Amy H. Ringwood

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.

Comparative sensitivity of gametes and early developmental stages of a sea urchin species (Echinometra mathaei) and a bivalve species (Isognomon californicum) during metal exposures

Bioassays were developed using sperm of a sea urchin (Echinometra mathaei), and sperm, embryos, and larvae of a bivalve species (Isognomon californicum). Sea urchin spawning was restricted to only a few months of the year and viability of sperm throughout the year varied from 5 to 75%. Sea urchin fertilization assays were affected by temporal variation in sperm viability. Spawning in this bivalve species occurs year-round and there is little temporal variation in sperm viability. Since stringent sperm:egg ratios are not required for the bivalve embryo and larval assays, these were less affected by variation in gamete quality. The relative sensitivity of the various assays were compared during exposure to three different metal pollutants: cadmium, copper, and tributyltin. Gametes and embryos were relatively resistant to cadmium toxicity, but larvae were very sensitive. With copper and tributyltin, sea urchin and bivalve fertilization assays were the least sensitive; and bivalve growth assays were the most sensitive, followed closely by the bivalve embryo assays. On the basis of sensitivity, ease and time required to conduct the assay, and salinity tolerance, the bivalve embryo assay was recommended as the overall single most reliable toxicity bioassay. However a multispecies, multidimensional approach using sperm fertilization assays as well as embryo assays should be employed, perhaps in a hierarchal manner. Larval growth assays were deemed too tedious and time consuming to be used routinely, but due to their high sensitivity, should still be considered as a valuable comparative tool.

Water Quality Variation and Clam Growth: Is pH Really a Non-issue in Estuaries?

A tandem deployment system was used to critically evaluate relationships between important water chemistry parameters (pH, salinity, dissolved oxygen) and biotic performance based on clam growth. The effects of environmental conditions on growth of juvenile clams,Mercenaria mercenaria, were determined after 7-day field deployments in cages at reference sites from 1998 to 2000. Continuous measurements of the overlying water chemistry parameters were monitored by deploying an in situ water quality instrument (Hydrolab Datasonde) at the same time. While salinity was identified as an important determinant of clam growth over wide salinity ranges (10–35‰), pH was also found to be a very important parameter, especially in low-salinity regimes (<25‰). Average pH measurements ranged from 7.2 to 7.8; minimal pHs ranged from 6.9 to 7.6. The results indicated that when average pH levels fell below 7.5 or minimum pH levels fell below 7.2, growth rates were <50% that of clams deployed under higher pH conditions. Estuarine systems are generally perceived as being well-buffered so pH is frequently assumed to be unimportant, but our results suggest that pH levels can decline in estuarine systems to levels that can adversely affect biological responses. The potential impacts on biological resources of even moderate decreases in pH, particularly in systems that naturally tend to have lower pH conditions, may be more important than previously realized.

Potential Indicators of Stress Response Identified by Expressed Sequence Tag Analysis of Hemocytes and Embryos from the American Oyster, Crassostrea virginica

Abstract: A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.

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

SUMMARY 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°C) and exposure to a trace metal, cadmium (50 μg 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°C but not at 28°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°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.

Metal-specific induction of metallothionein isoforms in the blue crab Callinectes sapidus in response to single- and mixed-metal exposure

Metallothioneins (MTs) play an important role in the metabolism of copper and zinc during the molt cycle of the blue crab. In this study we examined the hypothesis that MT expression in crabs is metal specific. Anion-exchange chromatography showed one major ZnCuMT (ZnMT-I) in control crabs, two MT isoforms in cadmium-treated crabs (CdMT-I, CdMT-II), and three forms (CuMT-I, CuMT-II, CuMT-III) in copper-treated animals. Amino acid analysis of the carboxymethylated apo-MTs, purified by reversed-phase HPLC, showed minor differences between ZnMT-I, CdMT-I, CdMT-II, CuMT-I, and CuMT-II, while CuMT-III was unique. After combined exposure to cadmium and copper, four MTs with differing copper/cadmium ratios were observed, equivalent to CdMT-II and the three CuMTs. We conclude that the blue crab has four genes that encode different MTs. Transcription of the CdMT-I gene is induced by cadmium, but inhibited by copper. CuMT-I, CdMT-II, and ZnMT-I may be the products of a single gene responsive to copper, cadmium, and zinc. Expression of the CuMT-II and CuMT-III genes is initiated by copper and not by cadmium. We believe that CdMT-I and CuMT-III are important in detoxification, whereas Zn/CuMT-I and CuMT-II are involved in regulatory functions. These results show the importance of the use of mixed-metal exposures in the study of the molecular mechanisms of metal regulation and function.

