Snapshot of Vibrio parahaemolyticus densities in open and closed shellfish beds in Coastal South Carolina and Mississippi.

Vibrio parahaemolyticus is a Gram negative, halophilic bacterium that is ubiquitous in warm, tropical waters throughout the world. It is a major cause of seafood-associated gastroenteritis and is generally associated with consumption of raw or undercooked seafood, especially oysters. This study presents a snapshot of total V. parahaemolyticus densities in surface waters and shellstock American oysters (Crassostrea virginica) from open and closed shellfish harvesting areas, as well as "more rural areas" on two different US coasts, the Atlantic and the Gulf. Sampling was conducted from 2001 to 2003 at five sites near Charleston/Georgetown, SC and at four locations in the Gulfport/Pascagoula, MS area. V. parahaemolyticus numbers were determined by a direct plating method using an alkaline-phosphatase-labeled DNA probe targeting the species-specific thermolabile hemolysin gene (tlh) that was used for identification of bacterial isolates. The greatest difference between the two coasts was salinity; mean salinity in SC surface waters was 32.9 ppt, whereas the mean salinity in MS waters was 19.2 ppt, indicating more freshwater input into MS shellfish harvesting areas during the study period. The mean V. parahaemolyticus numbers in oysters were almost identical between the two states (567.4 vs. 560.1 CFU/g). Bacterial numbers in the majority of surface water samples from both states were at or below the limit of detection (LOD = <10 CFU/mL). The bacterial concentrations determined during this study predict a low public health risk from consumption of oysters in shellfish growing areas on either the Gulf or the Atlantic US coast.

Isolation of faecal coliform bacteria from the American alligator (Alligator mississippiensis).

AIMS: To determine whether American alligators (Alligator mississippiensis) are an unrecognized poikilothermic source of faecal coliform and/or potential pathogenic bacteria in South Carolina's coastal waters. METHODS AND RESULTS: Bacteria from the cloaca of American alligators, as well as bacteria from surface water samples from their aquatic habitat, were isolated and identified. The predominant enteric bacteria identified from alligator samples using biochemical tests included Aeromonas hydrophila, Citrobacter braakii, Edwardsiella tarda, Escherichia coli, Enterobacter cloacae, Plesiomonas shigelloides and putative Salmonella, and these were similar to bacteria isolated from the surface waters in which the alligators inhabited. Based on most-probable-number enumeration estimates from captive alligator faeces, faecal coliform bacteria numbered 8.0x10(9) g(-1) (wet weight) of alligator faecal material, a much higher concentration than many other documented endothermic animal sources. CONCLUSIONS: A prevalence of enteric bacteria, both faecal coliforms and potential pathogens, was observed in American alligators. The high faecal coliform bacterial density of alligator faeces may suggest that alligators are a potential source of bacterial contamination in South Carolina coastal waters. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings help to increase our understanding of faecal coliform and potential pathogenic bacteria from poikilothermic reptilian sources, as there is the potential for these sources to raise bacterial water quality levels above regulatory thresholds.

F+ RNA coliphage typing for microbial source tracking in surface waters.

AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.

An evaluation of polycyclic aromatic hydrocarbon (PAH) runoff from highways into estuarine wetlands of South Carolina.

This study investigated the concentrations and potential toxicity of polycyclic aromatic hydrocarbons (PAHs) associated with highway runoff into adjacent estuarine wetlands from road segments representing three levels of average daily traffic (ADT): low (<5,000 ADT), moderate (10,000-15,000 ADT), and high use (>25,000 ADT) based on SC Department of Transportation data. Sediments from three estuarine wetland habitats (tidal creeks, Spartina marsh, and mud flats) adjacent to these road segments were sampled to represent nine highway use class/habitat type combinations. Surficial sediments were collected at 3, 25, and 50 meters from the upland/wetland interface along transects established perpendicular to the road at each site, with additional samples taken from the road berm. Average PAH concentrations, representing 25 compounds, ranged from 3.9 to 11,000 ng/g dry weight. Berm samples had significantly greater total PAH concentrations than samples taken in any of the wetland habitats. Average total PAH concentrations decreased with increasing distance from the road berm within the wetland habitats sampled, but the differences were not statistically significant. Average total PAH concentrations also were not significantly different among the wetland habitats compared. Analysis of PAH profiles indicated that the PAH source was dominated by pyrogenic combustion products rather than from petrogenic sources. This, combined with the presence of dibenzothio-phene, which is a tire oxidation product, indicated that the primary source of PAHs was related to vehicles. Two sites with total PAH concentrations exceeding published bioeffects levels were resampled for bioassay tests using the amphipod Ampelisca verrilli, the polychaete Streblospio benedicti, and the clam, Mercenaria mercenaria, with the first two assays conducted under UV lighting since previous studies had demonstrated enhanced UV toxicity of PAHs for these species. No toxicity was observed in the amphipod or polychaete assays. Toxicity was observed in the juvenile clam assay at one site, possibly due to the combined effects of PAHs and other contaminants present.

