Experiment 4 returned to shelter more quickly at low concentration than those exposed to 1000 mg ly1 ŽFig. 3.. A possible explanation for this pattern is that the exposure to a low concentration produces an alarm response from the lobster while the exposure to a high concentration produces an orientation or locomotion disorder due to the inhibitory effects of the pesticide. At the supplier-recommended application rates, the concentration reaching a shelter should be at most 100 mg ly1 . Thus, we might expect juvenile lobsters which are out of their shelter to quickly find refuge and interrupt whatever activity they were pursuing. Such a situation might occur if cages were treated at night when lobsters are out of shelter. While it seems that the pesticide would disrupt the lobster’s behavior, it does not seem that its survival would be affected in the short term. It is, however, unlikely that any lobster would ever be exposed to a concentration of 100 mg ly1 following a single treatment. The effects of chronic exposure to low concentrations of azamethiphos on the lobster’s shelter use and behavior in general are not known. Our experiments in laboratory have shown that higher concentrations of azamethiphos can affect the shelter use of juvenile lobsters. It is unlikely, however, that the shelter use of juvenile lobsters would be affected by azamethiphos released under operational conditions. When considering the impacts of chemicals on non-target organisms, it is important to include the sublethal impacts as these may have long term effects at the population level. This bioassay may prove to be an efficient way to look at behavioral effects and could, after standardization, be used as a screening procedure prior to the introduction of new pesticides. Acknowledgements The authors thank Dr. D.L. Kramer for his help in analysing the data and organizing the manuscript and Murray Humphries for assistance with software. Laboratory facilities and funding for the exposure trials were provided by the Department of Fisheries and Oceans through operating funds to L.E. Burridge and P. Lawton. We thank S. Waddy and W. Young-Lai for providing juvenile lobsters from the lobster hatchery they maintain at the Biological Station, St. Andrews. All experiments followed Canadian Council on Animal Care Guidelines. References Baticados, Ma. C.L., Tendencia, E.A., 1991. Effects of Gusathion A on the survival and shell quality of juvenile Penaeus monodon. Aquaculture 93, 9–19. Beveridge, M.C.M. 1987. Cage Aquaculture. Fishing News Books, England, 352 pp. Burridge, L.E., Haya, K., 1993. The lethality of ivermectin, a potential agent for treatment of salmonids against sea lice, to the shrimp Crangon septemspinosa. Aquaculture 117, 9–14. Burridge, L.E., Haya, K., Zitko, V., Waddy, S., 1999. The lethality of Salmosanw Žazamethiphos. to American lobster Ž Homarus americanus. larvae, post-larvae, and adults. Ecotoxicol. Environ. Saf. 43, 165–169.
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Cusack, R., Johnson, G., 1990. A study of dichlorvos ŽNuvan; 2,2 dichloroethenyl dimethyl phosphate., a therapeutic agent for the treatment of salmonids infected with sea lice Ž Lepeophtheirus salmonis .. Aquaculture 90, 101–112. Dobson, D.P., Tack, T.J., 1991. Evaluation of the dispersion of treatment solutions of dichlorvos from marine salmon pens. Aquaculture 95, 15–32. Egidius, E., Moster, B., 1987. Effect of Neguvon w and Nuvan w treatment on crabs Ž Cancer pagurus, C. maenas ., lobster Ž Homarus gammarus. and blue mussel Ž Mytilus edulis .. Aquaculture 60, 165–168. Eidt, D.C., 1975. The effect of fenitrothion from large-scale forest spraying on benthos in New Brunswick headwaters streams. Can. Ent. 107, 743–760. Flannagan, J.F., 1973. Field and laboratory studies of the effect of exposure to fenitrothion on freshwater aquatic invertebrates. Manit. Entomol. 