A multi-taxonomic framework for assessing relative petrochemical vulnerability of marine biodiversity in the Gulf of Mexico.

A fundamental understanding of the impact of petrochemicals and other stressors on marine biodiversity is critical for effective management, restoration, recovery, and mitigation initiatives. As species-specific information on levels of petrochemical exposure and toxicological response are lacking for the majority of marine species, a trait-based assessment to rank species vulnerabilities to petrochemical activities in the Gulf of Mexico can provide a more comprehensive and effective means to prioritize species, habitats, and ecosystems for improved management, restoration and recovery. To initiate and standardize this process, we developed a trait-based framework, applicable to a wide range of vertebrate and invertebrate species, that can be used to rank relative population vulnerabilities of species to petrochemical activities in the Gulf of Mexico. Through expert consultation, 18 traits related to likelihood of exposure, individual sensitivity, and population resilience were identified and defined. The resulting multi-taxonomic petrochemical vulnerability framework can be adapted and applied to a wide variety of species groups and geographic regions. Additional recommendations and guidance on the application of the framework to rank species vulnerabilities under specific petrochemical exposure scenarios, management needs or data limitations are also discussed.

20th Pollutant Responses in Marine Organisms (PRIMO 20): Global issues and fundamental mechanisms caused by pollutant stress in marine and freshwater organisms.

The 20th Pollutant Responses in Marine Organisms (PRIMO 20) conference provided a forum for scientists from around the world to communicate novel toxicological research findings specifically focused on aquatic organisms, by combining applied and basic research at the intersection of environmental and mechanistic toxicology. The work highlighted in this special issue of Aquatic Toxicology, a special issue of Marine Environmental Research, and presented through posters and presentations, encompass important and emerging topics in freshwater and marine toxicology. This includes multiple types of emerging contaminants including microplastics and UV filtering chemicals. Other studies aimed to further our understanding of the effects of endocrine disrupting chemicals, pharmaceuticals, and personal care products. Further research presented in this virtual issue examined the interactive effects of chemicals and pathogens, while the final set of manuscripts demonstrates continuing efforts to combine traditional biomonitoring, data from -omic technologies, and modeling for use in risk assessment and management. An additional goal of PRIMO meetings is to address the link between environmental and human health. Several articles in this issue of Aquatic Toxicology describe the appropriateness of using aquatic organisms as models for human health, while the keynote speakers, as described in the editorial below, presented research that highlighted bioaccumulation of contaminants such as PFOS and mercury from fish to marine mammals and coastal human populations such as the Gullah/GeeChee near Charleston, South Carolina, USA.

Bioreactivity and Sunlight Potentiation of Hybrid Polymer Nanoparticles in Oysters, Crassostrea virginica.

Polymer dynamic organic theranostic spheres (PolyDOTS) are being studied as a photothermal ablation treatment of cancer and are activated by blue or near-infrared light. PolyDOTS or related hybrid polymeric nanoparticle products could be mass-produced, and it is expected that they, like any other engineered nanoparticle (ENP), could be found in aquatic environments. The purpose of this study was to investigate the potential toxicity of these types of hybrid nanoparticles on nontarget marine organisms. To investigate the potential of even greater toxicity due to solar exposure, the PolyDOTS were exposed to the sun for 12 h and "charged". Once charged, hepatopancreas and gill tissues from oysters (Crassostrea virginica) were exposed to concentrations of both uncharged and charged PolyDOTS for 24 h in vitro. The effects of PolyDOTS on lysosomal destabilization, cell viability, and free radical damage were analyzed. Significant sublethal toxicity was observed with the charged particles, and reduced viability was observed with uncharged particles in vitro. In vivo imaging studies using IVIS (In Vivo Imaging System) indicated PolyDOTS uptake in tissues. These types of studies are important for understanding the potential effects of light sensitive ENPs in oysters and their broader ecological impacts on estuarine ecosystems.

Dietary Uptake of Cu Sorbed to Hydrous Iron Oxide is Linked to Cellular Toxicity and Feeding Inhibition in a Benthic Grazer.