Toxicological studies in tropical ecosystems: an ecotoxicological risk assessment of pesticide runoff in South Florida estuarine ecosystems.

A multiyear study in the C-111 canal system and associated sites in Florida Bay was undertaken to determine the potential pesticide risk that exists in South Florida. After the examination of extensive pesticide concentration data in surface water, tissues, and semipermeable membrane devices (SPMDs), canal contamination seems to be derived from the extensive agricultural production that drains into the C-111 canal. The results of this study indicate that runoff from agricultural processes led to quantifiable pesticide residues in both canal and bay surface water, which occasionally exceeded current water quality criteria. The major pesticide of concern was endosulfan, which was detected at 100% of the sites sampled. Endosulfan exposure did not cause any acute effects in fish and crustaceans deployed in field bioassays. Chronic effects were observed in copepods, clams, and oysters but could not be attributed to endosulfan exposure. The decision to alter the C-111 canal flow and allow increased freshwater flow into the adjacent Everglades National Park may result in discharges of pesticides into the Everglades. Continued monitoring in this area is needed during this change in flow regime.

Reproductive alterations in adult grass shrimp, Palaemonetes pugio, following sublethal, chronic endosulfan exposure.

Grass shrimp (Palaemonetes pugio) populations exposed to anthropogenic contaminant sources in South Carolina (SC) have reduced densities when compared with populations at SC-reference sites. This laboratory study examined the effects of a commonly used agricultural insecticide, endosulfan, on grass shrimp reproduction. Reproductively active grass shrimp were chronically exposed to sublethal concentrations of endosulfan (200 or 400 ng/l) for 43 days. The cumulative number of females that became gravid and the rate at which they became gravid were measured. Endosulfan exposure reduced the cumulative number of gravid females by 31% in the 200 ng/l exposure and 39% in the 400 ng/l exposure. The first appearance of gravid females in the population was significantly delayed in treated populations compared with the control treatment in a dose dependent manner. Clutch size in these gravid females was not significantly different among the treatments. Additionally, there was no difference in the onset of reproduction in the treated populations. These results implicate a population reduction due to a decrease in the overall number of females becoming gravid in a population over time, not a reduction in clutch size per individual. While the mechanisms of action have yet to be defined, these results indicate that sublethal endosulfan concentrations may have a negative effect on grass shrimp reproductive biology.

Ginsenosides Rb1 and Re decrease cardiac contraction in adult rat ventricular myocytes: role of nitric oxide.

1. Panax ginseng is used to enhance stamina and relieve fatigue as well as physical stress. Ginsenoside, the effective component of ginseng, regulates cardiovascular function. This study was to examine the effect of ginsenosides Rb1 and Re on cardiac contractile function at the cellular level. Ventricular myocytes were isolated from adult rat hearts and were stimulated to contract at 0.5 Hz. Contractile properties analysed included: peak shortening (PS), time-to-90%PS (TPS), time-to-90% relengthening (TR90), and fluorescence intensity change (DeltaFFI). Nitric oxide synthase (NOS) activity was determined by the 3H-arginine to 3H-citrulline conversion assay. 2. Both Rb1 and Re exhibited dose-dependent (1-1000 nM) inhibition in PS and DeltaFFI, with maximal inhibitions between 20-25%. Concurrent application Rb1 and Re did not produce any additive inhibition on peak shortening amplitude (with a maximal inhibition of 24.9+/-6.1%), compared to Rb1 or Re alone. Pretreatment with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished the effect of Rb1 and Re. Both Rb1 and Re significantly (P<0.05) stimulated NOS activity concentration-dependently. 3. This study demonstrated a direct depressant action of ginsenosides on cardiomyocyte contraction, which may be mediated in part through increased NO production.

Toxicity of atrazine to the estuarine phytoplankter Pavlova sp. (Prymnesiophyceae): increased sensitivity after long-term, low-level population exposure.

