Resmethrin immunotoxicity and endocrine disrupting effects in the American lobster (Homarus americanus) upon experimental exposure.

ABSTRACT A lobster die-off significantly reduced the 1999 fall landings in western Long Island Sound. The die-off corresponded in time with the application of pesticides for the control of mosquitoes that carried West Nile virus, a new emerging disease in North America at the time. To determine the possible implication of pesticide application as a direct cause or contributing factor in the die-off, we studied the effects of experimental exposure to resmethrin on the health of lobsters. Lobsters (Homarus americanus) were exposed in 80-L tanks, and the direct toxicity as well as sublethal effects on the immune and endocrine system were determined. The 96-h L[C.sub.50] for resmethrin on single exposure was greater than 1 [micro]g/L, the highest concentration tested in our experiments, whereas the 14-day L[C.sub.50] was 0.75 [micro]g/L. Phagocytosis was significantly decreased 5 days after a single exposure to initial water concentrations of 1 and 0.1 [micro]g/L, as well as after weekly exposure to 0.1 [micro]g/L (week 3 and 4) and 0.01 [micro]g/L (week 4). Cell counts varied widely and inconsistently upon exposure to resmethrin. Evaluation of phagocytosis is a sensitive indicator of subtle sublethal effects of resmethrin. Crustacean hyperglycemic hormone (CHH), a potential stress-related hormone in lobsters, was measured in the hemolymph of the chronically-exposed animals. Significant increases in CHH concentrations were observed after 4 wk of exposure to 0.1 [micro]g/L. Whereas it is yet unknown if the concentrations at which toxicity was documented were ever encountered by lobsters in Long Island Sound during the 1999 die off, exposure resulting in the modulation of their immunology and physiology could likely have contributed to increasing lobster susceptibility to infectious diseases.

KEY WORDS: resmethrin, lobster, Homarus americanus, immunotoxicity, endocrine disruption, crustacean hyperglycemic hormone, CHH

INTRODUCTION

An unusual mortality event was observed in Long Island Sound (LIS) lobsters (Homarus americanus H. Milne Edwards, 1837) in the fall of 1999, resulting in a reduction of 90% to 100% of landings in western LIS. The lobsters examined suffered from a Paramoeba sp. infection not previously described in lobsters, which mainly affected the nervous system (Mullen et al. 2004). Nevertheless, it is not known if this organism was solely responsible for the mortality event or represented a terminal opportunistic infection. The die-off corresponded in time with the application of pesticides, including resmethrin, for the control of mosquitoes that carried West Nile virus, a new emerging disease in North America at the time. Initial analyses for the presence of pesticides used for the control of West Nile virus-infected mosquitoes did not reveal detectable concentrations in randomly sampled lobsters, but these chemicals are relatively short lived and could have been metabolized. The assay results could not determine with certainty if LIS lobsters had been exposed to those pesticides. It is also possible that exposed lobsters could have suffered short- or long-term effects after the chemicals had been metabolized and were no longer detectable.

The immune system is central to health and resistance/ susceptibility to pathogens in all species. Interestingly, it is also one of the most susceptible and sensitive systems to the effects of xenobiotics (Tryphonas & Feeley 2001). It is, therefore, highly possible that environmental stressors (chemical or others) that would have affected the immune system of lobsters in LIS would have rendered them more susceptible to infections (for example with paramoeba) and have played a significant role in the 1999 LIS lobster die-off. Resmethrin, especially when synergized by piperonyl butoxide as is the case in commercial preparations (Scourge), is a highly effective mosquito adulticide. Nevertheless, concern over its toxicity had the United States Environmental Protection Agency impose a restricted use classification on Scourge. Whereas the toxic effects (lethal and sublethal, upon acute and chronic exposure) of resmethrin have been evaluated in a range of aquatic species (Rand 2002, Holck & Meek 1987, Johnson & Finley 1980, EPA 1988, Tietze et al. 1991, Paul & Simonin 1996), including vertebrates and invertebrates, the lethal and sublethal (immunotoxic and endocrine disruptor) effects of resmethrin on lobsters have never been determined. To determine the possible implication of resmethrin application as a direct cause or contributing factor in the die-off, we studied the effects of experimental exposure to resmethrin on aspects of the health and physiology of lobsters.

