William W. Walker

MACROBENTHIC RESPONSES TO NATURAL AND CONTAMINANT‐RELATED GRADIENTS IN NORTHERN GULF OF MEXICO ESTUARIES

Effects of pollution on biotic integrity are difficult to identify when correlations occur between environmental gradients and contaminant effects, as they do in estuaries. In this broad-scale study, we used canonical correspondence analysis (CCA) to distinguish influences of natural and contaminant-related gradients on macrobenthic community structure among 319 sites from estuaries throughout the northern Gulf of Mexico. Natural gradients in salinity, depth, and sediment composition obscured the detection of macrobenthic responses to sediment contamination. After adjusting for natural environmental variability, however, partial CCA revealed important macrobenthic variation in relation to sediment contamination. A rotated principal component analysis (PCA) distinguished five composite environmental factors, each largely reflecting contaminant or natural variation. Two complex gradients in sediment contamination identified by the PCA diverged in partial CCA space and correlated with different macrobenthic indicator taxa. Contaminant gradients represented variation in two different classes of sediment contaminants: trace metals and organic chemicals. Dispersion patterns of CCA site coordinates enabled cross validation of implied contamination-related variation in community function and the utility of several interpretive or management metrics. Trophic diversity decreased with sediment contamination, linking shifts in macrobenthic community function and community structure along contaminant gradients. The CCA model complemented an earlier benthic index developed from these data to examine biotic integrity, but the benthic index could not discern macrobenthic responses to the different contaminant gradients. Neither was the benthic index useful for showing transitions in macrobenthic community structure commensurate with different levels of contamination. Ampelisca amphipod sediment bioassays were inadequate for identifying contaminant effects on biotic integrity, whereas Mysidopsis mysid sediment bioassays conservatively reflected sediment contamination and associated macrobenthic indicators.

Carcinogenicity tests with small fish species

Abstract Development of carcinogenesis bioassays that utilize small fish species is important principally for investigating the causes of neoplasms in wild fishes and for providing alternative or supplementary models to rodent carcinogenicity tests. Availability, economy, latency of tumorigenic response, and ease of maintenance and exposure are commonly cited advantages of small fish species in carcinogenesis bioassays. Carcinogen metabolism and mechanisms of carcinogenesis in several small fish species appear similar to those processes in the more thoroughly studied large fish species as well as in rodents. Recent studies suggest that small fish species are appropriate test models for waterborne carcinogens having a variety of mechanisms. Several small fish species readily develop hepatic and non-hepatic neoplasms following brief static exposures to direct-acting genotoxic compounds such as methylazoxymethanol acetate (MAM-Ac) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Tumorigenic responses appear to be related to species and to various exposure factors. Indirect-acting genotoxic carcinogens such as nitrosamines and polynuclear aromatic hydrocarbons have not been thoroughly tested in small fish species but appear to require longer exposures than direct-acting ones to induce hepatic neoplasms. An especially important potential use of small fish carcinogenesis bioassays is in testing carcinogens that might have epigenetic mechanisms, especially complex mixtures that might affect man or the environment but are difficult to test in rodent models. Preliminary studies indicate that prolonged exposures of up to 6 months to a mixture of halogenated organic compounds result in hepatic neoplasms in two small fish species. To improve their usefulness and exploit small fish carcinogenesis models better, gaps in several areas need to be filled. These include (1) a better understanding of biological and nutritional requirements of test species as related to carcinogenesis, (2) a broader database on neoplastic responses of various species to various chemicals, and (3) development of special exposure methods and standardization of test protocols.

Decreased resistance of eastern oysters (Crassostrea virginica) to a protozoan pathogen (Perkinsus marinus) after sublethal exposure to tributyltin oxide

Abstract Anthropogenic environmental stress is a likely contributor to outbreaks of disease due to immunosuppression or increased host vulnerability. Estuarine organisms are exposed to variable concentrations of marine antifouling agents, such as tributyltin (TBT), with higher exposures existing near ports and marinas. Eastern oysters ( Crassostrea virginica ), inhabiting the northern Gulf of Mexico and the Atlantic coast of North America, are susceptible to a protozoan pathogen, Perkinsus marinus , which has devastated oyster populations and depleted oyster fisheries throughout its range. This study examined the effects of exposure to environmental levels of TBT on susceptibility and survival of oysters when subsequently challenged with P. marinus . Oysters were exposed to TBT (0, 30 and 80 ng/L) in flow-through aquaria for 9 weeks, then moved to static aquaria, where they were challenged with parasites and monitored for an additional 8 weeks for mortality and disease. Results indicated increased infection intensity and oyster mortality in the TBT-exposed treatments relative to unexposed controls. It is hypothesized that TBT exacerbates the infectious disease process and that exposed oysters succumb at lower levels of infection.