Phytoplankton are potentially more at risk to the adverse effects of herbicides than many other organisms in estuarine ecosystems. The focus of this study was to characterize the toxicity of a widely used herbicide, atrazine, to a single species of phytoplankton. The nanoplankter Pavlova sp. was grown under controlled laboratory conditions and was used to elucidate short-term, high-level population effects of atrazine. Secondly, a long-term, multigenerational population exposure was performed with a low level of atrazine followed by an additional short-term, high-level exposure on the same population with no recovery period. The 96-h growth rate of 50% effective concentration (EC50) for Pavlova sp. was 147 [microg/L (95% CI = 116.4-178.7 microg/L). Long-term exposure at 20 [microg/L for four (batch culture) growth cycles (approximately 20 generations) had no significant effect on the growth rate of Pavlova sp. except during the fourth growth cycle. However, a subsequent short-term, high-level 96-h exposure inoculated from the long-term, low-level exposed population showed increased sensitivity to atrazine (96-h growth rate EC50 = 96.0 microg/L, 95% CI = 90.2-103.7 microg/L). Multigenerational exposure to atrazine appeared to render phytoplankton significantly more sensitive (35%) to atrazine effects. Given the documented persistence of atrazine in surface waters, long-term exposure to low levels of atrazine without recovery may lead to shifts in species sensitivity and potential alterations in phytoplankton population dynamics.

Uptake and distribution of three PCB congeners and endosulfan by developing white leghorn chicken embryos (Gallus domesticus).

The distributions of PCB 105, 156, 189, and endosulfan in incubating, maternally exposed, viable white leghorn chicken eggs (Gallus domesticus) were investigated. Hens were subcutaneously injected every 4 days with a mixture of the above chemicals. One group of five eggs was removed from the incubator at each of 9, 14, and 19 days of incubation; dissected into three compartments (embryo, chorioallantoic membrane, and yolk + albumin); weighed; frozen; and then later analyzed for the dosing chemicals. Through 19 days of development (90% of incubation), greater than 70% of the total chemical mass in the whole egg remained within the yolk + albumin, whereas, depending on the chemical, 17% to 30% was absorbed by the embryo and 0.2% to 9% was transported into the chorioallantoic membrane. As a percentage of total PCB mass within the respective compartment, PCB 105 composition in the embryo and chorioallantoic membrane decreased significantly throughout development while PCB 156 and 189 composition increased significantly throughout development. Though endosulfan composition within any of the compartments was highly variable, it did not change significantly during development. The results of this study indicate that the majority of avian chick exposure to contaminants occurs posthatch as the chick continues to utilize the residual yolk.

Characterization of contractile function in diabetic hypertensive cardiomyopathy in adult rat ventricular myocytes.

Diabetes and hypertension both produce myocardial dysfunction that accelerates cardiovascular morbidity and mortality. Coexistence of the two often results in a more severe cardiomyopathy than either process alone. The purpose of this study was to characterize the contractile function of diabetic hypertensive cardiomyopathy at the single myocyte level. Adult spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were made diabetic with a single injection (55 mg/kg) of streptozotocin (STZ). Contractile properties of ventricular myocytes were evaluated, including peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR90) and maximal velocities of shortening/relengthening (+/-dL/d t). The experimental animals exhibited enlarged heart size, elevated blood glucose and systolic blood pressure. PS was unchanged (SHR), enhanced (WKY-STZ) or depressed (SHR-STZ) compared to control (WKY). Myocytes from all experimental groups displayed prolonged TPS and TR90 compared to the WKY group, although only those from the hypertensive groups (SHR, SHR-STZ) were associated with reduced +/-dL/d t. Additionally, myocytes from the WKY-STZ but not the SHR or the SHR-STZ groups exhibited impaired responsiveness to increased extracellular Ca2+. Myocytes from the SHR-STZ group displayed a leftward shift of the stimulus frequency-peak shortening response curve compared to the WKY group. These results confirmed observations at the multicellular levels that combination of diabetes and hypertension results in a greater impairment of cardiac contractile function than is seen with either disease alone.

Influence of ovariectomy on ventricular myocyte contraction in simulated diabetes.

We studied the effect of ovariectomy (OVX) on cardiac contraction in myocytes maintained under a 'diabetes-simulated high-glucose' environment. Female rats were ovariectomized or sham operated (SHAM) and kept for 6 weeks. Isolated myocytes were maintained in a diabetes-simulated high [glucose] medium (HG; 25.5 mM) for 24 h before mechanical properties were measured. Contractile indices analyzed included peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca2+ fura-2 fluorescence intensity and decay rate (tau). Nitric oxide synthase (NOS) activity was also evaluated. OVX myocytes displayed a longer TR(90), slower +/- dL/dt, lower fluorescence intensity and higher tau (slower decay rate) when compared to SHAM myocytes. In the SHAM group, HG exerted diabetes-like contractile dysfunctions, including depressed PS, prolonged TR90, reduced fluorescence intensity, higher tau and enhanced NOS activity when compared to myocytes maintained in low [glucose] medium (5.5 mM). Interestingly, the HG- induced mechanical alterations were significantly exaggerated (TPS, TR90 and tau), reversed (PS and NOS) or lost (+/- dL/dt and fluorescence intensity) in the OVX group. These data suggest that ovarian hormones play a role in the regulation of cardiac contractile function, and may have potentially protective effects against diabetes-associated cardiac dysfunction.

