Interactive effects of Louisiana Sweet Crude (LSC) thin oil sheens and ultraviolet light on mortality and swimming behavior of the larval Eastern oyster, Crassostrea virginica.

The Eastern oyster (Crassostrea virginica) is an important commercial bivalve species which also has numerous ecological roles including biogeochemical cycling, providing habitat for larval fish and crustaceans, and reducing the impacts of coastal storms. Oil may pose a threat to oyster larvae swimming in the water column, leading to potential negative effects on survival, growth, and development. Oil toxicity may be further enhanced by chemical changes in the presence of sunlight. This study determined the toxicity of thin oil sheens with and without ultraviolet (UV) light, then examined the latent effects of the short term exposure on longer term survival and swimming ability. Larval C. virginica were exposed to four different oil sheen thicknesses for 24 h with either no UV light or 2-h UV light. Following the exposure, larvae were transferred to clean seawater and no UV light for 96 h. The presence of a 2-h UV light exposure significantly increased oyster mortality, indicating photo-enhanced toxicity. The LC50 for a 24-h oil sheen exposure without UV was 7.26 µm (23 µg/L PAH50) while a 2 h-UV exposure lowered the sheen toxicity threshold to 2.67 µm (10 µg/L PAH50). A previous 24-h oil sheen exposure (≥0.5 µm) led to latent effects on larval oyster survival, regardless of previous UV exposure. Sublethal impacts to larval oyster swimming behavior were also observed from the previous oil sheen exposure combined with UV exposure. This study provides new data for the toxicity of thin oil sheens to a sensitive early life stage of estuarine bivalve.

Comparative Toxicity of Two Chemical Dispersants and Dispersed Oil in Estuarine Organisms.

Chemical dispersants can be a useful tool to mitigate oil spills. This study examined potential risks to sensitive estuarine species by comparing the toxicity of two dispersants (Corexit® EC9500A and Finasol® OSR 52) individually and in chemically enhanced water-accommodated fractions (CEWAFs) of Louisiana Sweet Crude oil. Acute toxicity thresholds and sublethal biomarker responses were determined in seven species (sheepshead minnow, grass shrimp, mysid, amphipod, polychaete, hard clam, mud snail). Comparing median lethal (LC50) values for the dispersants, Finasol was generally more toxic than Corexit and had greater sublethal toxicity (impaired embryonic hatching, increased lipid peroxidation, decreased acetylcholinesterase activity). The nominal concentration-based mean LC50 for all species tested with Corexit was 150.31 mg/L compared with 43.27 mg/L with Finasol. Comparing the toxicity of the CEWAFs using the nominal concentrations (% CEWAF), Corexit-CEWAFs appeared more toxic than Finasol-CEWAFs; however, when LC50 values were calculated using measured hydrocarbon concentrations, the Finasol-CEWAFs were more toxic. There was greater dispersion efficiency leading to greater hydrocarbon concentrations measured in the Corexit-CEWAF solutions than in equivalent Finasol-CEWAF solutions. The measured concentration-based mean LC50 values for all species tested with Corexit-CEWAF were 261.96 mg/L total extractable hydrocarbons (TEH) and 2.95 mg/L total polycyclic aromatic hydrocarbons (PAH), whereas the mean LC50 values for all species tested with Finasol-CEWAF were 23.19 mg/L TEH and 0.49 mg/L total PAH. Larval life stages were generally more sensitive to dispersants and dispersed oil than adult life stages within a species. These results will help to inform management decisions regarding the use of oil-spill dispersants.

Distribution and sources of PCBs (Aroclor 1268) in the Sapelo Island National estuarine research reserve.