7, 15–25. Johns, P.M., Mann, K.H., 1987. An experimental investigation of juvenile lobster habitat preference and mortality among habitats of varying structural complexity. J. Exp. Mar. Biol. Ecol. 109, 275–285. Kent, R.A., Caux, P.-Y., 1995. Sublethal effects of the insecticide fenitrothion on freshwater phytoplankton. Can. J. Bot. 73, 45–53. Kleiner, C.F., Anderson, R.L., Tanner, D.K., 1984. Toxicity of fenitrothion to fathead minnows Ž Pimephales promelas . and alternative exposure duration studies with fenitrothion and endosulfan. Arch. Environ. Contam. Toxicol. 13, 573–578. Kikuchi, R., Yasutaniya, T., Takimoto, Y., Yamada, H., Miyamoto, J., 1984a. Accumulation and metabolism of fenitrothion in three species of algae. J. Pestic. Sci. 9, 331–337. Kikuchi, R., Takimoto, Y., Yamada, H., Miyamoto, J., 1984b. Effect of fenitrothion on growth of a green alga, Chlorella Õulgaris. J. Pestic. Sci. 9, 325–329. Lawton, P., 1987. Diel activity and foraging behavior of juvenile American lobsters, Homarus americanus. Can. J. Fish. Aquat. Sci. 44, 1195–1205. Lawton, P., Lavalli, K.L., 1995. Chapter 4: Postlarval, juvenile, adolescent, and adult ecology. In: Factor, J.R. ŽEd.., Biology of the Lobster Homarus Americanus. Academic Press, pp. 47–88. Lockhart, W.L., Metner, D.A., Grift, N., 1973. Biochemical and residue studies on rainbow trout Ž Salmo gairdneri . following field and laboratory exposures to fenitrothion. Manit. Entomol. 7, 26–36. O’Brien, R.D., 1967. Insecticides: Action and Metabolism. Academic Press, New York, pp. 1–82. Pottle, R.A., Elner, R.W., 1982. Substrate preference behavior of juvenile American lobsters, Homarus americanus, in gravel and silt-clay sediments. Can. J. Fish. Aquat. Sci. 39, 928–932. Roth, M., Richards, R.H., Sommerville, C., 1993. Current practices in the chemotherapeutic control of sea lice infestations in aquaculture: a review. J. Fish Dis. 16, 1–26. Roth, M., Richards, R.H., Dobson, D.P., Gordon, H.R., 1996. Field trials on the efficacy of the organophosphorus compound azamethiphos for the control of sea lice ŽCopepoda: Caligidae. infestations of farmed Atlantic salmon Ž Salmo salar .. Aquaculture 140, 217–239. Sampath, K., Stanley, A., Sivakumar, V., 1990. Respiratory responses of Heteropneustes fossilis. Env. Ecol. 8 Ž1., 92–94. Sarkar, U.K., Konar, S.K., 1993. Sublethal effects of pesticide, heavy metal, detergent and petroleum product in three combinations on fish. Env. Ecol. 11 Ž3., 609–615. Siegel, S., Castellan, N.J., Jr., 1988. Nonparametric Statistics for the Behavioral Sciences, 2nd edn., McGraw-Hill, 399 pp. Sievers, G., Palacios, P., Inostroza, R., Dolz, ¨ H., 1995. Evaluation of the toxicity of 8 insecticides in Salmo salar and the in vitro effects against the isopode parasite, Ceratothoa gaudichaudii. Aquaculture 134, 9–16. Takimoto, Y., Miyamoto, J., 1976. Studies on accumulation and metabolism of sumithion in fish. J. Pestic. Sci. 1, 261–271. Takimoto, Y., Hagino, S., Yamada, H., Miyamoto, J., 1984. The acute toxicity of fenitrothion to killifish Ž Oryzias latipes . at twelve different stages of its life history. J. Pestic. Sci. 9, 463–470. Taylor, E.J., Morrison, J.E., Blockwell, S.J., Tarr, A., Pascoe, D., 1995. Effects of lindane on the predator-prey interaction between Hydra oligactis Pallas and Daphnia magna Strauss. Arch. Environ. Contam. Toxicol. 29, 291–296.