Harmful algal blooms in South Carolina residential and golf course ponds

The South Carolina coastal zone is among the fastest growing areas in the U.S., and population epicenters are marked by dense brackish water pond (lagoon) coverage associated with housing complexes and golf courses. Surveillance efforts in 2001–2002 documented the widespread occurrence of several types of potentially or measurably toxic harmful algal blooms (HABs) in these ponds. These man-made retention ponds were constructed in order to serve as a buffer between developed areas and open estuaries or for aesthetic reasons. However, the combination of restricted tidal flow and nutrient and/or contaminant deposition creates a stimulatory environment for potential HAB formation. These discoveries introduce the need to consider mitigation measures to existing ponds and HAB preventive strategies for future pond construction.

Biomarker studies with juvenile oysters (Crassostrea virginica) deployed in-situ

Hatchery-reared juvenile oysters (Crassostrea virginica) were deployed in situ for approximately 1 month from mid-May to mid-June of 1996 at sites that were classified as reference, agricultural, suburban, or urban/industrial. Cellular responses (lysosomal destabilization, glutathione concentrations, lipid peroxidation, heat shock proteins, metallothioneins, and multi-xenobiotic resistance proteins) were analysed, and their efficacy as biomarkers of stress was evaluated. Increased lysosomal destabilization, glutathione depletion, increased lipid peroxidation, and induction of heat shock proteins and metallothioneins were observed at many of the polluted sites, but increases in multixenobiotic resistance proteins were not. Significant correlations between sediment contaminants and lysosomal destabilization or glutathione concentrations were observed. Similarly, there were significant correlations between sediment cadmium and copper levels and metallothioneins. Although elevated lipid peroxidation products and heat shock proteins were observed at some of the contaminated sites, there were no significant correlations with contaminants. These studies suggest that lysosomal destabilization and glutathione depletion are sensitive, robust indicators of contaminant stress. Although lipid peroxidation and heat shock protein responses were not correlated with contaminants, they are still regarded as valuable indicators of stress. These studies demonstrate the value of using a suite of cellular biomarkers to identify and characterize stress responses related to anthropogenic perturbations.

The effects of copper exposures on cellular responses in oysters

Copper is an essential and toxic trace metal that can adversely affect metal-requiring enzymes and proteins, and cause oxidative damage. Oysters, Crassostrea virginica, bioconcentrate Cu from environments contaminated by a variety of point and non-point sources. The purpose of these studies was to evaluate a suite of cellular parameters (lysosomal destabilization, glutathione concentrations, lipid peroxidation, and metallothioneins) in Cu-exposed oysters (5 to 80 μgl−1 for two weeks) in order to distinguish between exposure and stress responses. Copper exposures caused increased lysosomal destabilization, increased lipid peroxidation, and induction of Cu metallothioneins, but no effects on glutathione were observed. Experiments were also conducted in which BSO was used to deplete glutathione to consider how other environmental factors that affect amelioration responses may potentiate Cu toxicity. Lysosomal destabilization rates were significantly higher in glutathione-depleted oysters. Lipid peroxidation was higher initially during the first four days of Cu exposures, but then the levels declined to control levels. This amelioration was associated with the increased expression of metallothioneins. During the first four days of Cu exposure, no significant increases in MTs were observed, but subsequent induction of MTs was associated with reduced lipid peroxidation. Some cellular responses to Cu exposures represent normal compensatory mechanisms that may effectively ameliorate the insult. Therefore, it is important to appreciate the significance of the response or use a suite of responses to determine when effects have progressed from an exposure response to really being a signal of stress.

Effects of glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica)

Glutathione is a tripeptide that plays an important role in ameliorating metal toxicity. Depletion of glutathione has been associated with an increased risk of metal toxicity in mammals. An understanding of the toxicological significance of glutathione depletion in oysters would be of considerable importance given the widespread use of bivalves in biological monitoring. Laboratory studies were conducted by using an inhibitor of glutathione synthesis (buthionine sulfoximine) to investigate the effects of glutathione depletion on metallothionein expression, lysosomal membrane destabilization, and lipid peroxidation in Cu-exposed oysters. In oysters exposed to Cu (20 and 80 µg/l) and buthionine sulfoximine (20 mg/l), metallothionein induction was suppressed and cellular stress responses were frequently higher than those observed in oysters exposed singly to Cu. Together, these results suggest that environmental conditions that cause glutathione depletion may increase the potential for adverse effects to pollutants during in situ exposures.