Field applications of the piscine anaphase aberration test: lessons from the Exxon Valdez oil spill.

Several large-scale genotoxicity assessments have been performed in coastal marine areas that have demonstrated either localized or widespread genetic effects resulting from human activity. One common assessment method is the anaphase aberration test, a measurement of abnormal chromosome division, using embryolarval fishes. It can be used to detect the presence of mutagens within a poorly characterized complex mixture or monitor specific genotoxins and is easily adapted for laboratory screening. One comprehensive marine genotoxicity assessment was conducted using Pacific herring (Clupea pallasi) following the Exxon Valdez oil spill (EVOS) in Prince William Sound (PWS), AK in late March 1989. In early May, genetic damage was detected at many sites within the oil trajectory and was correlated with concentrations of polycyclic aromatic hydrocarbons characteristic of Exxon Valdez oil (EVO) in intertidal mussels. Effects were related spatially and temporally to oil exposure. Anaphase aberration rates decreased throughout May and June 1989, and by 1991, genotoxicity was undetectable. The abundance of the 1989 herring year class in PWS is significantly reduced; this is the first reported example linking genotoxicity to subsequent population level effects. This review describes the methodology for the anaphase aberration test using fish eggs, its applications for large-scale assessments and supportive laboratory studies, and its limitations for prediction of higher level effects on populations.

Defining the role of pollutants in the disruption of reproduction in wildlife.

Although chemical exposure has been associated with reduced reproduction in certain North American fish, reptiles, and mammals, definitive cause-and-effect data are lacking in many instances. Because the increasing use and global transport of industrial chemicals pose significant risk to successful reproduction, methods should be developed that can define the geographic extent and magnitude of injury and risk to wildlife. Because industrial chemicals are articles of commerce, information about injury to wildlife has been contentious and too often ineffective in changing societal behavior. The following strategies are advocated for inferring causal relationships. First, a balanced and comprehensive assessment of the data is necessary to determine the geographic extent of exposure and reproductive effects associated with environmental pollution. Initial efforts to document reproductive injury should focus on specific ecosystems in which detrimental effects have been observed, but lack sufficient causal data. Model systems (including experimental mesocosms or field ecosystems) should be identified or designed that can adequately test multigenerational reproductive effects. Mechanistic data from supportive laboratory studies on reproductive toxicity, quantitative structure-activity relationships, and bioaccumulation can be used to predict effects of related pollutants and to determine risk. Such information is essential to prevent future injury to wildlife and to prioritize the numerous remediation decisions facing our society.

Large-scale genotoxicity assessments in the marine environment.

There are a number of techniques for detecting genotoxicity in the marine environment, and many are applicable to large-scale field assessments. Certain tests can be used to evaluate responses in target organisms in situ while others utilize surrogate organisms exposed to field samples in short-term laboratory bioassays. Genotoxicity endpoints appear distinct from traditional toxicity endpoints, but some have chemical or ecotoxicologic correlates. One versatile end point, the frequency of anaphase aberrations, has been used in several large marine assessments to evaluate genotoxicity in the New York Bight, in sediment from San Francisco Bay, and following the Exxon Valdez oil spill.

Genetic and molecular ecotoxicology: a research framework.

Participants at the Napa Conference on Genetic and Molecular Ecotoxicology assessed the status of this field in light of heightened concerns about the genetic effects of exposure to hazardous substances and recent advancements in our capabilities to measure those effects. We present here a synthesis of the ideas discussed throughout the conference, including definitions of important concepts in the field and critical research needs and opportunities. While there were many opinions expressed on these topics, there was general agreement that there are substantive new opportunities to improve the impact of genetic and molecular ecotoxicology on prediction of sublethal effects of exposure to hazardous substances. Future studies should emphasize integration of genetic ecotoxicology, ecological genetics, and molecular biology and should be directed toward improving our understanding of the ecological implications of genotoxic responses. Ecological implications may be assessed at either the population or ecosystem level; however, a population-level focus may be most pragmatic. Recent technical advancements in measuring genetic and molecular responses to toxicant exposure will spur rapid progress. These new techniques have considerable promise for increasing our understanding of both mechanisms of toxicity on genes or gene products and the relevance of detrimental effects to individual fitness.

Patterns of Hemocyte Production and Release Throughout the Molt Cycle in the Penaeid Shrimp Sicyonia ingentis.

The production and release of hemocytes was evaluated throughout the molt cycle in the shrimp Sicyonia ingentis. Hematopoiesis occurs in paired epigastric hematopoietic nodules (HPN) which consist of an extensive network of vessels. Hemocytes are produced within the walls of these tubules and released into the vessel lumens. During molt stage C (intermolt), few cells were present in the tubule wall; most of these were hematopoietic stem cells. Elevated mitotic rates during stages C to D1-2 (2-4%) led to the production and rapid release of individual hemocytes, primarily granulocytes. Although the mitotic rate progressively declined from stage D3-4 until after ecdysis (stage A1), the maturing hemocytes accumulated within the tubule walls. Around ecdysis, production of hyaline hemocytes exceeded that of granulocytes. Large groups of these hemocytes were channeled into the vessel lumens immediately after molting. Mitotic rates increased again during stages A2 and B with the number of hemocytes in the tubules reaching seven times that of stage C. Morphological stages in the transition of hematopoietic stem cells into hyaline hemocytes and granulocytes are described, and a model of decapod hemocyte maturation is presented.