Sublethal effects of azamethiphos on shelter use by juvenile lobsters žHomarus americanus / P. Abgrall b, R.W. Rangeley a , L.E. Burridge b
a,)
, P. Lawton
a
a Department of Fisheries and Oceans, Biological Station, St. Andrews, New Brunswick, Canada E0G 2X0 McGill UniÕersity, Department of Biology, 1205 Docteur Penfield AÕenue, Montreal, QC, Canada H3A 1B1
Accepted 13 May 1999
Abstract The use of pesticides to treat sea lice infestations in aquaculture may have negative impacts on non-target organisms such as the American lobster Ž Homarus americanus .. Juvenile lobsters spend most of their time in shelter to avoid predation. This study examined: Ž1. whether the organophosphate pesticide azamethiphos affected shelter use by juvenile lobsters; Ž2. whether leaving shelter was a form of azamethiphos avoidance; and Ž3. whether azamethiphos affected shelter re-entry. The experiments were performed on juvenile lobsters Ž6.5–8 mm carapace length. in individual aquaria with an artificial shelter placed on a sand substrate. Azamethiphos concentrations of 0, 100, 500 and 1000 mg ly1 were used. Ten-minute short-term pulsed exposures to azamethiphos mimicking field conditions resulted in no shelter exits or lobster deaths. Under continuous exposure to azamethiphos, all lobsters left their shelters and the time to shelter exit and death decreased with increasing azamethiphos concentration. Survival of lobsters placed in fresh seawater following shelter exit was 100% for the 100 mg ly1 treatment, 50% for the 500 mg ly1 treatment and 33% for the 1000 mg ly1 treatment. Time to re-enter the shelter following exposure to azamethiphos was significantly shorter than the control for lobsters exposed to 100 mg ly1 and significantly longer than the control for lobsters exposed to 1000 mg ly1. Shelter exit appears to be a form of avoidance behavior to high concentrations of azamethiphos. At concentrations used by the aquaculture industry Ž100 mg ly1 and short exposure times., azamethiphos would not affect lobster shelter use. However, if the concentration or exposure time increased, mortality could occur directly due to this pesticide or indirectly as a consequence of leaving shelter. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Homarus americanus; Azamethiphos; Aquaculture
)
Corresponding author. E-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 2 4 - 0
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1. Introduction Salmon aquaculture has recently grown into a leading industry in Eastern Canada. In 1996, preliminary statistics from the Department of Fisheries and Oceans established the value of salmon aquaculture to be $290 million. In New Brunswick, the aquaculture industry is valued at ) $110 million as compared to $31 million for commercial landings of pelagic fish, groundfish and shellfish. Aquaculture is often presented as a solution to the decline of wild fish stock ŽBeveridge, 1987.. However, problems arising from salmon aquaculture have resulted in a reassessment of its true value. Outbreaks of pests such as sea lice ŽCopepoda:Caligidae. represent a major problem facing the industry due to the potentially devastating economic losses they cause. Various treatments including formaldehyde, organophosphates Žmalathion, trichlorfon, dichlorvos and azamethiphos., ivermectin, pyrethrum, hydrogen peroxide and garlic and onions have been suggested in an attempt to control these infestations ŽRoth et al., 1993.. These treatments are tested for both their effectiveness and their effects on the infected fish ŽSievers et al., 1995; Roth et al., 1996.. There is, however, a growing concern about the effects of pesticides and insecticides on non-target organisms ŽFlannagan, 1973; Lockhart et al., 1973; Eidt, 1975; Takimoto and Miyamoto, 1976; Kikuchi et al., 1984a; Kleiner et al., 1984; Takimoto et al., 1984; Egidius and Moster, 1987; Cusack and Johnson, 1990; Burridge and Haya, 1993.. Most studies look at direct mortality caused by chemicals, but there is increasing interest in sublethal effects Že.g., Kikuchi et al., 1984b; Sampath et al., 1990; Baticados and Tendencia, 1991; Sarkar and Konar, 1993; Kent and Caux, 1995; Taylor et al., 1995.. Sublethal effects can have important consequences such as increased risk of predation or reduced reproduction. Azamethiphos targets arthropods by inhibiting cholinesterase thus causing the accumulation of acetylcholine and preventing the restoration of the sensitivity of the synapse ŽO’Brien, 1967.. The lethal effects of azamethiphos on lobsters have recently been studied ŽBurridge et al., 1999. since their harvest represents an important economic activity. It has recently been introduced for sea lice treatment in salmon aquaculture and has been registered for use by Health Canada. The juvenile phase of the American lobster is divided in three stages: shelter-restricted, emergent Žmostly shelter