Maternal transfer of contaminants: case study of the excretion of three polychlorinated biphenyl congeners and technical-grade endosulfan into eggs by white Leghorn chickens (Gallus domesticus).

Reported avian maternal transfer rates of organochlorine contaminants range from 1% to as much as 20% of maternal body burdens. However, to our knowledge, no investigation of factors governing maternal transfer has been reported. Here, we report an investigation of maternal transfer of 2,3,3',4,4'-pentachlorinated biphenyl (PCB 105), 2,3,3',4,4',5-hexachlorinated biphenyl (PCB 156), 2,3,3',4,4',5,5'-heptachlorinated biphenyl (PCB 189), and technical-grade endosulfan into eggs by white leghorn chickens (Gallus domesticus). Two experiments were performed to evaluate individual chemical excretion into eggs when hens were injected with each chemical individually (experiment one) or with a mixture of all four chemicals (experiment two). Each hen was injected subcutaneously every 4 d during a 21-d period with 100 microliters of the dosing solution during both experiments. The mass of each chemical excreted into the egg was compared among eggs and with the mass injected into hens to determine the influence of chemical structure (experiment one) and interaction (experiment two) on maternal transfer of those chemicals into eggs. Maternal transfer of PCBs was inversely related to congener chlorination. The congener mass in eggs, as a percentage of the mass injected into hens, was 0.42% for PCB 189, 0.54% for PCB 156, and 0.61% for PCB 105. In experiment two, absolute excretion of only PCB 189 and alpha-endosulfan into eggs was affected by the presence of other chemicals. Excretion of PCB 189 (0.51%) and alpha-endosulfan (0.03%) increased and decreased, respectively, compared with when they were individually injected into hens during experiment one. Lastly, much less of the more metabolically susceptible endosulfan (0.04-0.12% of the mass injected) was excreted into the egg relative to PCBs, despite being injected into the hens at concentrations comparable with those of PCBs, suggesting, at least in avian species, lower maternal transfer of more metabolically susceptible chemicals.

Toxicity of pesticides to aquatic microorganisms: a review.

Microorganisms contribute significantly to primary production, nutrient cycling, and decomposition in estuarine eco-systems; therefore, detrimental effects of pesticides on microbial species may have subsequent impacts on higher trophic levels. Pesticides may affect estuarine microorganisms via spills, runoff, and drift. Both the structure and the function of microbial communities may be impaired by pesticide toxicity. Pesticides may also be metabolized or bioaccumulated by microorganisms. Mechanisms of toxicity vary, depending on the type of pesticide and the microbial species exposed. Herbicides are generally most toxic to phototrophic microorganisms, exhibiting toxicity by disrupting photosynthesis. Atrazine is the most widely used and most extensively studied herbicide. Toxic effects of organophosphate and organochlorine insecticides on microbial species have also been demonstrated, although their mechanisms of toxicity in such nontarget species remain unclear. There is a great deal of variability in the toxicity of even a single pesticide among microbial species. When attempting to predict the toxicity of pesticides in estuarine ecosystems, effects of pesticide mixtures and interactions with nutrients should be considered. The toxicity of pesticides to aquatic microorganisms, especially bacteria and protozoa, is an area of research requiring further study.

Analysis of pesticide runoff from mid-Texas estuaries and risk assessment implications for marine phytoplankton.

During 1993, estuarine surface water samples were collected from the mid-Texas coast (Corpus Christi to Port Lavaca, TX). Agricultural watershed areas as well as tidal creeks immediately downstream were chosen as sampling sites along with adjoining bay sampling stations. Collections were made throughout the growing season (February to October 1993) before and after periods of significant (> 1.25 cm) rainfall. All samples were initially screened for the presence of pesticides using enzyme-linked immunosorbent assay (ELISA) test kits (EnviroGard) for triazine herbicides and carbamate insecticides. All samples were extracted and then analyzed using gas chromatography (GC) for quantification of atrazine. Only samples testing positive for carbamate insecticides via ELISA were further extracted for GC analysis to quantify aldicarb and carbofuran. Additionally, laboratory toxicity tests using phytoplankton were examined from published, peer-reviewed literature and compared with the atrazine field levels found in Texas. Results of ELISA screening indicated the presence of triazine herbicides in nearly all samples (>93%). GC analysis further confirmed the presence of atrazine concentrations ranging from <0.01-62.5 microg/L. Screening tests also found detectable levels of carbamate insecticides (aldicarb and carbofuran) that were also confirmed and quantified by GC. Comparison of measured concentrations of atrazine compared with published toxicity tests results indicated that there was a potential environmental risk for marine/estuarine phytoplankton in surface waters of Texas estuaries, particularly when the chronic nature of atrazine exposure is considered.