MATERIALS AND METHODS

Experimental exposures were performed in aerated 80-L tanks each containing three lobsters, with a total of nine lobsters per dose. Lobsters of both sexes from Maine were distributed randomly between groups and kept in the dark (except during sampling and water changes) in 14[degrees]C artificial seawater (Instant Ocean, Mentor, Ohio) at a salinity of 24 parts per thousand and exposed to technical grade resmethrin (ICN Biomedicals Inc., Aurora, Ohio) using different regimes. Standard L[C.sub.50] experiments were performed over the course of 96 h. L[C.sub.50] was also calculated after the 14-day exposure, for which exposure occurred on day 0 and 7. Acute exposure lasted 5 days, with serial sampling on day 1, 3 and 5 and consisted of a single dose of resmethrin on day 0. Subacute exposure was performed over the course of 4 wk, with weekly serial sampling, followed by a weekly water change and re-exposure to an identical concentration of resmethrin as the original dose. All experiments started with nine lobsters per dose, which were all serially sampled until the end of the study. All experiments were conducted in the dark (except for sampling and water changes) to minimize possible breakdown of resmethrin through photodegradation. The concentrations of resmethrin for each exposure group were 0, 0.01, 0.1 and 1.0 [micro]g/L and are referred to as the nominal (calculated) concentrations added to the water.

At the end of each exposure regimen, lobsters were sacrificed and a gross and histopathologic examination was performed to determine the presence/absence of pathologic lesions, and to determine whether they were associated with exposure to resmethrin. Lobsters were euthanized using potassium chloride (Battison et al. 2000), and a complete necropsy was performed. Tissues were fixed in 10% neutral buffered formalin. Select tissues were then transferred to Dietrichs with 5% trichloroacetic acid for 48 h for partial decalcification of exoskeleton. Tissues were then trimmed and processed for paraffin embedding. Tissues were sectioned at 4 [micro]m, routinely stained with hematoxylin and eosin and examined by light microscopy for the presence/absence of lesions.

Muscle and hepatopancreas were also collected and frozen during the postmortem exam to analyze for the presence of resmethrin. Water samples (1 L) were collected periodically using precleaned I-CHEM amber glass jars certified to meet or exceed "US EPA specifications and guidance for contaminant-free sample containers" (Nalgen Nunc International, Rochester, New York). Water samples were preserved with methylene chloride at the time of collection. Water and tissue samples were analyzed at the Environmental Research Institute (ERI) based on a modified form of EPA method 616. This EPA method is not validated for sediment and tissue analysis by the EPA Office of Pesticide Programs. The primary changes from the EPA method is the use of capillary column techniques in lieu of the packed column specified in the methods, and the use of GC/MS instead of a flame ionization detector. EPA method 616 is based on older techniques and the ERI improvements to the method allow for the identification and quantitation at lower levels. The practical quantitation limit for the analysis of the resmethrin in water was 0.5 [micro]g/L. The practical quantitation limit for the analysis in lobster tissues ranged from 0.2-1.8 mg/L.

The immunologic endpoints tested include evaluation of hemocyte counts and phagocytosis on hemolymph samples. Briefly, 2.0 mL of hemolymph was collected from the dorsal vasculature and immediately transferred to a Vacutainer tube (Becton Dickinson, Rutherford, N J) containing acid citrate dextrose (ACD). This proved to be the best anticoagulant for use with lobster hemolymph cells in preliminary studies in our laboratory. Cells were then counted using a hemocytometer and Trypan Blue to determine viability. Phagocytosis was evaluated as previously described (De Guise et al. 2004a). Briefly, hemolymph (with anticoagulant as described earlier) was incubated at room temperature (20[degrees]C to 25[degrees]C). Fluorescent latex beads (1-[micro]m diameter) (Molecular probes, Eugene, Oregon) were diluted l:10 in phosphate-buffered saline (PBS) and 5 [micro]l of the bead mixture was added for every 200 [micro]l of hemolymph. After a 1 h incubation in the dark, 200 [micro]l of each cell suspension was analyzed by flow cytometry. The fluorescence of approximately 10,000 hemocytes was evaluated with a FACScan (Becton Dickinson, Mountain View, California) flow cytometer, using a forward scatter threshold to eliminate free beads from the acquisition. Phagocytosis was evaluated as the proportion of hemocytes that had phagocytized 2 or more beads.

