Effects of laboratory exposure to sediments contaminated with polycyclic aromatic hydrocarbons on the hemocytes of the American oyster Crassostrea virginica

Marine Environmental Research 35 (1993) 131-135

~,1 Effects of Laboratory Exposure to Sediments Contaminated with Polycyclic Aromatic Hydrocarbons on the Hemocytes of the American Oyster Crassostrea virginica Susanne Sami, Mohamed Faisal & Robert J. Huggett Virginia Institute of Marine Science, School of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062, USA

ABSTRACT We have previously shown that hemoeytes of American oysters collected from polycyclic aromatic hydrocarbon (PAH)-contaminated areas in the Elizabeth River, Virginia, have a lower ability to phagocytose yeast particles and a decreased expression of concanavalin A-binding sites as compared to hemocytes of oysters collected from cleaner environments. In the present study, we exposed healthy oysters to PAH-contaminated sediments of the Elizabeth River under laboratory conditions for up to 11 weeks. The data obtained indicate that exposure to the contaminated sediments induced a significant suppression in the number of Con A-binding sites as early as four weeks post exposure, but had no significant effects on the phagocytosis. On the other hand, depuration of the Elizabeth River oysters was accompanied by a steady increase in the number of hemocytes expressing Con A and phagocytosis was slightly enhanced. The sum of our data indicates that phenotypic changes are probably more sensitive to P A H exposure than functional parameters.

Fully functional defense mechanisms in aquatic organisms are necessary for protection against disease. ~ These mechanisms are often influenced by external and internal factors. For instance, heavy metals, pesticides, and organic compounds have suppressive effects on the activity of Crassostrea virginica hemocytes. 2-4 In a previous report, s we described the effects of exposure to pollutants in an environment heavily contaminated with polycyclic aromatic hydrocarbons (PAH) on some morphological and functional properties of C. virginica hemocytes. Our previous results 131 Marine Environ. Res. 0141-1136/92/$05.00 © 1992 ElsevierSciencePublishers Ltd, England. Printed in Great Britain

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RETENTION TIME (MINUTES) Fig. 1. Chromatograms of PAH in sediments of (A) the York River, Gloucester Point, VA, and (B) the Elizabeth River at Hospital Point, VA: • = standard peak, l: phenanthrene, 2: fluoranthene, 3: pyrene, 4: chrysene, 5: benzofluoranthene, 6: benzo[a]pyrene, 7: ideno(1,2,3cd)pyrene, 8: benzo[ghi]perylene.

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indicated that oysters collected from Hospital Point (HP), a site in the Southern Branch of the Elizabeth River (ER), VA, where sediments are known to be contaminated with high levels of total PAH, 6 had fewer concanavalin A (Con A) binding sites (11-2% + 4-7) on the hemocyte-surface membrane than oysters collected from the relatively unpolluted Rappahannock River (RR) (60"5% ___19-86) at Bowlers Rock (820-1200 ppb total PAH), 7 Tappahannock, VA. Transplanting R R oysters to HP for a period of up to eight weeks suppressed Con A binding by 80%, while maintaining ER oysters in the relatively unpolluted York River (YR), VA, induced an augmentation of Con A binding sites, which was 5.7 times as high as that in the original ER oysters. However, phagocytic activity of the hemocytes was shown not to be closely correlated with the site of origin of the oysters or the transplantation conditions. We planned studies to assess whether the results obtained from the field investigation could be reproduced by exposing oysters to PAH-contaminated sediments under laboratory conditions. The investigation involved the collection of sediments from HP and from the YR at Gloucester Point, VA. The sediments were analyzed for the presence of PAH (Fig. l) by standard procedures. 6 Each sediment was thoroughly mixed by using a fiberglass-lined cement mixer and then evenly spread on the floor of a designated glass aquarium (80 liter) to form a 5-10 cm thick layer. Oysters were maintained on a plastic grid just above the sediment surface. Oysters were collected from the R R (where oysters are known to be parasite-free, J. LaPeyre, personal communication) and from the HP site in the ER. Baseline Con A binding and phagocytic responses for both groups of oysters were determined. R R oysters were then maintained over the ER sediments (12000ppb total PAH) for up to eleven weeks, while ER oysters were maintained over the YR sediment (20ppb total PAH) for the same time period. As controls, groups of the RR oysters and ER oysters were kept in the YR and ER sediments, respectively. Samples were taken after four, eight, and eleven weeks. Oysters were maintained in a controlled, static-renewal system and were fed with Isochrysisgalbana algae (Tahitian strain) regularly. Each sample group consisted of 25 oysters. Hemolymph was collected from notched oysters. For the Con A assay, hemocytes were fixed with modified Karnovsky solution (1"25% glutaraldehyde, 2% formaldehyde in 0"025M sodium-cacodylate-buflered natural sea water) and then incubated with 1:20 fluorescein isothiocyanate labeledCon A for 30 min at 4°C in the dark. Hemocytes were washed twice with phosphate-buffer saline (0-2M) containing 2% fetal calf serum and supplemented with 0-2% sodium azide. The cells (10 000 cells/sample) were analyzed by using a Coulter EPICS-C flow cytometer. To assess phagocytosis, 1 × 104 hemocytes (suspended in 0"7 ml hemolymph) were