A Decapod Hemocyte Classification Scheme Integrating Morphology, Cytochemistry, and Function.

We have examined the hemocytes of three decapod crustaceans (Homarus americanus, Panulirus interruptus, and Loxorhynchus grandis) and propose a classification of these cells based on morphology, cytochemistry, and studies of cell functions. In all species, hyaline cells and granulocytes were identified. Although we have retained the widely used names for these cells, we show that traditional morphological features alone do not accurately differentiate between these categories. Historically, the term hyaline cell refers to hemocytes that contain no or only a few cytoplasmic granules, whereas granulocytes contain abundant granules. However, the size and number of granules in hyaline cells vary greatly between species and therefore are not useful criteria for identifying these cells. Since morphological identification alone is inadequate and misleading, especially with regard to hyaline cells, a combination of morphological, cytochemical and functional methods is necessary to identify decapod hemocytes. Features of hyaline cells include: a higher nucleocytoplasmic ratio than that of granulocytes, the presence of abundant small ({approx}50 nm), round, electron-dense deposits in the cytoplasm, and their accumulation of trypan blue dye prior to cytolysis. Granulocytes do not take up trypan blue or lyse during a 5-min incubation, and they contain prophenoloxidase and hydrolases. Hyaline cells are involved in the initiation of hemolymph coagulation whereas granulocytes are involved in defense against foreign material by phagocytosis and encapsulation. We propose that these criteria be applied to other crustacean species and expect that they will facilitate our understanding of the physiological roles of their hemocytes.

Reproductive impairment in a fish inhabiting a contaminated coastal environment off Southern California.

White croaker (Genyonemus lineatus), collected from a highly contaminated site in San Pedro Bay and from a reference site 80 km away (Dana Point), were induced to spawn in the laboratory. Forty-one per cent of San Pedro Bay females and 54% of Dana Point females spawned. Examination of the ovaries of non-spwaning females revealed that spawning was imminent in the remainder of Dana Point fish but only in 16% of the San Pedro Bay fish. The remainder of the San Pedro Bay fish (43%) contained only immature, yolky oocytes. No croakers containing more than 3.8 ppm ovarian total DDT could be induced to spawn whereas 36% of a contemporaneous San Pedro Bay sample had ovarian total DDT residues in excess of 4 ppm. This suggests that the inability to induce spawning in white croaker may be associated with an ovarian total DDT threshold of about 4 ppm. These data, coupled with observed decreases in fecundity (32%), fertility (14%), and early oocyte loss (30%) relative to reference fish, could partially explain the population declines observed for many southern California fishes since the 1940s.

Evaluation of acute bioassays for assessing toxicity of polychlorinated biphenyl-contaminated soils.

Proposed State of California regulations use fish toxicity information as one criterion in municipal or industrial waste hazard evaluation. Static 96-hr bioassays were performed using fathead minnows (Pimephales promelas), blacksmith (Chromis punctipinnis), and glass shrimp (Palaemonetes kadiakensis) exposed to soil experimentally contaminated with up to 500 ppm polychlorinated biphenyl (PCB) capacitor fluid added at a concentration of 500 mg liter-1. Other bioassays were conducted with a 6-day mixing period prior to the bioassay or with acetone added to solubilize the PCBs. No mortality attributable to PCB toxicity was observed in definitive bioassays using the two fish and one invertebrate species. PCB levels leached from soil containing 500 ppm Aroclor 1242 ranged from less than 0.6 to 3.4 ppb in freshwater tests to 3.5 ppb in seawater bioassays. Using these data as the basis for waste classification, soils contaminated with up to 500 ppb PCBs during capacitor spills would be designated nonhazardous. PCBs are known to be environmentally persistent and to bioaccumulate. Acute toxicity tests, therefore, do not adequately evaluate the general toxicity of PCB-contaminated soils. Hazardous waste regulations for hydrophobic compounds such as PCBs should instead be based upon chronic toxicity data and should also consider bioaccumulation potential.

Potential uses of sea urchin embryos for identifying toxic chemicals: description of a bioassay incorporating cytologic, cytogenetic and embryologic endpoints.