Hemolymph was obtained via syringe before addition of resmethrin and after chronic exposure. Crustacean hyperglycemic hormone (CHH) was quantified by enzyme-linked immunosorbent assay as previously described (Chang et al. 1999). Standards were from the crayfish Orconectes limosus and corrected for lobster equivalents. The polyclonal antisera were rabbit antiCHH.

Results are reported as mean standard deviation. A separate 1-way analysis of variance (ANOVA) with Dunnett test was used to compare the different experimental groups to the unexposed control group at each time point, using P < 0.05 for statistical significance.

RESULTS

The direct toxicity of resmethrin in lobster was determined through a standard 96-h L[C.sub.50], the calculated concentration that killed 50% of the animals. Mortalities were not recorded in lobsters exposed to the highest concentrations of resmethrin used in this study (1 [micro]g/L). It was then concluded that the 96-h L[C.sub.50] of resmethrin in lobsters was greater than 1 [micro]g/L. The 14-day L[C.sub.50] calculated based on the cumulative mortality was 0.75 [micro]g/L (Fig. 1).

[FIGURE 1 OMITTED]

The concentration of resmethrin was measured in the water on day 0 (initial concentration), as well as on day 1, 3 and 5 after a single initial exposure, to evaluate its degradation. On day 0, the resmethrin concentration measured in water was 1.08 [micro]g/L. Resmethrin degradation in our system could not be measured accurately because concentrations were not detectable in most instances; most concentrations used were under the practical quantitation limit of 0.5 [micro]g/L (data not shown). Nominal concentrations were then used throughout this study.

Phagocytosis was significantly decreased 5 days (but not 1 or 3) after a single exposure to an initial water concentration of 1 and 0.1 [micro]g/L resmethrin, the two highest concentrations tested (Fig. 2). No resmethrin was detected in lobster tissues at the end of the 5-day exposure.

[FIGURE 2 OMITTED]

Phagocytosis was affected significantly in the course of the subacute (month-long) exposure (Fig. 3). The highest concentration used in this study (1 [micro]g/L) was omitted from week 2, 3 and 4 because mortality in that exposure group on those time points was 67%, 78% and 89%, respectively. There was a significant reduction of phagocytosis after exposure to 0.1 [micro]g/L resmethrin (3 and 4 wk after initial exposure), and 0.01 [micro]g/L (4 wk after initial exposure). No resmethrin was detected in lobster tissues at the end of the 4-wk exposure.

[FIGURE 3 OMITTED]

Cell counts varied widely and inconsistently in controls and upon exposure to resmethrin, with no significant differences from controls except for a significant reduction at the highest concentration on day 7 of the monthly exposure. Gross or histologic lesions were not observed when exposed to resmethrin.

Hemolymph concentrations of CHH were elevated significantly after 4 wk of exposure at 0.1 [micro]g/L (Fig. 4). There seems to be a dose-response relationship, however the data for 0.01 [micro]g/L are not significantly different from the controls.