confined. and vagile ŽLawton and Lavalli, 1995.. During the shelter-restricted juvenile stage, the lobsters have been found to be nocturnally active ŽPottle and Elner, 1982; Lawton, 1987. and prefer to shelter in more complex habitats ŽPottle and Elner, 1982; Johns and Mann, 1987.. These behaviors have been suggested to be adaptations to minimize risk of predation. Juvenile American lobsters are known to use more than one shelter ŽPottle and Elner, 1982. and thus, do not have an exclusive bond to a particular shelter. The introduction of a chemical could change the lobster’s normal behavior and result in the lobster leaving its shelter and becoming subject to increased predation risk. If azamethiphos additions inhibited the lobster’s physical ability to then return to shelter, this would also increase the risk of predation. We tested the hypothesis that the shelter use of shelter-restricted juvenile lobsters would be modified if exposed to short-term pulsed exposures ŽExperiment 1. or continuous exposure ŽExperiment 2. to azamethiphos. We predicted that Ž1. lobsters
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would leave their shelters, Ž2. the latency to exit shelter would decrease, and Ž3. the number of exits would increase with increasing azamethiphos concentration. Latency to death and between shelter exit and death were also expected to decrease with increasing concentration. We then tested whether leaving the shelter was an avoidance behavior ŽExperiment 3.. If it was an avoidance behavior, the lobster should survive if transferred to fresh seawater following shelter exit. In addition, we tested whether the exposure to azamethiphos affected the lobster’s ability to re-enter the shelter ŽExperiment 4.. We predicted that the time spent before the lobster re-entered the shelter would increase as the concentration of azamethiphos increased.
2. Materials and methods Artificial shelters Žsections of PVC pipe 50 mm long = 10 mm in diameter. were placed on the sand covered bottom of glass aquaria Ž24 cm = 19 cm = 20.5 cm deep.. Seawater was pumped directly from the Bay of Fundy surrounding the Biological Station in St. Andrews, New Brunswick Žtemperature 9.0–10.98C, salinity ; 31‰.. In Experiments 1 and 4, a through flow of seawater insured aeration and constant water temperature. Experiments 2 and 3 were performed in static seawater where air stones insured proper aeration and the aquaria were placed in a 10-cm high flowing seawater bath to insure constant water temperature. Water depth in aquaria was 20.5 cm. The light level reaching the shelters averaged 1.36 LUX. Lobster culture facilities in use at the Biological Station included individual housing of juvenile lobsters Žfollowing mass rearing. in small cells within plexiglass holding trays. These cells Ž; 5 cm = 6.5 cm = 6 cm deep with a seawater flow. contained neither substrate, nor shelter materials. Randomly picked, non-moulting laboratory reared shelter-restricted juvenile lobsters Ž6.5–8 mm carapace length. were transferred from individual holding cells to aquaria the evening before the experiment and left overnight to acclimatize. There was one lobster per aquarium. All lobster transfers in every experiment were done using a net. In Experiments 1, 2 and 3, the observer was standing on an elevated platform approximately 1 m away from the closest aquarium and avoided any disturbance to the lobsters. In Experiment 4, the observer was again standing 1 m away, but on the same level as the aquaria to facilitate the numerous lobster transfers required. The shelters were always placed perpendicularly in relation to the observer’s vision to allow simultaneous observation of both shelter openings. The latency measurements were taken to the nearest second using a stopwatch. 2.1. Experiment 1 The purpose of this experiment was to test whether short pulses of azamethiphos would modify the shelter use of juvenile lobsters. Three pulses of azamethiphos lasting 10 min each were added to a continuous ambient seawater flow Žflow rate s 0.4 lrmin.. at 1 h intervals. The seawater inflow was placed on the bottom of the aquarium 4 cm from the shelter and directed toward it. The azamethiphos was added by drops using marriott bottles to produce the desired concentration. Four concentrations were tested: 0,
