William S. Fisher

Strategies for evaluating indicators based on guidelines from the Environmental Protection Agency's Office of Research and Development

The Environmental Protection Agency’s Office of Research and Development (ORD) has prepared technical guidelines to evaluate the suitability of ecological indicators for monitoring programs. The guidelines were adopted by ORD to provide a consistent framework for indicator review, comparison and selection, and to provide direction for research on indicator development. The guidelines were organized within four evaluation phases: (1) conceptual relevance; (2) feasibility of implementation; (3) response variability; (4) interpretation and utility. Three example indicators were analyzed to illustrate the use of the guidelines in an evaluation. The examples included a direct chemical measurement (dissolved oxygen concentration), an estuarine benthic community index, and a stream fish community index of biotic integrity. Comparison of the three examples revealed differences in approach, style and types of information used to address each guideline. The Evaluation Guidelines were intended to be flexible within a consistent framework and the various strategies used in the examples demonstrate that the process can be useful for a wide variety of indicators and program objectives. Published by Elsevier Science Ltd.

A survey of oysters Crassostrea virginica from Tampa Bay, Florida: associations of internal defense measurements with contaminant burdens

Oysters from 16 sites in Tampa Bay, Florida, were collected during a 6-week period in winter 1993 and analyzed for both biological characteristics and tissue chemical concentrations. Using previous sediment contamination and toxicity data, oyster tissues from the selected sites were expected to exhibit a wide range in both quantity and type of chemicals. Chemical analysis showed tissue concentrations at some of these sites to be greater than national averages, as reported by the National Status and Trends Mussel Watch Program, for total PAH, total PCB, total chlordanes, DDT, Cu, Pb and Zn. Measures of oyster internal defense, including hemocyte density, rate of locomotion and superoxide generation, varied significantly among sites and were generally higher at sites with higher tissue concentrations of xenobiotic chemicals. Potential associations between oyster defense characteristics and accumulated chemical contaminants, either singly or in chemical classes, were explored using correlation analysis and a composited ranking procedure. Positive relationships were found for hemocyte characteristics with certain trace metal (Cu, Sn and Zn) and PAH analytes, whereas negative relationships were found with certain PCB and pesticide analytes. Heightened defenses in contaminated conditions may reflect a hemocyte process for sequestration and detoxification of environmental contaminants. Oysters from four of the 16 sites were additionally collected in June and September 1993 and site-related differences did not closely parallel those obtained in winter. Seasonal environmental factors may have altered contaminant-related differences among sites.

Mitigating Local Causes of Ocean Acidification with Existing Laws

Even as global and national efforts struggle to mitigate CO2 emissions, local and state governments have policy tools to address “hot spots” of ocean acidification. As the level of atmospheric carbon dioxide (CO2) continues to rise, so too does the amount of CO2 in the ocean (1, 2), which increases the oceans acidity. This affects marine ecosystems on a global scale in ways we are only beginning to understand: for example, impairing the ability of organisms to form shells or skeletons, altering food webs, and negatively affecting economies dependent on services ranging from coral reef tourism to shellfish harvests to salmon fisheries (3–5). Although increasing anthropogenic inputs drive acidification at global scales, local acidification disproportionately affects coastal ecosystems and the communities that rely on them. We describe policy options by which local and state governments—as opposed to federal and international bodies—can reduce these local and regional “hot spots” of ocean acidification.

Relationships between tissue contaminants and defense-related characteristics of oysters (Crassostrea virginica) from five Florida bays.

Evidence linking bivalve defense responses with pollutant exposure is increasing. Contaminant effects on immune or defense responses could influence the ability of an organism to resist infectious disease. This study explored relationships between xenobiotic chemicals accumulated in oyster (Crassostrea virginica) tissue and various measures of putative oyster internal defense activities and physiological condition. Defense-related and physiological measurements were made on individual oysters collected from 22 sites at five Florida bays and pooled oyster tissue from each site was analyzed for polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and certain pesticides. Chemical concentrations, physiological condition, and hemocyte and hemolymph characteristics varied across bays and among sites within a bay. Within-bay comparisons showed that sites with high oyster defense-related activities often had accompanying high tissue concentrations of one or more classes of xenobiotic chemicals. Correlation analysis performed across bays demonstrated significant positive relationships between most defense-related characteristics and at least one contaminant, including various PAH, PCB and trace metal analytes. In combination with other recent studies, these results strengthen the hypothesis that certain xenobiotic chemicals may be associated with elevated oyster hemocyte activities, even though the ultimate influence on disease resistance remains unknown.