[FIGURE 4 OMITTED]

DISCUSSION

The acute and chronic toxicity of resmethrin on aquatic organisms has been reviewed recently, with the conclusion that it was acutely toxic to fish and invertebrates in the 0.22-15.0 [micro]g/L range (Rand 2002). This is in agreement with the findings of several previous studies in invertebrates (Holck & Meek 1987) and a wide range of fish species (Johnson & Finley 1980, EPA 1988, Tietze et al. 1991), with the exception of channel catfish in which acute toxicity occurred at a slightly higher concentration (16.6 [micro]g/L) (Johnson & Finley 1980). The lack of acute lethal toxicity in a 96-h assay suggests that lobsters are not much more sensitive to resmethrin than other aquatic species. On the other hand, the toxicity of resmethrin became more evident over time, with a 14-day LC50 of 0.75 [micro]g/L. It is important to note that our study tested the toxicity of technical grade resmethrin, and not the commercial preparations that are synergized with piperonyl butoxide, which was approximately four times more toxic (LC50 0.23 [micro]g/L) than the technical grade compound (LC50 1.3 [micro]g/L) in pink shrimp (Rand 2002). It is also possible that the present study underestimated the acute toxicity of resmethrin in lobsters because a static exposure was chosen, to better mimic pesticides application. Flowthrough exposure resulted in a LC50 (0.22 [micro]g/L) more than 10 times lower than static exposure (LC50 3.7 [micro]g/L) in Daphnia magna (Rand 2002). Whereas other studies reported that acute toxicity occurred within 24 h (Rand 2002), significant deaths in this study only occurred at the highest concentration tested and after more than 1 wk. It is unlikely that the toxicity of resmethrin in lobsters was overestimated in the present study for several reasons, including (1) the use of technical grade resmethrin, rather than the more toxic formulation synergized with piperonyl butoxide; (2) a static exposure rather than constant concentrations via a flow through system and (3) the use of environmental conditions (such as temperature and salinity) favorable to lobsters.

Our data suggest that evaluation of phagocytosis using flow cytometry is a sensitive indicator of subtle sublethal effects of resmethrin, and that transient exposure to low, sublethal concentrations of resmethrin can affect lobster defense mechanisms. To our knowledge, this is the first report of immunomodulatory effects associated with exposure to resmethrin in any species. These effects were not immediate, appeared dose-dependant and exacerbated on repeated exposure. Immunotoxicity was observed at concentrations 10-100 times lower than those that failed to kill lobsters in the traditional 96-h LC50 assay. Whereas other authors claim that chronic toxicity to aquatic organisms will not be an issue for resmethrin (Rand 2002), the present demonstration of delayed, sublethal effects associated with exposure to resmethrin clearly warrants concern over the long-term effects of resmethrin on lobsters. In addition, the sublethal effects on chronic exposure of invertebrate and fish reproduction and larval growth did not occur below 0.59 [micro]g/L (Rand 2002), whereas swimming performance was not altered at concentrations below 3.2 [micro]g/L (synergized) and 6.3 [micro]g/L (nonsynergized) (Paul & Simonin 1996), suggesting that either reproduction, larval growth and swimming performance are far less sensitive than the immune system to the toxic effects of resmethrin or that lobsters are far more sensitive than Daphnia and the fish species tested in other studies. Another interesting finding is the fact that phagocytosis was altered in lobsters exposed to 0.1 and 1 [micro]g/L resmethrin 5 days after exposure (in the acute exposure study) but not 7 days after exposure (subacute exposure study). This suggests that either the effects are transient, or exacerbated by the stress associated with handling and water changes (on day 1, 3 and 5 in the acute exposure study, and on day 7, 14, 21 and 28 in the subacute study). The onset of effects on phagocytosis on exposure to 0.1 [micro]g/L resmethrin on wk 3, the third time the lobsters are handled, as for day 5 in the acute study, suggests that stress may in fact affect the immunotoxicity of resmethrin in lobsters. Similarly, the stress associated with repeated handling seems to result in a small but steady increase in phagocytosis in the control group for the acute subacute exposure, highlighting the importance of stressors on the immune system. The use of appropriate controls nevertheless allows the detection of the effects of resmethrin in time.