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100, 500 and 1000 mg ly1 . Six trials were performed at each concentration. The number of times that the lobsters completely exited the shelter, the latency to exit shelter and the latency to death were measured relative to the time of pesticide addition. Death was defined as the time at which no movement of any part of the lobster could be observed. 2.2. Experiment 2 The purpose of this experiment was to test whether continuous exposure to azamethiphos would modify the shelter use of juvenile lobsters. Lobsters were continuously exposed to a known concentration of azamethiphos. Azamethiphos Žor seawater for the control. was added to the sea water using a pipette and stirred to produce the desired azamethiphos concentration in the aquarium. Four concentrations were tested: 0, 100, 500 and 1000 mg ly1 . Six trials were performed at each concentration. The number of times that the lobsters completely exited the shelter, the latency to exit shelter and the latency to death were measured relative to the time of pesticide addition. 2.3. Experiment 3 The purpose of this experiment was to test whether the shelter exit was an avoidance behavior. The same methodology as in Experiment 2 was used with the exception that the lobsters were taken out of the aquaria immediately after their first shelter exit. They were then placed in the holding cells from which they were initially taken. These holding cells had seawater flowing through them at the same temperature and salinity as the water in the aquaria. Three concentrations were tested: 100, 500 and 1000 mg ly1 . Twelve trials were performed at each concentration. The survival of the lobsters was assessed 1 and 24 h following their removal from aquaria. 2.4. Experiment 4 The purpose of this experiment was to test whether the exposure to azamethiphos affected the lobster’s ability to return in shelter. Lobsters were taken from their aquaria and placed individually in a 25-ml beaker containing a fixed concentration of azamethiphos for a 10-min time period. After this time, the lobsters were removed, dipped in fresh seawater, and returned to the same aquaria which all had a fresh seawater input. Four concentrations were tested: 0, 100, 500 and 1000 mg ly1 . Six trials were done at each concentration. The latency to re-enter shelter was measured relative to the time at which they were put back in the aquaria. The latency to re-enter shelter was defined as the time it took for the entire lobster to re-enter the shelter. 2.5. Statistical analysis Wilcoxon–Mann–Whitney tests were performed to test the significance of the difference of latency to exit shelter, latency to death or latency between shelter exit and death between the different concentrations in Experiments 2 and 4. Fisher exact tests were used to test the difference in the number of deaths between various concentrations
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of azamethiphos and to compare lobster survival at different concentrations between 1 and 24 h in Experiment 3. Finally, a Kruskal–Wallis one-way analysis of variance by rank was used to test the relationship between the latencies to re-enter shelter in Experiment 4. All statistical tests were performed according to Siegel and Castellan Ž1988..
3. Results 3.1. Experiment 1 Three 10-min pulses of azamethiphos at 1 h intervals did not affect shelter use of juvenile lobsters. The lobsters did not leave their shelter during the first two 10-min
Fig. 1. ŽA. Latency of lobsters to exit shelter in relation to concentration of azamethiphos during constant exposure ŽA.. ŽB. Latency of lobsters to death in relation to concentration of azamethiphos during constant exposure ŽB.. For both A and B, six lobsters were exposed to each concentration and were numbered from 1 to 18.
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exposures at any of the four treatment concentrations. Only one exit was observed during the third exposure. No deaths were observed at any concentration. 3.2. Experiment 2 All the lobsters continuously exposed at 100, 500 and 1000 mg ly1 of azamethiphos left their shelters once and subsequently died. No shelter exits or deaths were observed in the controls. As azamethiphos concentration increased, the latency to exit shelter ŽFig. 1A. and to death ŽFig. 1B. decreased. The difference of latency to exit shelter and latency to death between all concentrations was significant ŽWilcoxon–Mann–Whitney tests, m s n s 6, C L F 22 and p - 0.005 for all.. There was a significant difference of latency between shelter exit and death between 100 and 500 mg ly1 and 100 and 1000 mg ly1 ŽWilcoxon–Mann–Whitney tests, m s n s 6, C L s 23 and p - 0.05 for both., but not between 500 and 1000 mg ly1 ŽWilcoxon–Mann–Whitney tests, m s n s 6, C L s 38 and p ) 0.05.. 3.3. Experiment 3 No deaths were observed in lobsters following their removal from the aquaria with a 100 mg ly1 solution of azamethiphos. However, 67% and 50% of the lobsters died within 1 h of shelter exit after having been exposed to a 500 and 1000 mg ly1 solution of azamethiphos, respectively ŽFig. 2.. One hour following transfer to fresh seawater, there was a difference in the proportion of deaths between 100 and 500 mg ly1 ŽFisher
Fig. 2. Survival of lobsters put in fresh seawater following shelter exit. Comparison of survival after 1 Žopen bar. and 24 h Žshaded bar. in fresh seawater. Sample sizes12 lobsters in each treatment.