Whereas feeding inhibition caused by exposure to contaminants has been extensively documented, the underlying mechanism(s) are less well understood. For this study, the behavior of several key feeding processes, including ingestion rate and assimilation efficiency, that affect the dietary uptake of Cu were evaluated in the benthic grazer Lymnaea stagnalis following 4-5 h exposures to Cu adsorbed to synthetic hydrous ferric oxide (Cu-HFO). The particles were mixed with a cultured alga to create algal mats with Cu exposures spanning nearly 3 orders of magnitude at variable or constant Fe concentrations, thereby allowing first order and interactive effects of Cu and Fe to be evaluated. Results showed that Cu influx rates and ingestion rates decreased as Cu exposures of the algal mat mixture exceeded 10(4) nmol/g. Ingestion rate appeared to exert primary control on the Cu influx rate. Lysosomal destabilization rates increased directly with Cu influx rates. At the highest Cu exposure where the incidence of lysosomal membrane damage was greatest (51%), the ingestion rate was suppressed 80%. The findings suggested that feeding inhibition was a stress response emanating from excessive uptake of dietary Cu and cellular toxicity.

Cellular responses of eastern oysters, Crassostrea virginica, to titanium dioxide nanoparticles.

Because of the continued development and production of a variety of nanomaterials and nanoparticles, their uptake and effects on the biota of marine ecosystems must be investigated. Filter feeding bivalve molluscs are highly adapted for capturing particles from the external environment and readily internalize nano- and micro-sized particles through endocytosis, so they are commonly used as valuable indicator species for nanoparticle studies. In these studies, adult eastern oysters, Crassostrea virginica, were exposed to a range of titanium dioxide nanoparticle (TiO2-NP) concentrations (5, 50, 500, and 5000 µg/L) in conjunction with natural sunlight. Isolated hepatopancreas tissues were also exposed to the same TiO2-NP concentrations using particles exposed to similar light and dark conditions. Dose-dependent decreases in lysosomal stability were observed in the adult oyster studies as well as in the isolated tissues, at exposures as low as 50 µg/L. Titanium accumulation in isolated hepatopancreas tissue studies was directly correlated to lysosomal destabilization. Based on measurements of lipid peroxidation as an indicator of oxidative stress, TiO2-NPs toxicity was not related to increased ROS production over the short-term course of these exposures. Analysis of particle size using dynamic light scattering (DLS) indicated that concentration had a significant impact on agglomeration rates, and the small agglomerates as well as individual particles are readily processed by oysters. Overall, this study illustrates that low concentrations of TiO2-NPs may cause sublethal toxicity on oysters, which might be enhanced under natural sunlight conditions. In estuarine environments, where these nanomaterials are likely to accumulate, agglomeration rates, interaction with organics, and responses to sunlight are critical in determining the extent of their bioreactivity and biological impacts.

Resuspended contaminated sediments cause sublethal stress to oysters: A biomarker differentiates total suspended solids and contaminant effects.

Resuspended contaminated sediments represent an important route of contaminant exposure for aquatic organisms. During resuspension events, filter-feeding organisms are exposed to contaminants, in both the dissolved form (at the gills) and the particulate form (in the digestive system). In addition, these organisms must manage the physical stress associated with an increase in total suspended solids (TSS). To date, few studies have experimentally compared the contributions to biological stress of contaminated and clean suspended solids. The authors mixed field-collected sediments (<63 µm) from clean and contaminated field sites to create 4 treatments of increasing metal concentrations. Sydney rock oysters were then exposed to sediment treatments at different TSS concentrations for 4 d, and cellular biomarkers (lysosomal membrane stability, lipid peroxidation, and glutathione) were measured to evaluate sublethal toxicity. Lysosomal membrane stability was the most sensitive biomarker for distinguishing effects from resuspended contaminated sediments, as increasing amounts of contaminated TSS increased lysosomal membrane destabilization. The authors' results illustrate the importance of considering contaminant exposures from resuspended sediments when assessing the toxicity of contaminants to aquatic organisms.

Short-term acute hypercapnia affects cellular responses to trace metals in the hard clams Mercenaria mercenaria.

