George R. Gardner

Vitellogenin-induced pathology in male summer flounder (Paralichthys dentatus)

Male summer flounder (Paralichthys dentatus) were given two injections (initially and 2 weeks later) of 17beta-estradiol (E2) totaling 0.2 (2 x 0.1), 2.0 (2 x 1.0) or 20.0 (2 x 10.0) mg E2/kg body weight. Blood and tissue samples were collected 4, 6 and 8 weeks after the initial injection in the (2 x 0.1) mg/kg treatment, 4, 6, 8, and 15 weeks after the first injection in the (2 x 1.0) mg/kg treatment and at 4 weeks only in the (2 x 10.0) mg/kg treatment. Five of the 12 fish injected twice with 10.0 mg/kg were moribund before the first sampling period. Circulating levels of vitellogenin (VTG) in the blood of all E2-injected fish from all treatments were comparable with those concentrations found in the blood of wild male carp (Cyprinus carpio) and walleye (Stezostedion vitreum) previously collected near a sewage treatment plant (0.1-10.0 mg VTG/ml plasma). Excessive hyalin material accumulated in the livers, kidneys and testes of the treated fish. A portion of that material was identified as VTG by immunohistochemistry. The accumulation of VTG, and possibly other estrogen-inducible proteins, resulted in hepatocyte hypertrophy, disruption of spermatogenesis, and obstruction or rupture of renal glomeruli.

Carcinogenicity of Black Rock Harbor sediment to the eastern oyster and trophic transfer of Black Rock Harbor carcinogens from the blue mussel to the winter flounder.

The eastern oyster (Crassostrea virginica) developed neoplastic disorders when experimentally exposed both in the laboratory and field to chemically contaminated sediment from Black Rock Harbor (BRH), Bridgeport, Connecticut. Neoplasia was observed in oysters after 30 and 60 days of continuous exposure in a laboratory flow-through system to a 20 mg/L suspension of BRH sediment plus postexposure periods of 0, 30, or 60 days. Composite tumor incidence was 13.6% (49 neoplasms in 40, n = 295) for both exposures. Tumor occurrence was highest in the renal excretory epithelium, followed in order by gill, gonad, gastrointestinal, heart, and embryonic neural tissue. Regression of experimental neoplasia was not observed when the stimulus was discontinued. In field experiments, gill neoplasms developed in oysters deployed in cages for 30 days at BRH and 36 days at a BRH dredge material disposal area in Central Long Island Sound, and kidney and gastrointestinal neoplasms developed in caged oysters deployed 40 days in Quincy Bay, Boston Harbor. Oysters exposed to BRH sediment in the laboratory and in the field accumulated high concentrations of polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), and chlorinated pesticides. Chemical analyses demonstrated high concentrations of PCBs, PAHs, chlorinated pesticides, and heavy metals in BRH sediment. Known genotoxic carcinogens, co-carcinogens, and tumor promoters were present as contaminants. The uptake of parent PAH and PCBs from BRH sediment observed in oysters also occurs in blue mussels (Mytilus edulis). Winter flounder fed BRH-contaminated blue mussels contained xenobiotic chemicals analyzed in mussels. The flounder developed renal and pancreatic neoplasms and hepatotoxic neoplastic precursor lesions, demonstrating trophic transfer of sediment-bound carcinogens up the food chain. ImagesFIGURE 2.FIGURE 3.FIGURE 4.FIGURE 5.FIGURE 6.FIGURE 7.FIGURE 8.FIGURE 9.FIGURE 10.

Germinomas and teratoid siphon anomalies in softshell clams, Mya arenaria, environmentally exposed to herbicides.