Carcinogenic effects of some polycyclic aromatic hydrocarbons on the Japanese medaka and guppy in waterborne exposures

In this paper, we review studies on the carcinogenic effects of two polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene (BaP) and 7,12-dimethylbenz(a)anthracene (DMBA), on the Japanese medaka (Oryzias latipes) and the guppy (Poecilia reticulata). Exposure media were prepared by adding PAHs to water, with and without dimethylformamide (DMF) as a carrier, and passing this solution through various sized filters. The low exposure medium was a 0.45 micron filtrate without DMF that contained less than 5 micrograms L-1 PAH. The intermediate medium was a DMF-mediated 0.45 micron filtrate which contained 30-50 micrograms L-1 of PAH. The high medium was a DMF-mediated glass-fibre filtrate which contained 150-250 micrograms L-1 of PAH. Young fish specimens (6-10 d old) were given a 6 h exposure once weekly for 2 to 4 wk. Both BaP and DMBA induced hepatic neoplasms in the two species, with the medaka being more sensitive than the guppy, and DMBA being a stronger carcinogen than BaP. Liver neoplasms almost exclusively developed after exposure to BaP and the neoplasms were limited to the high concentration exposure, whereas DMBA caused a substantial number of extrahepatic neoplasms as well as hepatic ones, especially in the medaka. Furthermore, all three concentrations of DMBA induced hepatic neoplasms in the medaka and all but the low concentration induced neoplasms in the guppy. These studies demonstrate the carcinogenic effects of two waterborne PAHs on two small fish species following brief exposures to very low concentrations, and support the contention that environmental PAHs can contribute to the occurrence of cancer in wild fishes.

Dose-related carcinogenic effects of water-borne benzo [a] pyrene on livers of two small fish species

Benzo[a]pyrene (BaP) administered by water-borne exposures caused dose-related carcinogenic effects in livers of two small fish species, the Japanese medaka (Oryzias latipes) and the guppy (Poecilia reticulata). Medaka and guppies each were given two 6-h exposures. The first exposure was conducted on 6- to 10-day-old specimens. The second exposure was given 7 days later. The tests incorporated five treatment groups: (1) control, (2) carrier (dimethylformamide) control, (3) low BaP dose (not detectable--4 ppb), (4) intermediate BaP dose (about 8-47 ppb BaP), and (5) high BaP dose (200-270 ppb). Following the high-dose exposure, hepatocellular lesions classified as foci of cellular alteration (altered foci), adenomas, and hepatocellular carcinomas occurred in both species. In medaka, the lesions appeared to develop sequentially with the appearance of altered foci followed by adenomas and then hepatocellular carcinomas. Most lesions in guppies, however, were classified as altered foci although a few adenomas occurred in the early (24-week) sample and hepatocellular carcinomas occurred in the late (52-week) sample. When total lesions were combined, medaka had an 11% incidence at 24 weeks after the initial exposure and 36% incidence at 36 weeks. In guppies, 8% had liver lesions at 24 weeks, 19% at 36 weeks, and 20% at 52 weeks. A single extrahepatic neoplasm, a capillary hemangioma in a gill filament, occurred in a medaka from the 36-week high-dose sample. The results suggest that the medaka and guppy are capable of metabolizing water-borne BaP to carcinogenic metabolites which initiate hepatic tumor development.

Use of the Japanese medaka (Oryzias latipes) and guppy (Poecilia reticulata) in carcinogenesis testing under national toxicology program protocols.

A need exists for whole animal toxicity, mutagenesis, and carcinogenesis models that are alternative to the traditional rodent test models and that are economical, sensitive, and scientifically acceptable. Among small fish models, the Japanese medaka (Oryzias latipes) is preeminent for investigating effects of carcinogenic and/or toxic waterborne hazards to humans. The guppy (Poecilia reticulata ), although less widely used, is valuable as a comparison species. Both species are easy to maintain and handle in the laboratory and there is a large body of background information on their responsiveness to a range of classes of carcinogens. There are considerable data on the occurrence of background diseases and on spontaneous neoplastic lesions, both of which occur relatively rarely. With few modifications, the medaka and guppy are amenable to carcinogenicity testing under the rigid standards established by the National Toxicology Program (NTP) for rodent tests. The advantages of the small fish in carcinogenesis studies are best realized in long-term studies that involve environmentally realistic exposures. Studies to identify chronic effects can be conducted in about 12 months, near the life span of medaka in our laboratory. Practically, 9-month studies are optimal but shorter study cycles and a variety of exposure/growout and initiation/promotion scenarios are available. Studies on 3 compounds tested in medaka under NTP protocols are under review and preliminary analysis indicates that chronic carcinogenicity bioassays with medaka, guppy, and potentially with other small fish species are feasible and scientifically valid.