Estuarine and coastal habitats experience large fluctuations of environmental factors such as temperature, salinity, partial pressure of CO2 ( [Formula: see text] ) and pH; they also serve as the natural sinks for trace metals. Benthic filter-feeding organisms such as bivalves are exposed to the elevated concentrations of metals in estuarine water and sediments that can strongly affect their physiology. The effects of metals on estuarine organisms may be exacerbated by other environmental factors. Thus, a decrease in pH caused by high [Formula: see text] (hypercapnia) can modulate the effects of trace metals by affecting metal bioavailability, accumulation or binding. To better understand the cellular mechanisms of interactions between [Formula: see text] and trace metals in marine bivalves, we exposed isolated mantle cells of the hard clams (Mercenaria mercenaria) to different levels of [Formula: see text] (0.05, 1.52 and 3.01 kPa) and two major trace metal pollutants - cadmium (Cd) and copper (Cu). Elevated [Formula: see text] resulted in a decrease in intracellular pH (pHi) of the isolated mantle cells from 7.8 to 7.4. Elevated [Formula: see text] significantly but differently affected the trace metal accumulation by the cells. Cd uptake was suppressed at elevated [Formula: see text] levels while Cu accumulation has greatly accelerated under hypercapnic conditions. Interestingly, at higher extracellular Cd levels, labile intracellular Cd(2+) concentration remained the same, while intracellular levels of free Zn(2+) increased suggesting that Cd(2+) substitutes bound Zn(2+) in these cells. In contrast, Cu exposure did not affect intracellular Zn(2+) but led to a profound increase in the intracellular levels of labile Cu(2+) and Fe(2+). An increase in the extracellular concentrations of Cd and Cu led to the elevated production of reactive oxygen species under the normocapnic conditions (0.05 kPa [Formula: see text] ); surprisingly, this effect was mitigated in hypercapnia (1.52 and 3.01 kPa). Overall, our data reveal complex and metal-specific interactions between the cellular effects of trace metals and [Formula: see text] in clams and indicate that variations in environmental [Formula: see text] may modulate the biological effects of trace metals in marine organisms.

Tissue specific responses of oysters, Crassostrea virginica, to silver nanoparticles.

Silver nanoparticle (AgNP) toxicity in eastern oysters, Crassostrea virginica, was investigated in both gill and hepatopancreas tissues, and compared to dissolved Ag exposures. Oysters were exposed to varying concentrations of AgNPs prepared with citrate coatings and dissolved Ag (AgNO3) for 48 h and the effects on a suite of biomarkers of cellular stress were evaluated. Two biomarkers of cellular damage were evaluated-lysosomal destabilization rates of hepatopancreas cells as indicators of lysosomal damage, and lipid peroxidation as an indicator of oxidative damage. Total glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) assays were used as indicators of impacts on antioxidant capacity. The results indicated that gill tissues were more prone to oxidative damage following dissolved Ag exposures than AgNP exposures, while hepatopancreas tissues were more sensitive to AgNP exposures. Total protein levels increased in hepatopancreas tissues following AgNP exposures, but not dissolved Ag exposures. These tissue specific differences in toxicity and bioreactivity of Ag nanoparticles compared to dissolved metal ions reflect distinct nanoparticle effects. Oysters exposed to dissolved Ag would be more prone to cellular and tissue damage of gills, but oysters exposed to AgNPs could be more prone to hepatopancreas damage contributing to metabolic and reproductive impairment.

Indicators of environmental stress: cellular biomarkers and reproductive responses in the Sydney rock oyster (Saccostrea glomerata).

We measured a suite of common biomarker responses for the first time in the Sydney rock oyster Saccostrea glomerata to evaluate their utility as biological effects measures for pollution monitoring. To examine the relationship between biomarker responses and population level effects, fertilisation and embryo development assays were also conducted. Adult oysters were deployed in two contaminated estuaries and a reference estuary in Sydney, Australia. The concentrations of various contaminants (metals and polycyclic aromatic hydrocarbon, PAHs) were quantified in oyster's tissue from each site and both metals and total PAHs were significantly elevated in contaminated estuaries relative to the reference estuary. Lysosomal membrane destabilisation, lipid peroxidation levels and glutathione (GSH) concentrations were measured in the digestive gland of oysters. Of all biomarkers measured, lysosomal membrane destabilisation proved to be the most useful indicator of oysters facing anthropogenic stress and we suggest this may be an especially useful biomarker for incorporation into local environmental monitoring programs. Moreover, lysosomal membrane destabilisation showed good correlations with fertilisation, normal embryo development and estuary status. GSH and lipid peroxidation were not as valuable for distinguishing between estuaries exposed to differing levels of anthropogenic stress, but did provide additional valuable information regarding overall health status of the oysters.