Seminomas and dysgerminomas are epizootic in softshell clams, Mya arenaria, from three Maine estuaries contaminated with herbicides. The first epizootic was discovered in 22% of clams collected as Searsport near Long Cove Brook and three culverts that conveyed heating oil and jet fuel spilled from a tank farm in 1971. Data from subsequent epizootiological studies and a series of long-term experimental exposures of softshell clams to no. 2 fuel oil, JP-4, and JP-5 jet fuel at the U.S. EPA, Environmental Research Laboratory in Narragansett, Rhode Island, and in the field did not support an etiology by these petroleum products. In the two recent epizootics reported here, the germinomas have been observed in 3% of the softshell clams collected from Roque Bluffs near Machiasport and from 35% of softshell clams collected from Dennysville. Mya collected at Dennysville had pericardial mesotheliomas and teratoid siphon anomalies in addition to gonadal neoplasms. Estuaries at Dennysville had been contaminated by herbicides in a 1979 accidental spray overdrift during aerial application of Tordon 101 to adjacent forests. Further investigation determined widespread use of the herbicides Tordon 101, 2,4-D,2,4,5-T, and other agrochemicals in an extensive forestry and blueberry industry in both the Roque Bluffs and the Dennysville areas. Herbicide applications at Searsport were confirmed for railroad property bordering Long Cove estuary and for Long Cove Brook adjacent to the estuary where a highway department reportedly cleans its spray equipment. Herbicide contamination is the only common denominator identified at all three sites where Mya have been found with gonadal neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS) ImagesFIGURE 5.FIGURE 6.

Acute toxicology of sodium nitrilotriacetic acid (NTA) and NTA-containing detergents to marine organisms

Abstract Static acute toxicity tests were conducted at 20°C and 20% salinity with (CH2COONa)3N-H2O (NTA) and two NTA-containing commercial household synthetic detergents (syndets), using adults or juveniles from eleven species of marine fishes and invertebrates. Concentrations of NTA in the medium allowing 50 per cent survival of individual test species in 168 h, = TL50 (168 h) values, ranged between 1800 mg 1−1 for grass shrimp, Palaemonetes vulgaris, and > 10,000 mg 1−1 for quahaug clam, Mercenaria mercenaria. Intermediate in sensitivity to NTA were hermit crab, Pagurus longicarpus (1875 mg 1−1); scup (fish), Stenotomus chrysops (2200 mg 1−1); common starfish, Asterias forbesi (3000 mg 1−1); American lobster, Homarus americanus (3150 mg 1−1); bay mussel, Mytilus edulis (3400 mg 1−1); eastern mud snail, Nassarius obsoletus (5100 mg 1−1); sandworm, Nereis virens (5500 mg 1−1); mummichog (fish), Fundulus heteroclitus (5500 mg 1−1); and striped bass, Roccus saxatilis (5500 mg 1−1). The two syndets tested were considerably more toxic than NTA to marine organisms under identical assay conditions. Fishes were the least resistant group of organisms tested to syndets; sptl50 (168 h) values for teleosts ranged between 4.6 and 36.0 mg 1−1 total packaged product. Histopathology was investigated for grass shrimp, hermit crab, quahaug clam, sandworm, scup, striped bass and mummichog that survived high concentrations of NTA or syndets for 168 h. Among invertebrates, pathological changes were confined to digestive diverticula and kidney of shrimp exposed to 1000 mg 1−1 of NTA. With fishes, pathology in striped bass and mummichog was observed in proximal kidney tubules at NTA levels > 3000 mg 1−1. However, significant intestinal lesions were noted in mummichog at NTA levels as low as 1 mg 1−1. No pathology was observed among fish and invertebrates subjected to syndets. The effect of biomass, water temperature and salinity on acute toxicity of NTA and syndets to mummichogs was also investigated. Increasing the biomass between 0.5 and 10.0 g 1−1 of test medium was associated with increasing survival at any given concentration of syndet during a 48 h period; the reverse was observed for NTA. Within the salinity range 5–35%, mummichogs were most sensitive to NTA at comparatively low salinities; but syndets were most toxic at higher salinities. Water temperatures of 5° and 20°C had negligible influence on NTA- or syndet-induced mortality patterns. Mixtures of NTA and salts of cadmium or mercury were evaluated for toxicity to mummichog. An observed decrease in biocidal properties of Cd2+ was observed with increasing levels of NTA; concentrations as low as 10 mg 1−1 NTA effected a measurable depression in Cd2+ lethality. Results of studies with NTA—Hg2+ mixtures were inconclusive. Tests with mixtures of syndet and Cd2+ or Hg2+ demonstrated that toxicity to mummichog of these mixtures could be expressed as a simple summation of the toxicity of individual components. It is concluded that NTA might be hazardous to marine fishes and macroinvertebrates when used as a partial replacement for sodium tripolyphosphate in household syndets.

