ABSTRACT
Malathion immunotoxicity in the American lobsters (Homarus americanus) upon experimental exposure.
Sylvain De Guise,
Ph.D and Jennifer Maratea, Department of Pathobiology and Veterinary Science,
University of Connecticut.
Christopher Perkins, Environmental Research Institute, University of Connecticut
Long Island Sound Lobster Research Initiative Working Meeting, Groton CT, 2003.
Introduction
A lobster die-off reduced the 1999 fall landings in western Long Island Sound by up to more than 99%. 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. The lobsters examined suffered from a Paramoeba sp. infection that mainly affected the nervous system. In order 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 malathion on the health of lobsters.
Material and methods
Experimental exposures were performed in aerated 20 gallon tanks each containing 3 lobsters, with a total of 9 lobsters (in 3 tanks) per dose. Lobsters were kept at 10°C in artificial sea water and exposed to malathion using different regimes. Standard LC50 experiments were performed in the course of 96 hours. Acute exposure lasted 5 days, with sampling on day 1, 3 and 5, and consisted of either a single dose of malathion or repeated doses through daily water changes. Subacute exposure was performed over the course of 4 weeks, with weekly sampling. At the end of each study, lobsters were sacrificed and tissues sampled for the presence of gross and histological lesions, and for determination of concentrations of the chemical used in pooled muscle, hepatopancreas and hemolymph, in comparison to water concentrations.
Water and tissue samples were analyzed at the environmental Research Institute (ERI) based upon a modified form of EPA Method 616. This EPA method is not validated for sediment and tissue from the EPA Office of Pesticide Programs (personal communication). 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 upon older techniques and the ERI improvements to the method allow for the identification and quantitation at lower levels.
The endpoints tested include evaluation of the immune system using hemocyte counts and phagocytic index on hemolymph samples. Briefly, hemolymph was collected and immediately transferred to Vacutainer tubes (Becton Dickinson, Rutherford, NJ) containing acid citrate dextrose (ACD). This proved to be the best anticoagulant for use with lobster hemolymph cells in preliminary studies in our lab. Cells were then counted using a hemocytometer and Trypan blue to determine viability. Phagocytosis was evaluated as previously described (De Guise et al. 1995) with some variations. Hemocytes were incubated in their hemolymph at room temperature (20-25°C) and compared to samples incubated on wet ice (0°C), which reduces metabolic activity and phagocytosis. One µm diameter fluorescent latex beads (Molecular probes, Eugene, OR) were diluted 1:10 in PBS and 5 µl of the bead mixture was added for every 200 µl of helolymph. After a 1 hour incubation in the dark, 200 µl 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, CA) flow cytometer. Phagocytosis was evaluated as the proportion of hemocytes that had phagocytized 1 or more beads and the mean fluorescence of hemocytes. The results were reported as the phagocytic index, which represents the ratio of phagocytosis at room temperature to that on ice. A ratio higher than 1 represents active phagocytosis, and the higher the ratio is, the more effective phagocytosis.
At the end of all studies, lobsters were be sacrificed and a gross and histopathological examination will be performed to determine the presence/absence of pathological conditions. Tissues were fixed in Bouin's fixative for 48 hours, then in 70% ethanol for 24 hours, and further trimmed and processed for paraffin embedding. Tissues were sectioned at 4µm, routinely stained with hematoxylin and eosin, and examined by light microscopy for the presence/absence of lesions.
Results
The direct toxicity was determined through a standard 96-hour LC50, the calculated concentration that killed 50% of the animals. To do so, lobster mortality was recorded daily and cumulated for a 4 day exposure. The cumulative mortality was then plotted against the concentrations of malathion used and a linear regression curve was determined using the Microsoft Excel software. The LC50 was calculated using the equation determined by the software for the regression curve. The 96 hour LC50 was 33.5 µg/L (or ppb) upon single exposure (Figure 1).
Figure 1: LC50 of malathion in lobsters.
Malathion degraded rapidly in our system, with 65-77% lost after one day and 83-96% after three days (Figure 2). No malathion was detectable in lobster tissues at the end of the 5 day exposure.
Figure 2: Concentrations of malathion in water decreased rapidly in our system.
Relatively high concentrations
of malathion, upon repeated exposure, initially (day 1) increase phagocytosis,
with no effects on day 3 and 5. Phagocytosis was significantly decreased
3 days (but not 1 or 5) after a single exposure to water concentration
as low as 5 ppb (the lowest concentration tested), when water concentrations
were as low as 0.55 ppb (Figure 3). Phagocytosis was not significantly
affected at any time point in the course of the month long exposure. Cell
counts did not differ significantly upon exposure to malathion.
Figure 3: Phagocytosis of lobster cells after a single exposure to increasing concentrations of malathion.
Discussion
Malathion has a wide range of toxicities in fish, extending from very highly toxic in the walleye (96-hour LC50 of 0.06 mg/L) to highly toxic in brown trout (0.1 mg/L) and the cutthroat trout (0.28 mg/L), moderately toxic in fathead minnows (8.6 mg/L) and slightly toxic in goldfish (10.7 mg/L) and mosquitofish (12.68 mg/L) (Johnson and Finley 1980, Tietze et al. 1991,Kidd and James 1991, U.S. Public Health Service 1995). Various aquatic invertebrates are extremely sensitive, with EC50 values from 1 ug/L to 1 mg/L (Menzie 1980). Lobsters, with a LC50 of 33.5 ppb, appear to be very sensitive to the acute lethal effects of malathion compared to other aquatic species.
The very rapid breakdown of malathion in our system suggests that failure to measure malathion in water samples does not necessarily mean lack of exposure. At day 3 of our acute exposure study, the concentrations of malathion in the water were very low, yet effects on phagocytosis were demonstrated in lobsters.
Our data suggest that evaluation of phagocytosis using flow cytometry is a sensitive indicator of subtle sub-lethal effects of malathion, and that transient exposure to relatively small concentrations of malathion (6-7 times lower than the LC50) can affect lobsters defense mechanisms, even with rapidly decreasing water concentrations. Those results are not surprising given that the immunotoxicity of malathion has been documented in several species of laboratory animals including effects on both humoral and cellular immune responses of mice, rats and rabbits (Banerjee et al. 1998). Malathion was also documented to affect the natural and acquired immunity of fishes (Japanese medaka), in addition to decreasing resistance to a common pathogen (Beaman et al. 1999). Nevertheless, it is interesting to note that the initial water concentrations that resulted in immunotoxicity in lobsters (5 ppb or 5 µg/L) are 40 times lower that those which resulted in reduction in immune functions and 20 times lower than those which resulted in reduced resistance to a pathogen in the fish study (Beaman et al. 1999).
In conclusion, our results suggest that lobsters are highly sensitive to both the lethal and sub-lethal toxicity of malathion in sea water. A reduction 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.
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