The effects of silver nanoparticles on oyster embryos.

Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release following their use in various commercial formulations and biologic applications. Filter-feeding bivalve mollusks such as oysters are valuable model species for characterizing nanoparticle bioavailability and interactions with basic cellular processes. The adults release their gametes into the environment, so their embryos and larvae are also likely targets of nanoparticles. The purpose of these studies was to characterize the toxicity of metal nanoparticles on embryonic development of oysters, Crassostrea virginica and to compare the relative sensitivity of embryos to adults. Newly-fertilized oyster embryos were exposed to silver nanoparticles (AgNP) and then the percent normal development after 48h was assessed. Studies were conducted with adult oysters in which they were also exposed to AgNP for 48h, and the effects on lysosomal destabilization were determined. The expression of metallothionein (MT) gene expression was also assessed in both embryos and adults. Adverse effects on embryonic development were observed at concentrations similar to those that caused both statistically and biologically significant effects on lysosomal destabilization of adults. Significant increases in MT mRNA levels were observed in both embryos and adult oysters, and MT levels were highly induced in embryos. While we do not know whether the toxicity and gene expression responses observed in this study were due to the nanoparticles themselves or the Ag ions that dissociated from the nanoparticles, these kinds of basic studies are essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as well as aquatic organisms.

Fullerene exposures with oysters: embryonic, adult, and cellular responses.

Oysters are an ecologically important group of filter-feeders, and a valuable toxicology model for characterizing the potential impacts of nanoparticles to marine organisms. Fullerene (C60) exposure studies with oysters, Crassostrea virginica, were conducted with a variety of biological levels, e.g., developmental studies with embryos, whole organism exposures with adults, and isolated hepatopancreas cells. Significant effects on embryonic development and lysosomal destabilization were observed at concentrations as low as 10 ppb. Moreover, based on our extensive experience with the lysosomal assay, the lysosomal destabilization rates at fullerene concentrations > or = 100 ppb were regarded as biologically significant as they are associated with reproductive failure. Interestingly, there was no significant increase in lipid peroxidation levels in hepatopancreas tissues. Oyster hepatopancreas tissues are composed of lysosomal rich cells, and confocal microscopy studies indicated thatthe fullerene particles readily accumulated inside hepatopancreas cells within 4 h. Fullerene aggregates tended to be localized and concentrated into lysosomes. The microscopic work in conjunction with the lysosomal function assays supports the premise that endocytotic and lysosomal pathways may be major targets of fullerenes and other nanoparticles. Nanoparticles that affect normal lysosomal and autophagic processes may contribute to long-term, chronic problems for individual health as well as ecosystem health.

Combined effects of temperature acclimation and cadmium exposure on mitochondrial function in eastern oysters Crassostrea virginica gmelin (Bivalvia: Ostreidae).