34

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Weeks Fig. 2. C o n c a n a v a l i n A a n d phagocytic responses at four, eight, a n d eleven weeks. D a t a are represented as m e a n s (circles)+ s t a n d a r d deviation (vertical bars). - - O - - , C o n A; - - O - - , phagocytosis.

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incubated with 0.5 ml suspended yeast cells (4 x 107 cells/ml) for 2 h at 22°C. The incubated preparations were washed three times with PBS, air-dried onto slides, stained, and examined microscopically with an oil-immersion lens. Figure 2 shows that the percentage of Con A positive hemocytes in the R R oysters, maintained over ER sediment, was decreased by 16%, 40%, and 84% after four, eight, and eleven weeks, respectively. R R oysters kept in the YR sediment maintained a relatively constant level of the percentage of hemocytes expressing Con A binding sites. In addition, Con A positive hemocytes in the ER oysters (19% + 10) maintained over the YR sediment increased significantly, up by 250% at eight weeks and 278% at eleven weeks. ER oysters maintained over the ER sediments maintained very low expression of Con A binding sites. No consistent pattern was observed in the phagocytic capability of hemocytes under any of the exposure conditions. The findings of this study corroborate with our previous data obtained from field samples, i.e. the percentage of Con A positive hemocytes but not the phagocytic activity was negatively correlated with levels of PAH in the sediment. Since the effects of other toxic chemicals in the sediments cannot be ruled out, we plan to study the effects of exposure to purified, individual PAH on the immune response of oysters.

ACKNOWLEDGEMENT The authors wish to thank Dr Morris H. Roberts for his valuable advice in the design of this experiment. This is Virginia Institute of Marine Science Contribution Number 1685.

REFERENCES 1. 2. 3. 4. 5.

Fisher, W. S. (ed.). Am. Fish. Soc. Spec. Pup., 18, 225-237 (1988). Cheng, T. C. 3". Invertebr. Pathol., 51, 207-14 (1988). Cheng, T. C. J. Invertebr. PathoL, 51, 215-20 (1988). Larson, K. G., Roberson, B. S. & Hetrick, F. M. Dis. Aquat. Org., 6, 131-6 (1989). Sami, S., Faisal, M. & Huggett, R. J. Eastern Regional Symposium on Mechanisms of Immunotoxicity, Williamsburg, Virginia, USA, October 1990. 6. Bieri, R. H., Hein, C., Huggett, R. J., Shou, P., Slone, H., Smith, C. L. & Su, C. W. Int. J. Environ. Anal Chem., 26, 97-113 (1986). 7. Smith, C. Virginia Institute Marine Science Toxics Database, Gloucester Point, VA 23062 (unpublished data).