Appraisal of prospective bivalve immunomarkers.

Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental conditions. This has stimulated the need for development and application of select biological and ecological measurements, or indicators, that are responsive to environmental stress. Measures of bivalve mollusc defence activities, such as haemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules; and haemolymph constituents, such as agglutinins and lysozyme, have potential as indicators and appear to be responsive to xenobiotic chemical insults in the aquatic environment. However, basic research on the relevance of these measurements in inferring resistance to disease or enhanced survival is currently insufficient, reducing their value as potential biomarkers to address environmental objectives. In addition, variation in defence activities caused by seasonal temperature and reproductive cycling, salinity changes, nutritional status, diseases ...Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental conditions. This has stimulated the need for development and application of select biological and ecological measurements, or indicators, that are responsive to environmental stress. Measures of bivalve mollusc defence activities, such as haemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules; and haemolymph constituents, such as agglutinins and lysozyme, have potential as indicators and appear to be responsive to xenobiotic chemical insults in the aquatic environment. However, basic research on the relevance of these measurements in inferring resistance to disease or enhanced survival is currently insufficient, reducing their value as potential biomarkers to address environmental objectives. In addition, variation in defence activities caused by seasonal temperature and reproductive cycling, salinity changes, nutritional status, diseases and parasites, and genetic stocks is high and may limit applicability of bivalve defence-related measurements as indicators. This review examines these sources of variability and their possible implications for interpreting changes in bivalve defence activity as an indicator of stress. Examples of contaminant-induced changes in bivalve defence functions are described.

Occurrence of agglutinins in the pallial cavity mucus of oysters

Abstract Mucus and fluid from the pallial (mantle) cavity of eastern oysters Crassostrea virginica Gmelin from Chesapeake Bay and Galveston Bay were found to agglutinate a variety of vertebrate erythrocytes (RBC) and bacteria. Agglutinating activity of pallial cavity fluid was probably due to dissociation of agglutinins from mucus on the external surfaces of organs. Agglutination titers of pallial cavity fluid from individual Galveston Bay oysters for six different RBC were positively correlated with high significance, indicating a strong interdependence regardless of RBC specificity. The relative agglutinating activity for different RBC by tissue explants and homogenized tissues (mantle, gill, digestive gland and adductor muscle) was similar to the relative activity of the pallial cavity fluid; mantle and gill tissue had the greatest agglutinating capacity over all RBC, whereas the adductor muscle had the least. Positive correlation was detected between agglutination titers of pallial cavity fluid (for different RBC) and titers of hemolymph agglutinins from the same oysters. It is hypothesized that agglutinins in the pallial cavity and hemolymph have a common source. The results of the explant and homogenate assays can be interpreted to suggest that the source is mantle and gill tissues.

A simple test for toxicity of Number 2 fuel oil and oil dispersants to embryos of grass shrimp, Palaemonetes pugio

Abstract A simple test, using embryos of the grass shrimp Palaemonetes pugio, was employed to determine the toxicity of two commercial oil dispersants (Corexit 7664 and Corexit 9527) and toxicity of the water soluble fraction of Number 2 fuel oil (WSFoil) prepared with and without the addition of the dispersants. Tests revealed P. pugio embryos were similar to previously measured life stages in their sensitivity to WSFoil prepared without dispersants. They were approximately ten times more sensitive to water soluble fractions of dispersed oil, which may have been due to the increases in total hydrocarbons (measured analytically). Both temperature and salinity of the sea water affected toxicity of WSF prepared with dispersants, the most obvious effect being earlier onset of mortalities at higher temperatures. Differences observed in the onset of mortalities with WSF prepared with and without dispersants implicated egg-casing permeability as a factor in toxicity. The shrimp embryo toxicity test, described here for the first time, exhibited highly significant results, outstanding reproducibility and virtually 100% response within a narrow time interval.

