Michael A. Unger

GC determination of butyltins in natural waters by flame photometric detection of hexyl derivatives with mass spectrometric confirmation

Abstract Butyltin compounds in the 1–100 ng Sn/Liter range are solvent extracted from estuarine waters and derivatized with n-hexyl magnesium bromide. This forms tetraalkyltins that are quantified by gas chromatography with flame photometric detection and confirmed by gas chromatography mass spectrometry. The n-hexyl derivatives of methyltin and butyltin species are easily separated, and quantified relative to an internal standard, tripentyltin chloride.

Enhancement of Perkinsus marinus disease progression in TBT-exposed oysters (Crassostrea virginica)

In vitro exposure of Crassostrea virginica hemocytes to tributyltin (TBT) can inhibit antimicrobial defense mechanisms dependent on reactive oxygen intermediates, as measured by chemiluminescence (CL). However, tissue TBT concentrations attained in this in vivo study (~ 7500 ppb, dry weight) did not inhibit hemocyte recruitment, phagocytosis, CL activity or serum lysozyme. This TBT level was nevertheless effective in enhancing progression and lethality of Perkinsus marinus infections. Immune stimulation was found to be associated with advanced P. marinus infections, as indicated by increased hemocyte recruitment into the circulation and increased CL activity. This played no apparent role in controlling the infection and was insensitive to TBT. Using this battery of standard assays of defense-related functions, the exacerbation of P. marinus infections by TBT could not be attributed to its putative immunosuppresive activity.

Genetic structure of Fundulus heteroclitus from PAH-contaminated and neighboring sites in the Elizabeth and York Rivers

Population genetic characteristics of mummichog, Fundulus heteroclitus, from the heavily industrialized Elizabeth River and nearby York River (Virginia USA) were assessed relative to sediment PAH concentrations. Allozyme genotype frequencies for all loci were consistent with random mating expectations at each locality and age class. Fish from all sites had comparable levels of enzyme polymorphism and heterozygosity regardless of the associated sediment PAH concentrations. Allozyme frequencies for 12 of 15 loci were homogeneous for mummichog from all localities except that allozyme frequencies were significantly different for the Idh-2 locus of (adult and juvenile) mummichog at the heavily-contaminated Atlantic Wood site relative to all other sites. Additionally, allele frequency differences were noted for Ldh-C and Gpi-1 among juvenile mummichog. Values for F(st) were 0.0254 and 0.0141 in the juvenile and adult samples, respectively, indicating greater among-locality genetic differentiation for juvenile mummichog than for adults. Juvenile mummichog are more likely to remain in their natal area while adult samples reflect movement of fish during two or more winter seasons. Correlation analysis suggested that genetic differentiation was not correlated with geographic distance at the spatial scale studied here; however, there was a significant correlation between genetic distance and differences among sites in organic carbon-normalized PAH concentrations. Mummichog collected at the heavily PAH-contaminated AW locality were genetically distinct from those at neighboring sites.

A Probabilistic Ecological Risk Assessment of Tributyltin in Surface Waters of the Chesapeake Bay Watershed