Cadmium and temperature have strong impacts on the metabolic physiology of aquatic organisms. To analyze the combined impact of these two stressors on aerobic capacity, effects of Cd exposure (50 microg/L) on mitochondrial function were studied in oysters (Crassostrea virginica) acclimated to 12 and 20 degrees C in winter and to 20 and 28 degrees C in fall. Cadmium exposure had different effects on mitochondrial bioenergetics of oysters depending on the acclimation temperature. In oysters acclimated to 12 degrees C, Cd exposure resulted in elevated intrinsic rates of mitochondrial oxidation, whereas at 28 degrees C, a rapid and pronounced decrease of mitochondrial oxidative capacity was found in Cd-exposed oysters. At the intermediate acclimation temperature (20 degrees C), effects of Cd exposure on intrinsic rates of mitochondrial oxidation were negligible. Degree of coupling significantly decreased in mitochondria from 28 degrees C-acclimated oysters but not in that from 12 degrees C- or 20 degrees C-acclimated oysters. Acclimation at elevated temperatures also increased sensitivity of oyster mitochondria to extramitochondrial Cd. Variation in mitochondrial membrane potential explained 41% of the observed variation in mitochondrial adenosine triphosphate synthesis and proton leak between different acclimation groups of oysters. Temperature-dependent sensitivity of metabolic physiology to Cd has significant implications for toxicity testing and for extrapolation of laboratory studies to field populations of aquatic poikilotherms, indicating the importance of taking into account the thermal regime of the environment.

Regulation of metallothionein genes in the American oyster (Crassostrea virginica): ontogeny and differential expression in response to different stressors.

Metallothioneins (MTs) are typically low molecular weight (6-7 kDa), metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-Xn-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in metals homeostasis, detoxification and the stress response. The present study, combined with previous observations (e.g., Jenny et al., Eur. J. Biochem. 2005; 271:1702-1712) defines an unprecedented diversity of MT primary structure and domain organization in the American oyster, Crassostrea virginica. Two novel molluscan MT families are described. One of these (CvMT-III) is characterized by the presence of two beta-domains and the absence of alpha-domains. This family exhibits constitutive expression during larval development and is the dominant CvMT isoform expressed in larvae. CvMT-III displays low basal levels of expression in adult tissues and only moderate responsiveness to metal challenges in both larvae and adults. A second novel MT isoform (CvMT-IV) was isolated from hemocytes by subtractive hybridization techniques following a 4-hour immune challenge with heat-killed bacteria (Vibrio, Bacillus, Micrococcus spp. mixture). Based on conservation of the cysteine motifs, this isoform appears to be a sub-family related to the molluscan alphabeta-domain MTs. A series of amino acid substitutions has resulted in four additional cysteines which give rise to a Cys-Cys motif and three Cys-Cys-Cys motifs. Northern blot analyses demonstrate that CvMT-IV is down-regulated upon sterile wounding and immune challenge, displays moderate expression in larvae and adults and differential gene induction in response to metals exposure.

Effects of acclimation temperature and cadmium exposure on cellular energy budgets in the marine mollusk Crassostrea virginica: linking cellular and mitochondrial responses.

In order to understand the role of metabolic regulation in environmental stress tolerance, a comprehensive analysis of demand-side effects (i.e. changes in energy demands for basal maintenance) and supply-side effects (i.e. metabolic capacity to provide ATP to cover the energy demand) of environmental stressors is required. We have studied the effects of temperature (12, 20 and 28 degrees C) and exposure to a trace metal, cadmium (50 microg l(-1)), on the cellular energy budget of a model marine poikilotherm, Crassostrea virginica (eastern oysters), using oxygen demand for ATP turnover, protein synthesis, mitochondrial proton leak and non-mitochondrial respiration in isolated gill and hepatopancreas cells as demand-side endpoints and mitochondrial oxidation capacity, abundance and fractional volume as supply-side endpoints. Cadmium exposure and high acclimation temperatures resulted in a strong increase of oxygen demand in gill and hepatopancreas cells of oysters. Cd-induced increases in cellular energy demand were significant at 12 and 20 degrees C but not at 28 degrees C, possibly indicating a metabolic capacity limitation at the highest temperature. Elevated cellular demand in cells from Cd-exposed oysters was associated with a 2-6-fold increase in protein synthesis and, at cold acclimation temperatures, with a 1.5-fold elevated mitochondrial proton leak. Cellular aerobic capacity, as indicated by mitochondrial oxidation capacity, abundance and volume, did not increase in parallel to compensate for the elevated energy demand. Mitochondrial oxidation capacity was reduced in 28 degrees C-acclimated oysters, and mitochondrial abundance decreased in Cd-exposed oysters, with a stronger decrease (by 20-24%) in warm-acclimated oysters compared with cold-acclimated ones (by 8-13%). These data provide a mechanistic basis for synergism between temperature and cadmium stress on metabolism of marine poikilotherms. Exposure to combined temperature and cadmium stress may result in a strong energy deficiency due to the elevated energy demand on one hand and a reduced mitochondrial capacity to cover this demand on the other hand, which may have important implications for surviving seasonally and/or globally elevated temperatures in polluted estuaries.

Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches.

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X(n)-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan alphabeta-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of alphabeta-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the alpha-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four alpha-domains and that lack a beta-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg).

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.

Differential accumulation of heavy metals in the sea anemone Anthopleura elegantissima as a function of symbiotic state.

The accumulation of metals by the North American Pacific Coast temperate sea anemone Anthopleura elegantissima, and its dinoflagellate-algal symbiont Symbiodinium muscatinei was examined following laboratory metal exposures. Both, naturally occurring symbiotic and symbiont-free (aposymbiotic) anemones were used in this study to investigate differences in metal uptake due to the symbiotic state of the animal. The effects of metal exposures on the anemone-algal symbiosis were determined using measures of algal cell density and mitotic index (MI). Anemones were exposed to either cadmium, copper, nickel or zinc chloride (0, 10, 100 microg l(-1) for Cd, Cu and Ni; 0, 100, 1000 microg l(-1) for Zn) for 42 days followed by a 42-day recovery period in ambient seawater. Anemones were analyzed for metal content using inductively coupled plasma mass spectroscopy (ICP-MS) at various time points during the study. Symbiotic anemones accumulated Cd, Ni and Zn to a greater extent than aposymbiotic anemones. A dramatically different pattern of Cu accumulation was observed, with aposymbiotic anemones accumulating higher levels than symbiotic anemones. Following recovery in ambient seawater, all tissue metal levels were reduced to near pre-exposure control levels in most cases. No changes in algal cell density or MI were observed in symbiotic anemone tentacle clips at any dose or time point in the Cd and Cu exposures. However, significant reductions in algal cell densities were observed in the Ni-exposed and some Zn-exposed animals, although levels returned to control values following recovery. There were no changes in mitotic index (MI) following Ni or Zn exposures. These results demonstrate that the extent of heavy metal accumulation depends upon cnidarian symbiotic state and the heavy metal in question.

Seasonal variation in lysosomal destabilization in oysters, Crassostrea virginica.

Lysosomal destabilization assays have been used as valuable biomarkers of pollutant exposures in a variety of bivalve and fish species. The responses of oysters, Crassostrea virginica, deployed at and native to various reference and degraded sites were evaluated for lysosomal destabilization during both summer and winter seasons. In both native and deployed oysters, lysosomal destabilization rates tended to be higher during the winter at both reference and polluted sites. There are at least two hypothetical explanations. Greater lysosomal destabilization rates may be related to physiological changes associated with mobilization of nutrient reserves during the winter and gametogenesis. However, lysosomal destabilization in deployed oysters was correlated with tissue metal concentrations. These data also support a second hypothesis that seasonal differences in physico-chemical factors (such as reduced levels of acid volatile sulfides) may increase the bioavailability of metals during the winter so that adverse effects are more pronounced.

Comparative in situ and laboratory sediment bioassays with juvenile Mercenaria mercenaria.

Sediment toxicity assays were conducted with juvenile Mercenaria mercenaria to compare the results of laboratory assays and in situ deployments. Juvenile clams were deployed for one week at a variety of degraded and undegraded sites in Charleston Harbor, South Carolina. USA, during the summers of 1998, 1999, and 2000. Parallel laboratory assays were conducted with sediments collected from the deployment sites. Mortality and a sublethal endpoint, seed clam growth rate, were used to compare toxicity between reference and degraded sites. Growth rates of field-deployed clams tended to be higher than growth rates for laboratory assays, especially at the reference sites. Field studies indicated a higher potential for toxicity than did the laboratory studies at degraded sites. These studies suggest that laboratory assays may underestimate potential sediment toxicity at degraded sites. However, field growth rates may be affected by natural environmental factors (e.g., pH, dissolved oxygen, and salinity), so regression normalization techniques were used to distinguish the effects of these variables from those of contaminants.