A rapid tetrazolium dye reduction assay to assess the bactericidal activity of oyster (Crassostrea virginica) hemocytes against Vibrio parahaemolyticus

Abstract An assay was developed to assess the ability of oyster, Crassostrea virginica , hemocytes to kill the human pathogenic bacterium, Vibrio parahaemolyticus (ATCC 17802). Bacterial killing was estimated colorimetrically by the enzymatic reduction of a tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium (MTS), and phenylmethasulfazone (PMS). The assay proposed here provides an indicator of immunocompetence of oysters against V. parahaemolyticus . The assay involved: (1) exposure of plasma-free oyster hemocytes to a streptomycin (SM)-resistant mutant strain of V. parahaemolyticus in a 96-well plate for 3 h at 17°C in SM-augmented sea water; (2) growout of surviving bacteria in nutrient broth for 2 h at 37°C; (3) addition of MTS and PMS; and (4) measurement of MTS/PMS reduction product (formazan) at 490 nm using a microplate reader. Advantages of this assay include the absence of radio-isotopes used in some killing assays and requirement of low volumes of plasma and numbers of hemocytes. In addition, we demonstrated greater precision than traditional, plate counting methods for bacterial estimation. This technique has the potential to evaluate oyster capacity to eliminate microbial agents and to assess effects of environmental changes and pollutant stress on defense capabilities of oysters.

Evaluation of stony coral indicators for coral reef management

Colonies of reef-building stony corals at 57 stations around St. Croix, US Virgin Islands were characterized by species, size and percentage of living tissue. Taxonomic, biological and physical indicators of coral condition were derived from these measurements and assessed for their response to gradients of human disturbance-a requirement for indicators used in regulatory assessments under authority of the Clean Water Act. At the most intensely disturbed location, five of eight primary indicators were highly correlated with distance from the source of disturbance: Coral taxa richness, average colony size, the coefficient of variation of colony size, total topographic coral surface area, and live coral surface area. An additional set of exploratory indicators related to rarity, reproductive and spawning mode and taxonomic identity were also screened. The primary indicators demonstrated sufficient precision to detect levels of change that would be applicable in a regional-scale regulatory program.

Greater hemocyte bactericidal activity in oysters (Crassostrea virginica) from a relatively contaminated site in Pensacola Bay, Florida

Bivalve mollusks such as Crassostrea virginica inhabiting polluted estuaries and coastal areas may bioaccumulate high concentrations of contaminants without apparent ill effects. However, changes in putative internal defense activities have been associated with contaminant accumulation in both experimental and long-term field exposures. In an effort to elucidate these relationships, 40 oysters were collected from Bayou Chico (BC) and East Bay (EB) in Pensacola Bay, FL, two estuaries known to differ in the type and magnitude of chemical contaminants present. Oyster tissue concentrations of metals, tri- and dibutyltin (TBT, DBT), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were measured in individual oysters, as were hemocyte counts (HCs), hemocyte bacterial killing indices (KI), serum lysozyme (LYS) and serum protein (PRO) levels. Average HC, KI, LYS and PRO were significantly higher in BC oysters, which also had significantly higher tissue concentrations of total trace metals, butyltins (BTs), PAHs, PCBs, pesticides, and Mn, Cu, Zn and Sn. EB oysters had low organic contaminant levels and no detectable BTs, but significantly higher concentrations of Al, Cr, Fe, Ag, Cd, and Hg. Simple correlation analysis between specific defense measurements and specific chemical analytes showed specific positive relationships that corroborated previous findings in other FL estuaries. Canonical correlation analysis was used to examine relationships between defense measurements and tissue metals using linearly combined sets of variables. Results were also consistent with previous findings-the highest possible canonical correlation was positive: r=0.864, P<0.0019 among canonical variables composed of HC, KI and LYS for defense, and Fe, Cu, Ag, Cd, Sb, Sn, Ni, Pb and Hg for metals.