A method for evaluating pollutant genotoxicity, embryotoxicity and teratogenicity using sea urchin embryos has been developed and was tested using benzo(a)pyrene (BP). Initial results suggested that the bioassay may be a sensitive indicator of pollutant toxicity and mutagenicity since several endpoints can be simultaneously assessed. The bioassay is rapid, inexpensive and appears applicable to a variety of toxicants and delivery methods. The test is based upon the standard 48 h sea urchin development assay and incorporates cytologic-cytogenetic analysis of embryos. Following toxic exposure of gametes, fertilization success is assessed. Embryos then develop for 48 h at which time survival and teratogenesis are evaluated. A subsample of embryos is stained and dissociated into monolayers and mitotic configurations are examined using light microscopy. Embryo mitotic rates are used as an indicator of overall embryonic health. Cytotoxic effects are concomitantly evaluated. Genotoxicity is measured using two methods: (1) anaphase aberration analysis, a technique which assesses abnormalities in the chromosome configurations (such as bridges and fragments) as the groups of chromosomes move to opposite poles and (2) micronucleus formation, a procedure examining the incidence of smaller, secondary nuclei composed of whole chromosomes or chromatid fragments. These two measurements preclude the need to examine individual chromosomes for deletions and exchanges, a laborious process in most aquatic organisms which possess numerous relatively small chromosomes. This genotoxicity-teratogenicity test appears promising for laboratory evaluations of individual substances or of complex chemical mixtures as well as for environmental monitoring of nearshore areas. The standard development assay has been used to screen pharmaceuticals and environmental contaminants and some recent investigations have included mitotic aberration analysis. Experiments in our laboratory suggest that the genotoxicity-teratogenicity test may be a feasible approach to field monitoring. Mutagen loads of spawning adult urchins could be assessed by conducting cytologic-cytogenetic analysis of resulting embryos although initial studies suggest that this method is less sensitive than direct embryo exposures.

Oxygen consumption rates of grunion (Leuresthes tenuis) embryos exposed to the petroleum hydrocarbon, benzo[a]pyrene.

Bioconcentration of [14C]benzo[a]pyrene and effects of unlabeled benzo[a]pyrene (BaP) accumulation on the routine oxygen consumption of embryonic grunion (Leuresthes tenuis) were studied. At Day 15, bioconcentration factors over dissolved BaP levels ranged from 249 to 466. Weight-specific respiration rates at Days 14-15 were significantly increased (P less than or equal to 0.05) at a mean BaP body burden of 0.51 ppm wet wt. Oxygen consumption rates of embryos containing 0.70 to 12.80 ppm BaP were not significantly different from control rates. Because in a previous study embryos containing 0.51 ppm BaP exhibited hatching and developmental rates similar to those of controls, their metabolic response to low-level hydrocarbon exposure may be an example of hormesis, an overcompensating metabolic regulation to inhibitory challenges.

Effects of benzo(a)pyrene on early development of flatfish.

The ontogenetic effects of the environmental carcinogen benzo(a)pyrene (BAP) on three species of larval flatfish were investigated using concentrations (from 0.10 to 4.2 ppb) which were comparable to levels found in polluted harbors. BAP-treated sand sole (Psettichthys melanostichus) eggs displayed a significant decline in hatching success and a significantly higher incidence of developmental anomalies than did control eggs. Flathead sole (Hippoglossoides elassodon) eggs exposed to a single dose of a water-soluble BAP-bovine serum albumin complex demonstrated evidence of toxic injury with pycnotic nuclei present in the integument and, more commonly, in ocular and neural tissues. An increased incidence of morphological anomalies in English sole (Parophyrs vetulus) eggs and larvae exposed to BAP was not detected.

Vibrio damsela, a Marine Bacterium, Causes Skin Ulcers on the Damselfish Chromis punctipinnis.

A previously undescribed marine bacterium, Vibrio damsela, was isolated from naturally occurring skin ulcers on a species of temperate-water damselfish, the blacksmith (Chromis punctipinnis). Laboratory infection of the blacksmith with Vibrio damsela produced similar ulcers. Vibrio damsela was pathogenic for four other species of damselfish but not for members of other families of fish. The bacterium has also been isolated from water and from two human wounds and may be a cause of human disease.

Uptake of benzo[a]pyrene by gonadal tissue of flatfish (family Pleuronectidae) and its effects on subsequent egg development.

Accumulation of benzo[a]pyrene (BaP) by sexually mature flatfish gonad, its transfer to developing gametes, and its subsequent effects on developing embryos were studied. Thin-layer chromatography revealed both unmetabolized BaP and polar metabolites in the ovary, wolffian ducts, oocytes, and semen of English sole 24 h after ip injection with 200 microCi [3H]BaP. Concentrations of BaP and its metabolites were 3-11 times higher in oocytes and semen than in gonadal tissue. Fertilized eggs from flathead sole that had been fed 4.0 mg BaP 5 h before spawning demonstrated a significantly lower (p less than 0.001) hatching success (11.9%) than eggs from control fish (56.6%). Morphological abnormalities were found in only 1.6% of control embryos but in 5.6% of embryos from treated females.