Mammalian studies comparing the relationship between immune functions and host resistance showed a good correlation between changes in the immune tests and altered host resistance, with no instances where host resistance was altered without affecting an immune test (Luster et al. 1993). We have documented immunomodulatory effects in lobsters on phagocytosis, reported as the most important defense mechanism in all phyla of the animal kingdom (van Oss 1986). Given that defective phagocyte function is an important cause of increased susceptibility to opportunistic pathogens in mammalian species (Rotrosen & Gallin 1987), and the above referenced data in mammalian immunotoxicology (Luster et al. 1993), it is likely that resmethrin-induced modulation of phagocytosis in lobsters could result in a significant decrease in lobster resistance to disease. It should nevertheless be noticed that the effects of resmethrin on other lobster defense mechanisms have not been evaluated, and the relative importance of and redundancy between different defense mechanisms in lobsters on encounter with pathogens are not well documented.

Elevated hemolymph glucose is required for lobsters to deal with any number of physiologic challenges. Because CHH regulates hemolymph glucose concentration, this hormone acts as a crustacean stress hormone. Elevations in hemolymph CHH have been shown to occur in lobsters following a number of environmental stresses. These include elevated temperature, hypoxia, osmotic stress (Chang et al. 1998, Chang et al. 1999) and parasitism (Stentiford et al. 2001).

An accepted definition of an endocrine disrupter is "an exogenous substance that causes adverse health effects in an intact organism, or its progeny, secondary to changes in endocrine function" (Holmes et al. 1997). Our data demonstrate that resmethrin does indeed alter the circulating concentration of the hormone CHH. Whether this alteration results in adverse health effects remains to be seen. Resmethrin, a pyrethroid insecticide, has not previously been shown to be an endocrine disrupter in crustaceans. However, other pyrethroids have been implicated as potential endocrine disrupters through the use of vertebrate hormone binding assays (Kojima et al. 2004). The decline in the CHH concentrations in the control lobsters was likely caused by the acclimation of the animals to their aquaria after 4 wk.

The current study assessed the toxicity of resmethrin in lobsters in view of its use around Long Island Sound to control West Nile virus-infected mosquitoes and its potential role in the 1999 lobster die-off. We documented that adult lobsters did not appear more sensitive than other aquatic species to the acute lethal toxic effects of resmethrin but much more sensitive than other aquatic species to the sublethal effects of resmethrin, although those effects were documented on one immune function and hormone, not on reproduction, development and growth. However, to the authors' knowledge, no data have documented the actual exposure of lobsters to resmethrin on environmental application, and it is not known if the concentrations that we documented to be toxic to lobsters were ever found in their environment in the course of the die-off. It is therefore not possible to determine with any certitude the role of resmethrin in the 1999 lobster die-off, but the risk of sublethal effects should be further explored, because they appeared at low concentrations compared with malathion (De Guise et al. 2004a) and methoprene (De Guise et al. 2004b), the 2 other pesticides that were applied for the control of mosquitoes around Long Island Sound and under investigation for their possible role in the lobster die-off.

CONCLUSION

In conclusion, our results suggest that adult lobsters are not more sensitive than other aquatic species to the lethal effects of resmethrin, but are very sensitive to its sublethal effects compared with other aquatic species, at least the immune and endocrine endpoints tested. A modulation in immune functions could likely result in an increase susceptibility to infectious agents, and could have contributed to the mass mortality if exposure was sufficient.

ACKNOWLEDGMENTS

The authors thank S. A. Chang for technical assistance. This research was supported by the Connecticut Sea Grant College Program, Grants No. LR/LR-3 (De Guise) and LR/LR-1 (Chang), through the U.S. Department of Commerce, National Oceanic and Atmospheric Administration (NOAA), Award No. NAI 6RG1364.

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SYLVAIN DE GUISE, (1) * JENNIFER MARATEA, (1) ERNEST S. CHANG (2) AND CHRISTOPHER PERKINS (3)

(1) Department of Pathobiology and Veterinary Science, University of Connecticut, 61 North Eagleville Road, U-89, Storrs, Connecticut 06269; (2) Bodega Marine Laboratory, University of California-Davis, PO Box 247, Bodega Bay, California 94923; (3) Environmental Research Institute, University of Connecticut, 270 Middle Turnpike, Unit 5210, Storrs, Connecticut 06269

* Corresponding author. E-mail: [e-mail]