Determination of acute mortality in adults and sublethal embryo responses of Palaemonetes pugio to endosulfan and methoprene exposure.

Adult grass shrimp (Palaemonetes pugio) were exposed to endosulfan or methoprene for 96 h and LC(50) values were calculated. Male and female P. pugio cohorts were also exposed to endosulfan for 96 h in an attempt to determine potential differences in sensitivity between the sexes. Results from the methoprene exposure indicated that this pesticide was not acutely toxic to adult grass shrimp at 1 mg l(-1). Due to the lack of sensitivity, sex specific tests with methoprene were not performed. The calculated LC(50) for a population of grass shrimp, including both males and females exposed to endosulfan, was 0.62 microg l(-1). The LC(50) determinations for the sex specific tests were 0.92 microg l(-1) for males and 1.99 microg l(-1) for females. Following these acute exposures, reproductively active grass shrimp were chronically exposed to 200 ng l(-1) endosulfan or 1 mg l(-1) methoprene and were allowed to produce embryos. The resulting embryos were assessed for potential sublethal toxicity. There were no observed differences in the percent successfully hatching or larval mortality 3-days post hatch among the treatments. However, endosulfan treated embryos had a significantly increased hatching time (9.76 days compared to 8.72 days in controls). Methoprene treated embryos also took longer to hatch (9.55 days), but this delay was not significantly different from controls. These findings suggest that endosulfan may preferentially affect male grass shrimp and exposed female grass shrimp may produce embryos with delayed hatching times.

Use of Metabolic Inhibitors to Characterize Ecological Interactions in an Estuarine Microbial Food Web.

Understanding microbial food web dynamics is complicated by the multitude of competitive or interdependent trophic interactions involved in material and energy flow. Metabolic inhibitors can be used to gain information on the relative importance of trophic pathways by uncoupling selected microbial components and examining the net effect on ecosystem structure and function. A eukaryotic growth inhibitor (cycloheximide), a prokaryotic growth inhibitor (antibiotic mixture), and an inhibitor of photosynthesis (DCMU) were used to examine the trophodynamics of microbial communities from the tidal creek in North Inlet, a salt marsh estuary near Georgetown, South Carolina. Natural microbial communities were collected in the spring, summer, and fall after colonization onto polyurethane foam substrates deployed in the tidal creek. Bacterial abundance and productivity, heterotrophic ciliate and flagellate abundance, and phototrophic productivity, biomass, and biovolume were measured at five time points after inhibitor additions. The trophic responses of the estuarine microbial food web to metabolic inhibitors varied with season. In the summer, a close interdependency among phototrophs, bacteria, and protozoa was indicated, and the important influence of microzooplanktonic nutrient recycling was evident (i.e., a positive feedback loop). In the fall, phototroph and bacteria interactions were competitive rather than interdependent, and grazer nutrient regeneration did not appear to be an important regulatory factor for bacterial or phototrophic activities. The results indicate a seasonal shift in microbial food web structure and function in North Inlet, from a summer community characterized by microbial loop dynamics to a more linear trophic system in the fall. This study stresses the important role of microbial loops in driving primary and secondary production in estuaries such as North Inlet that are tidally dominated by fluctuations in nutrient supply and a summer phytoplankton bloom.

Polycyclic aromatic hydrocarbons in storm runoff from urban and coastal South Carolina.

Stormwater runoff was collected in urbanized areas of South Carolina to investigate the levels and sources of polycyclic aromatic hydrocarbons (PAHs). Mean concentrations of total PAHs in runoff (sum(PAHs), 14 compounds), determined by gas chromatography-mass spectrometry, were 5590 ng/l in the city of Columbia and 282 ng/l in the coastal community of Murrells Inlet. Lower concentrations were found in estuarine water at Murrells Inlet (mean = 35 ng/l) and at undeveloped North Inlet estuary (13 ng/l). The PAH profiles in Columbia and Murrells Inlet runoff were similar to those of atmospheric particulate matter and unlike those in used crankcase oil. Examination of the aliphatic fraction of Columbia runoff samples by gas chromatography with flame ionization detection showed patterns that were more similar to used crankcase oil than to urban aerosols.