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Fig. 3. Latency to re-enter shelter following a 10-min exposure to azamethiphos. Six lobsters were exposed to each concentration.
exact test, p - 0.001. and between 100 and 1000 mg ly1 ŽFisher exact test, p - 0.01., but not between 500 and 1000 mg ly1 ŽFisher exact test, p s 0.89.. There was no significant increase in the number of deaths between 1 and 24 h following transfer to fresh seawater ŽFisher exact test, p - 0.05 for all concentrations.. No additional deaths were observed in the following two weeks. 3.4. Experiment 4 There was an overall increase in the lobsters’ latency to re-enter shelter as the concentration of azamethiphos to which it had previously been exposed increased ŽKruskal–Wallis one-way analysis of variance by ranks, H s 10.27, df s 3, p - 0.05. ŽFig. 3.. The latency to re-enter shelter was significantly lower following exposure to 100 mg ly1 and higher following exposure to 1000 mg ly1 than in the control ŽWilcoxon–Mann–Whitney tests, m s n s 6, C L F 27 and p - 0.05 for both.. The difference between 500 mg ly1 and the control was not significant ŽWilcoxon–Mann– Whitney test, m s n s 6, C L s 34, p s 0.24.. 4. Discussion Experiment 1 which was designed to mimic field applications through pulsed exposures showed that azamethiphos had no effect on the shelter use of juvenile lobsters
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at the four treatment concentrations used. Water samples collected from inside the shelter during additional trials have, however, indicated that the concentration to which the lobsters were actually exposed to was lower than expected by an average of 27.4% Žrange: 19.5–35.2%, n s 2.. The concentration values reported in this experiment are the ones initially entering the aquaria. These concentrations were all well controlled. The observed dilution can be explained by the 4-cm distance between the outflow and the shelter opening. The fact that the concentrations of azamethiphos reaching the shelter were partly diluted is not of great importance in this case because we still obtain a maximum exposure concentration averaging 726 mg ly1 . This is more than seven times greater than the concentration used to treat the cages. Experiments using a dye to follow the dispersion of chemicals ŽDobson and Tack, 1991. have shown that the concentration of pesticide reaching the sea floor was much lower than the initial concentration released at the surface. Therefore, under natural conditions, it does not seem that azamethiphos would constitute a threat to juvenile lobster’s through increased predation risk from shelter exit. Additional tests also demonstrated a residue of 7.2% of the original concentration Žrange: 1.9–16.7%, n s 3. left in the aquaria following the 50-min period separating the azamethiphos pulses. This means that the lobsters were not actually exposed to independent pulses of azamethiphos and were in fact exposed to the pesticide continuously at lower doses with periods of high doses. This does not affect the conclusion since there were no effects observed in any concentration even with this prolonged exposure to azamethiphos. However, if the concentration reaching the shelters lasted long enough, it could represent a risk to the lobster as shown in Experiment 2 where all of the lobsters only exited their shelters once and died due to their continuous exposure to azamethiphos. None of these deaths occurred in shelter and, as predicted, the latency to exit shelter and latency to death both decreased with increasing azamethiphos concentration ŽFig. 1A and B.. From this experiment, it seems that the duration of exposure is the critical factor to control when applying azamethiphos in the field. Since the exact concentration reaching the lobsters in the field is not known, we can determine a maximum tolerance time to azamethiphos exposure using the treatment concentration Ž100 mg ly1 .. We can predict that exposures of less than 200 min should not affect the lobster’s shelter use ŽFig. 1B.. We then tested if this shelter exit affected post-exit survival and potentially allowed the lobster to escape the noxious area. If