Comparative histopathological effects of chemically contaminated sediment on marine organisms

Comparative pathological effects in three species of fish and six species of invertebrates were investigated using a chemically contaminated sediment from Black Rock Harbor (BRH), Connecticut, USA. Substances contained in BRH sediment are known to be genotoxic, carcinogenic, co-carcinogenic and tumor-promoting. Proliferative lesions were observed in winter flounder (Pseudopleuronectes americanus) external, oral and esophageal epithelial surfaces, renal vascular and nephroblastic elements, and the pancreatic islets. Islet lesions included cystic adenomas and a diffuse islet proliferative condition, nesidioblastosis. In addition, pathology was enhanced when winter flounder were fed mussels (Mytilus edulis) previously exposed to BRH sediment. Neoplastic lesions developed in kidney tubule epithelia, gills, some regions of the gastrointestinal tract, neural elements and heart of oysters (Crassostrea virginica) exposed to BRH sediment for 30 days in the laboratory. In addition, renal carcinomas in three oysters had metastasized to the viseral ganglion. Lesions were also found in oysters exposed in situ in BRH and Long Island Sound for 30 or 36 days. Digestive diverticula, gills and kidneys appeared to be the prime organs affected in mussels, soft-shelled clams (Mya arenaria) and oysters exposed to BRH sediment. Mucous cell activity increased greatly in corals (Astrangia densa) while no microscopic lesions were detectable in lobster (Homarus americanus) exposed in the laboratory. Myxomas were also found in the hearts of < 5% of the mussels exposed in the laboratory.

A comparison of both neoplastic and non-neoplastic disorders in winter flounder (Pseudopleuronectes americanus) from eight areas in New England

Abstract Distribution patterns of liver disease observed in winter flounder indigenous to the northeastern USA indicated that hepatocytic neoplasms were absent in populations from uncontaminated offshore areas and endemic in populations from moderately to highly contaminated inshore areas. Liver neoplasms in winter flounder collected from eight different locations ranged from 0% in animals collected offshore from Cape Cod to 32% in the nearshore area of New Bedford, MA. Similarly, an array of other hepatic lesions ranged from 9% in Marthas Vineyard to 79% in Boston Harbor. Proliferate lesions in endocrine, exocrine, respiratory, sensory, excretory and digestive organs and alteration of plasma protein were also characteristic of winter flounder populations residing in the nearshore environment. The concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), other organic compounds and trace metals associated with marine sediment were elevated in urban embayments as compared with offshore locations. Degree of sediment chemical contamination and disease suggest a causal interrelationship.

MORPHOLOGICAL ANOMALIES IN ADULT OYSTER, SCALLOP, AND ATLANTIC SILVERSIDES EXPOSED TO WASTE MOTOR OIL

ABSTRACT Waste motor oil concentrations of 20 ppm and higher induced lesions in the vascular systems of Atlantic silversides and oyster. These lesions were associated with the pseudobranch, the hea...