The goal of this study was to conduct a probabilistic ecological risk assessment for tributyltin (TBT) in surface waters of the Chesapeake Bay watershed. Ecological risk was characterized by comparing the probability distributions of environmental exposure concentrations with the probability distributions of species response data determined from laboratory studies. The overlap of these distributions was a measure of risk to aquatic life. Tributyltin exposure data from the Chesapeake Bay watershed were available from over 3600 water column samples from 41 stations in nine basins from 1985 through 1996. Most of the stations were located in the Virginia waters of Chesapeake Bay, primarily the James, Elizabeth and York Rivers. In Maryland waters of the Bay, various marina, harbor and river systems were also sampled. As expected, the highest environmental concentrations of tributyltin (based on 90th percentiles) were reported in and near marina areas. The sources of TBT causing these high concentrations were primarily boat hulls and painting/depainting operations. Lower concentrations of TBT were reported in open water areas, such as the Potomac River, Choptank River and C and D Canal, where the density of boats was minimal. Temporal data from a ten year data base (1986-1996) from two areas in Virginia showed that TBT water column concentrations have declined since 1987 legislation prohibited the use of TBT paints on recreation boats (<25 m). Acute saltwater and freshwater TBT toxicity data were available for 43 and 23 species, respectively. Acute effects for saltwater species were reported for concentrations exceeding 420 ng/L; the lowest acute value for a freshwater species was 1110 ng/L. The acute 10th percentiles for all saltwater and freshwater species were 320 and 103 ng/L, respectively. The order of sensitivity from most to least sensitive for saltwater trophic groups and corresponding acute 10th percentiles were as follows: zooplankton (5 ng/L), phytoplankton (124 ng/L), benthos (312 ng/L) and fish (1009 ng/L). For freshwater species, the order of sensitivity from most to least sensitive trophic groups and corresponding acute 10th percentiles were: benthos (44 ng/L), zooplankton (400 ng/L), and fish (849 ng/L). Chronic data for both saltwater and freshwater species were limited to a few species in each water type. Based on these limited data, the saltwater and freshwater chronic 10th percentiles were 5 and 102 ng/L, respectively. Limited mesocosm and microcosm studies in saltwater suggested that TBT concentrations less than 50 ng/L did not impact the structure and function of biological communities. The saltwater acute (320 ng/L) and chronic (5 ng/L) 10th percentiles were used to determine ecological risk because all exposure data were from saltwater areas of the Chesapeake Bay watershed. Highest ecological risk was reported for marina areas in Maryland waters of Chesapeake Bay and for areas in Virginia such as the Elizabeth River, Hampton Creek and Sarah Creek. Low ecological risk was reported for areas such as the Potomac River, Choptank River, C and D Canal and Norfolk Harbor. Regulation of TBT on recreational watercraft in 1987 has successfully reduced water column concentrations of this organometallic compound. However, various studies have showed that TBT may remain in the sediment for years and continue to be source for water column exposures.

Organotin Concentrations in the Southern Chesapeake Bay

A monitoring program designed to determine the concentrations of tributyltin in waters surrounding marinas in the southern Chesapeake Bay started in January, 1986. In addition, water samples were analyzed from the Elizabeth River to ascertain the tributyltin concentrations in this highly industrialized estuary. The data show that tributyltin concentrations vary considerably over both space and time. Concentrations, an order of magnitude apart, were found at the same location within the same week and levels a factor of two different were found in samples collected from different locations at the same marina, at the same time.

Equilibrium Sorption of Tributyltin Chloride by Chesapeake Bay Sediments

Sorption of tributyltin chloride (TBT) on selected sediments from Chesapeake Bay and its tributaries has been measured to establish ranges for TBT sorption coefficients. Twenty-four hour sorption isotherms were linear and gave sorption coefficients on the order of10^{3}. Twenty-four hour desorption isotherms indicate that the sorption is reversible. To examine the dependence of TBT sorption on salinity, sediment was equilibrated with TBT solutions that spanned estuarine salinity conditions. The resulting sorption coefficients decreased with increasing salinity and varied by a factor of two over the salinity range.

The development and evaluation of monoclonal antibodies for the detection of polycyclic aromatic hydrocarbons

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of 3- to 5-ring polycyclic aromatic hydrocarbons (PAHs) has been developed. A functional derivative of dibenzothiophene was synthesized and covalently linked to carrier proteins that were used to produce monoclonal antibodies (mAbs). During the conjugation step, the conjugation efficiency was improved by the presence of 25% N,N-dimethylformamide (DMF). Antibodies were selected based on a competitive inhibition assay to isolate those with the highest sensitivity for free PAHs. When using the mAb in an ELISA format, free PAHs were detected at a concentration as low as 0.1 microg/L (0.1 ppb) in aqueous samples.

Near real‐time, on‐site, quantitative analysis of PAHs in the aqueous environment using an antibody‐based biosensor

Rapid, on-site, quantitative assessments of dissolved polycyclic aromatic hydrocarbons (PAHs) were demonstrated for two field applications. The platform, a KinExA Inline Sensor (Sapidyne Instruments), employed the monoclonal anti-PAH antibody, 7B2.3, which has specificity for 3- to 5-ring PAHs. A spatial study was conducted near a dredging site where contaminated sediments were being removed, and a temporal study was performed during a rainfall event. Most importantly, the generation of near real-time data guided management decisions in the field and determined proper sampling protocols for conventional analyses. The method was able to determine PAH concentrations as low as 0.3 µg/L, within 10 min of sample acquisition, and to assess 80+ samples (not including standards and blanks) in less than 3 d. These results were compared with a laboratory-based gas chromatography-mass spectrometry method in which a wide array of PAHs, including alkylated homologs, were examined. This system shows great promise as a field instrument for the rapid monitoring of PAH pollution.