the lobsters always die following shelter exit, the shelter exit would not represent an important behavior in terms of increasing survival and death through predation could not really be considered as an indirect mortality due to the exposure to azamethiphos. A 100% survival was observed at the field treatment concentration Ž100 mg ly1 ., but only 33–50% of the lobsters survived at 500 and 1000 mg ly1 , respectively ŽFig. 2.. This shows that if lobsters exit and get to clean water they can survive, in the absence of predators. If the lobster exits the shelter, but is not physically able to move out of the range of the pesticide, the shelter exit will not increase its survival. The important factors needed in this case to further understand the risk facing the lobsters will be the juvenile lobster’s mobility, the concentration surrounding the lobster and the range over which the
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pesticide is found. These factors will allow the determination of the duration of exposure following shelter exit. Further studies are necessary to determine the interaction between the dispersal and action of the pesticide and the behavioral responses of the lobsters. Lobsters in Experiment 4 returned to shelter more quickly at low concentration than those exposed to 1000 mg ly1 ŽFig. 3.. A possible explanation for this pattern is that the exposure to a low concentration produces an alarm response from the lobster while the exposure to a high concentration produces an orientation or locomotion disorder due to the inhibitory effects of the pesticide. At the supplier-recommended application rates, the concentration reaching a shelter should be at most 100 mg ly1 . Thus, we might expect juvenile lobsters which are out of their shelter to quickly find refuge and interrupt whatever activity they were pursuing. Such a situation might occur if cages were treated at night when lobsters are out of shelter. While it seems that the pesticide would disrupt the lobster’s behavior, it does not seem that its survival would be affected in the short term. It is, however, unlikely that any lobster would ever be exposed to a concentration of 100 mg ly1 following a single treatment. The effects of chronic exposure to low concentrations of azamethiphos on the lobster’s shelter use and behavior in general are not known. Our experiments in laboratory have shown that higher concentrations of azamethiphos can affect the shelter use of juvenile lobsters. It is unlikely, however, that the shelter use of juvenile lobsters would be affected by azamethiphos released under operational conditions. When considering the impacts of chemicals on non-target organisms, it is important to include the sublethal impacts as these may have long term effects at the population level. This bioassay may prove to be an efficient way to look at behavioral effects and could, after standardization, be used as a screening procedure prior to the introduction of new pesticides. Acknowledgements The authors thank Dr. D.L. Kramer for his help in analysing the data and organizing the manuscript and Murray Humphries for assistance with software. Laboratory facilities and funding for the exposure trials were provided by the Department of Fisheries and Oceans through operating funds to L.E. Burridge and P. Lawton. We thank S. Waddy and W. Young-Lai for providing juvenile lobsters from the lobster hatchery they maintain at the Biological Station, St. Andrews. All experiments followed Canadian Council on Animal Care Guidelines. References Baticados, Ma. C.L., Tendencia, E.A., 1991. Effects of Gusathion A on the survival and shell quality of juvenile Penaeus monodon. Aquaculture 93, 9–19. Beveridge, M.C.M. 1987. Cage Aquaculture. Fishing News Books, England, 352 pp. Burridge, L.E., Haya, K., 1993. The lethality of ivermectin, a potential agent for treatment of salmonids against sea lice, to the shrimp Crangon septemspinosa. Aquaculture 117, 9–14. Burridge, L.E., Haya, K., Zitko, V., Waddy, S., 1999. The lethality of Salmosanw Žazamethiphos. to American lobster Ž Homarus americanus. larvae, post-larvae, and adults. Ecotoxicol. Environ. Saf. 43, 165–169.