A CONTINUOUS FLOW BIOASSAY SYSTEM FOR THE EXPOSURE OF MARINE ORGANISMS TO OIL

ABSTRACT A continuous flow-through bioassay system is described for exposing marine test organisms and their various life stages to oil. The apparatus consists of two principal stages—one for administering the water-accommodated fractions of oil (WAF) and the second for administering “whole” oil fractions (WF), including the soluble as well as nonsoluble fractions. The oil-contaminated effluent from the system first passes through a chamber to skim off separated oil and finally through a filter which lowers the oil concentration to below one part per million (1 ppm). Short-term lethal and chronic sublethal bioassays of several months duration were conducted in the system and are summarized briefly herein. Biological effects observed during bioassays have ranged from lethal toxicity at oil concentrations of approximately 10 parts per million (ppm) to sublethal behavioral modifications at approximately 10 parts per billion (ppb).

Identification of dioxin-specific binding proteins in marine bivalves

Epidemiological investigations of germ cell tumors of Maine soft shell clams (Mya arenaria) and hard shell clams (Mercenaria spp.) from Florida demonstrate the prevalence of histogenically similar gonadal cancers as high as 40 and 60%, respectively. Human mortality rates due to ovarian cancer from the same areas are significantly greater than the national average. Since further investigation revealed that there was a heavy use of herbicides at these sites, we proposed to investigate their possible role in the etiology of the clam tumors. One avenue of investigation was to determine whether clams had a mechanism similar to the vertebrate Ah receptor which could be activated by halogenated aromatic hydrocarbons (HAH). We used the TCDD photoaffinity analog [125-I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin to detect two cytosolic proteins (28 and 39 kDa) in Mercenaria mercenaria and one (35 kDa) in Mya arenaria which specifically bound this ligand. Expression of both proteins in Mercenaria is tissue specific with the highest levels observed in cytosols from gill and gonad. Females exhibit higher levels of the 39 kDa protein in gonadal tissue than do the males. Studies to determine the relationship of these proteins to vertebrate Ah receptors, as well as their possible role in gonadal tumor development, are in progress.

Potential alterations in gene expression associated with carcinogen exposure in Mya arenaria

Gonadal cancers in soft-shell clams (Mya arenaria) have been found at high prevalences (20-40%) in populations in eastern Maine. The aetiology of these tumours is unknown. We hypothesized that gene expression would be altered in gonadal tumours and that examination of gene expression patterns would provide some information as to the mechanism of tumour development. To investigate this hypothesis, we initiated a broad search for differentially expressed genes using differential display polymerase chain reaction (dd-PCR) to compare RNA from tumour and normal gonadal tissue. We identified two classes of genes whose expression may be altered in the gonadal tumours: genes involved in biosynthetic processes and genes with possible roles in signal transduction. We also investigated the hypothesis that environmental contaminants, such as tetrachlorodibenzo-p-dioxin (TCDD), may play a role in the development of these tumours. To investigate this hypothesis, we performed a short-term exposure of M. arenaria to [(3)H]TCDD. Tissues were sampled up to 2 weeks after a 24-h exposure to 10 pptr or 2000 pptr of [(3)H]TCDD in the water. Using dd-PCR, we identified potential alterations in expression of genes associated with cell proliferation: heparan sulphate proteoglycan, E3 ubiquitinating enzyme and p68 RNAhelicase/initiation factor eIEF-4A. There were no observable histopathological alterations in gonadal or gill tissue from exposed animals. These results suggest possible early changes in gene expression indicative of environmental exposures.Gonadal cancers in soft-shell clams (Mya arenaria) have been found at high prevalences (20-40%) in populations in eastern Maine. The aetiology of these tumours is unknown. We hypothesized that gene expression would be altered in gonadal tumours and that examination of gene expression patterns would provide some information as to the mechanism of tumour development. To investigate this hypothesis, we initiated a broad search for differentially expressed genes using differential display polymerase chain reaction (dd-PCR) to compare RNA from tumour and normal gonadal tissue. We identified two classes of genes whose expression may be altered in the gonadal tumours: genes involved in biosynthetic processes and genes with possible roles in signal transduction. We also investigated the hypothesis that environmental contaminants, such as tetrachlorodibenzo-p-dioxin (TCDD), may play a role in the development of these tumours. To investigate this hypothesis, we performed a short-term exposure of M. arenaria to [3H]...