Carcinogenicity Tests Using Aquarium Fish

In this chapter, the rationale, guidelines, and approaches for conducting small-fish carcinogenicity tests were presented. Goals were to demonstrate that the tests are relatively easy to conduct, that they require little in equipment or expertise in addition to that already available in a modern aquatic toxicology laboratory, and that the tests can approach the power and relevance of rodent carcinogenicity bioassays. Because the development of neoplasms following exposure to a carcinogenic substance is a latent or chronic response, a whole-animal carcinogenicity test using a fish model can be considered an elaboration or extension of routine aquatic toxicity tests that might otherwise have mortality or physiological perturbations as the principal end points.

Carcinogenic effects of 7,12-dimethylbenz[a]anthracene on the guppy (Poecilia reticulata)

Abstract The carcinogenic effects of 7,12-dimethylbenz[a]anthracene (DMBA) on the king cobra strain of guppy (Poecilia reticulata) were examined. Specimens were exposed for 6 h once weekly for 4 wk to DMBA. Guppies were 6–11 days old at the initial exposure. Treatment groups included the following: (1) untreated control; (2) dimethylformamide (DMF) carrier control; (3) low, water-mediated DMBA concentration (about 1–3 μg/l or ppb DMBA); (4) intermediate, DMF-mediated DMBA concentration (about 20 ppb DMBA); and (5) high, DMF-mediated DMBA concentration (about 35 ppb DMBA). Hepatic neoplasms developed in guppies exposed to the intermediate and high concentrations. Both of these exposure media contained the same concentrations of soluble DMBA. The high exposure medium, however, also contained an insoluble, particulate fraction of DMBA. Hepatic neoplasm incidences in fish exposed to the intermediate concentrations were 10% at 24 wk and 19% at 37 weeks after the initial exposure. In samples from the high concentration group, 47% had hepatic neoplasms at 24 weeks and 46% at 37 wk. Histologically, the hepatic lesions were categorized as altered foci, hepatocellular adenomas, and hepatocellular carcinomas. In addition to liver neoplasms, several other types of lesions developed in DMBA-exposed guppies. These included two undifferentiated sarcomas, a rhabdomyosarcoma, a renal adenocarcinoma, and a neurilemmoma. Only one control specimen (representing 0.38% of all controls) had a neoplastic lesion, a small hepatocellular adenoma in a 37-wk specimen. This study demonstrates that the guppy develops hepatic and extrahepatic neoplasms rapidly following brief water-borne exposures to those rather low levels of DMBA. Furthermore, the study showed that the particulate fraction of DMBA contributes to the carcinogenicity of the compound.

Trophic accumulation and depuration of mercury by blue crabs (Callinectes sapidus) and pink shrimp (Penaeus duorarum)

Mercury concentrations in blue crabs (Callinectes sapidus) collected from an area of mercury-contaminated sediments in Lavaca Bay, TX, USA, are more than an order of magnitude greater than concentrations in penaeid shrimp from the same area. Laboratory feeding experiments using mercury-contaminated fish as food showed that both blue crabs and pink shrimp (Penaeus duorarum) could accumulate mercury concentrations comparable to those in their food in 28 days. Calculated mercury assimilation efficiencies averaged 76% for blue crabs and 72% for pink shrimp. Significant depuration of mercury by blue crabs was not observed during a subsequent 28-day period, but pink shrimp lost mercury at a rate of about 0.012 day-1. Model calculations predict biomagnification factors of mercury of about two to three at steady state for both species. The large difference in observed concentrations of mercury in field-collected blue crabs and penaeid shrimp does not result from differences in efficiency of mercury assimilation from their food or from differences in excretion rates. It is more likely the result of differences in residence times in the contaminated area and of differences in feeding habits.

EXPOSURE METHODOLOGIES AND SYSTEMS FOR LONG-TERM CHEMICAL CARCINOGENICITY STUDIES WITH SMALL FISH SPECIES

Testing waterborne chemical carcinogens in fish models requires accurate, reliable, and reproducible exposures. Because carcinogenesis is a chronic toxicological process and is often associated with prolonged latency periods, systems must accommodate lengthy in-life test periods in which compound concentrations and environmental conditions must be carefully maintained. Here, systems and approaches are described for continuous long-term exposures with small fish species. The exposure system employed has enclosed chambers with internal walls of water-resistant materials, sliding doors for easy access, a water bath to maintain temperature, and exterior venting for air. Timer-controlled, overhead fluorescent lights provide a controlled photoperiod with dusk and dawn. Test aquaria sit within water baths that maintain temperature in the exposure tanks. Exhaust vents in the exposure chambers maintain a slightly negative air pressure in each system. Treatment media are provided by multicompartmented water partiti...