Multistressor interactions in the zebrafish (Danio rerio): Concurrent phenanthrene exposure and Mycobacterium marinum infection.

The simultaneous exposure of organisms to toxicants and disease causing agents poses a serious risk to important stocks. Worldwide, aquatic animal disease outbreaks have been increasing in both frequency and severity, and many have been associated with anthropogenic environmental change. Little is known about the complex interactions of the immune system and biotransformational pathways of vertebrates; however, urbanization and coastal development create a scenario in which a wide range of species are exposed to chemical pollutants in conjunction with a wide spectrum of ubiquitous, opportunistic pathogens. These interactions can severely compromise organismal health. Potential effects include decreased fitness, increased predation, decreased fecundity, reduced metabolic activity, suppressed immune function and mortality. Recent attention has been paid to immunomodulation in toxicant exposed fishes. In our current study we investigated the effects of the common polycyclic aromatic hydrocarbon phenanthrene in conjunction with Mycobacterium marinum infection in the zebrafish, Danio rerio. The goal of our study was to elucidate the interactions between stressors in the host organism. Fish were exposed to either a high or low dose of phenanthrene, infected with M. marinum or received a combination exposure of toxicant and bacteria. Results of our study were evaluated using survivorship analysis, toxicant body burden, and histology. Our data show an interaction between M. marinum infection and exposure to a high dose of phenanthrene in the zebrafish. Survivorship was significantly reduced for animals only exposed to the high dose of phenanthrene as compared to all other experimental groups. The increased survivorship for fish exposed to both Mycobacterium and a high dose of phenanthrene suggests an antagonistic interaction between stressors. Body burden data, which show significant differences in the ratio of phenanthrene:metabolites between experimental groups, suggests a disruption of the biotransformational pathway. We postulate that the inflammatory response, initiated by bacterial infection, is impeding the ability of the zebrafish to completely metabolize phenanthrene. In addition, the correlation between reduced metabolite production and increased survival indicates that phenanthrene metabolites are more toxic than the parent compound. Our study underscores the importance of investigating multiple stressor interactions as a way to better understand complex environmental interactions.

Runoff from tomato cultivation in the estuarine environment: biological effects of farm management practices

The use of plastic row covers (plastic mulch) on vegetable farms increases runoff of pesticides after rainfall events and has been linked to toxic events in adjacent tidal waters. In coastal Virginia, USA, runoff from tomato fields with plastic mulch was suspected of causing mortality of commercial hard clam larvae at a hatchery located downstream of farming operations. Concern about the putative impacts of this practice on local waters resulted in a collection of studies to: (1) determine the sensitivity of early life stages of bivalves to copper, a commonly used fungicide; (2) examine acute and chronic biological effects of runoff on tidal creeks; and (3) examine the efficacy of management practices designed to reduce the delivery of pesticides to adjacent creeks. Laboratory bioassays revealed that 48-h LC50 values for embryonic clams Mulinia lateralis and Mercenaria mercenaria were 38 and 20 μg/l, respectively. In situ bioassays with Palaemonetes pugio showed that pulsed toxic conditions sometimes occur downstream of some tomato farms in plastic mulch following rainfall events. Growth, mortality rates and bioaccumulation of copper and organic pesticides in oysters were not correlated with the use of plastic mulch in watersheds. Sediment bioassays indicated potential toxicity in sediment collected downstream of some tomato fields in plastic mulch, but the effects were not consistent between years. Closer examination of management practices on the farms suggests that controlling runoff can prevent toxic impacts. Elevated levels of crop protectants measured at the outflow of farm ponds suggested that they may do little to reduce loadings of some pesticides. However, forested buffer zones and ephemeral sedimentation basins appeared to be effective in reducing pesticide concentrations in runoff and pulsed toxicity in tidal creeks.