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Cusack, R., Johnson, G., 1990. A study of dichlorvos ŽNuvan; 2,2 dichloroethenyl dimethyl phosphate., a therapeutic agent for the treatment of salmonids infected with sea lice Ž Lepeophtheirus salmonis .. Aquaculture 90, 101–112. Dobson, D.P., Tack, T.J., 1991. Evaluation of the dispersion of treatment solutions of dichlorvos from marine salmon pens. Aquaculture 95, 15–32. Egidius, E., Moster, B., 1987. Effect of Neguvon w and Nuvan w treatment on crabs Ž Cancer pagurus, C. maenas ., lobster Ž Homarus gammarus. and blue mussel Ž Mytilus edulis .. Aquaculture 60, 165–168. Eidt, D.C., 1975. The effect of fenitrothion from large-scale forest spraying on benthos in New Brunswick headwaters streams. Can. Ent. 107, 743–760. Flannagan, J.F., 1973. Field and laboratory studies of the effect of exposure to fenitrothion on freshwater aquatic invertebrates. Manit. Entomol. 7, 15–25. Johns, P.M., Mann, K.H., 1987. An experimental investigation of juvenile lobster habitat preference and mortality among habitats of varying structural complexity. J. Exp. Mar. Biol. Ecol. 109, 275–285. Kent, R.A., Caux, P.-Y., 1995. Sublethal effects of the insecticide fenitrothion on freshwater phytoplankton. Can. J. Bot. 73, 45–53. Kleiner, C.F., Anderson, R.L., Tanner, D.K., 1984. Toxicity of fenitrothion to fathead minnows Ž Pimephales promelas . and alternative exposure duration studies with fenitrothion and endosulfan. Arch. Environ. Contam. Toxicol. 13, 573–578. Kikuchi, R., Yasutaniya, T., Takimoto, Y., Yamada, H., Miyamoto, J., 1984a. Accumulation and metabolism of fenitrothion in three species of algae. J. Pestic. Sci. 9, 331–337. Kikuchi, R., Takimoto, Y., Yamada, H., Miyamoto, J., 1984b. Effect of fenitrothion on growth of a green alga, Chlorella Õulgaris. J. Pestic. Sci. 9, 325–329. Lawton, P., 1987. Diel activity and foraging behavior of juvenile American lobsters, Homarus americanus. Can. J. Fish. Aquat. Sci. 44, 1195–1205. Lawton, P., Lavalli, K.L., 1995. Chapter 4: Postlarval, juvenile, adolescent, and adult ecology. In: Factor, J.R. ŽEd.., Biology of the Lobster Homarus Americanus. Academic Press, pp. 47–88. Lockhart, W.L., Metner, D.A., Grift, N., 1973. Biochemical and residue studies on rainbow trout Ž Salmo gairdneri . following field and laboratory exposures to fenitrothion. Manit. Entomol. 7, 26–36. O’Brien, R.D., 1967. Insecticides: Action and Metabolism. Academic Press, New York, pp. 1–82. Pottle, R.A., Elner, R.W., 1982. Substrate preference behavior of juvenile American lobsters, Homarus americanus, in gravel and silt-clay sediments. Can. J. Fish. Aquat. Sci. 39, 928–932. Roth, M., Richards, R.H., Sommerville, C., 1993. Current practices in the chemotherapeutic control of sea lice infestations in aquaculture: a review. J. Fish Dis. 16, 1–26. Roth, M., Richards, R.H., Dobson, D.P., Gordon, H.R., 1996. Field trials on the efficacy of the organophosphorus compound azamethiphos for the control of sea lice ŽCopepoda: Caligidae. infestations of farmed Atlantic salmon Ž Salmo salar .. Aquaculture 140, 217–239. Sampath, K., Stanley, A., Sivakumar, V., 1990. Respiratory responses of Heteropneustes fossilis. Env. Ecol. 8 Ž1., 92–94. Sarkar, U.K., Konar, S.K., 1993. Sublethal effects of pesticide, heavy metal, detergent and petroleum product in three combinations on fish. Env. Ecol. 11 Ž3., 609–615. Siegel, S., Castellan, N.J., Jr., 1988. Nonparametric Statistics for the Behavioral Sciences, 2nd edn., McGraw-Hill, 399 pp. Sievers, G., Palacios, P., Inostroza, R., Dolz, ¨ H., 1995. Evaluation of the toxicity of 8 insecticides in Salmo salar and the in vitro effects against the isopode parasite, Ceratothoa gaudichaudii. Aquaculture 134, 9–16. Takimoto, Y., Miyamoto, J., 1976. Studies on accumulation and metabolism of sumithion in fish. J. Pestic. Sci. 1, 261–271. Takimoto, Y., Hagino, S., Yamada, H., Miyamoto, J., 1984. The acute toxicity of fenitrothion to killifish Ž Oryzias latipes . at twelve different stages of its life history. J. Pestic. Sci. 9, 463–470. Taylor, E.J., Morrison, J.E., Blockwell, S.J., Tarr, A., Pascoe, D., 1995. Effects of lindane on the predator-prey interaction between Hydra oligactis Pallas and Daphnia magna Strauss. Arch. Environ. Contam. Toxicol. 29, 291–296.