Aroclor 1268 is a highly chlorinated PCB mixture that was released into the aquatic environment near Brunswick, GA (BR), as a result of decades of local industrial activity. This extensive contamination has led to US EPA Superfund designation in estuarine areas in and around Purvis Creek, GA. Roughly 50 km to the northeast is the Sapelo Island National Estuarine Research Reserve (SI) where previous studies have documented unexpectedly high Aroclor 1268-like PCB levels in blubber and plasma samples of resident bottlenose dolphins. This result led to a collaborative effort to assess the PCB patterns and concentrations in SI sediment and fish (as potential vectors for PCB transfer to SI resident dolphins). Thirty SI randomly assigned stations were sampled for sediment PCB levels. Additionally, fish were collected and analyzed from SI (n = 31) and BR (n = 33). Results were pooled with regional assessments of PCB concentrations from South Carolina and North Carolina in an effort to determine the association of Aroclor 1268 levels in SI samples. Results indicated that PCB levels in sediment and fish are much lower in the SI estuary compared to BR sediment and fish concentrations. However, PCB congener profiles for both sediments and fish were similar between the two locations and consistent with the Aroclor 1268 signature, indicating possible transport from the Brunswick area. A likely source of Aroclor 1268 in dolphins from SI is contaminated fish prey.

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.

Relationship between land use classification and grass shrimp Palaemonetes spp. population metrics in coastal watersheds.

Estuaries in the southeastern USA have experienced increased loading of contaminants from nonpoint source runoff as well as changes in habitat (e.g., loss of wetlands) due to urbanization. These changes may pose significant risks to estuarine fauna, including crustaceans. Several studies have shown relationships between land use classification and levels of stress in estuarine populations. The grass shrimp of the genus Palaemonetes is one of the dominant species found in estuarine tidal creeks, accounting for more than 50 % of all macropelagic fauna. Grass shrimp populations were sampled monthly for 3 years at six estuarine creeks on Kiawah Island, SC. Creek watersheds were estimated using National Aerial Photograph Program color infrared and low-altitude true color aerial photography combined with in situ differentially corrected global positioning system mapping of engineered features. Land classifications delineated included water, marsh, buildings, roads, and lawns. Pairwise comparisons for grass shrimp densities among sites showed significant differences on an annual and seasonal basis. Significant relationships (p < 0.05) between land class variables and grass shrimp density were identified both annually and seasonally. These findings suggest an influence of land use on Palaemonetes spp. populations.

Daily or thrice weekly handling of eland antelope (Taurotragus oryx): effects on serum cortisol level.

We evaluated the cortisol response of adult female eland (n=8) that were handled in hydraulic chute daily or 3×/week. Females were divided into two groups and each group (n=4) successively received two estrous cycle synchronization treatments: (1) two injections of prostaglandin (PG-PG) F2α at 11 day intervals and (2) oral administration of altrenogest for 7 days and an injection of PGF2α on day 7 (Alt-PG). Blood samples were collected 3×/week during the synchronization (Synch) and expected luteal phase (Nonintensive) periods, and daily during the expected time of induced (Intensive 1) or natural (Intensive 2) estrus. Overall, mean cortisol levels were highest during Intensive 1, followed by Intensive 2, Synch and Nonintensive periods. Individual eland were the most significant source of variation for cortisol level. The frequency of handling and the synchronization treatment significantly affected cortisol levels in 3/8 and 4/8 females, respectively. In conclusion, in response to increased frequency of handling, eland cortisol levels rose transiently and returned to baseline within few days after more intensive handling. Thus, the eland females were tolerant to and recovered from the effects of repeated daily handling.

Effects of chemically spiked sediments on estuarine benthic communities: a controlled mesocosm study.

Ambient sediments were collected from a reference site in the North Edisto River, SC and transferred to a laboratory facility to investigate effects of chemical contaminants on estuarine infaunal communities under controlled mesocosm conditions. Sediment contaminant slurries were prepared using dried sediments collected from the reference site and spiked with a metal (copper), a polycyclic aromatic hydrocarbon (pyrene), and a pesticide (4,4(')-dichlorodiphenyltrichloroethane) to yield nominal mean effects range-median (ERM) quotients of <0.01 (no addition), 0.1, and 1.0 and applied to control, low dose (TRT A), and high dose (TRT B) treatment groups, respectively. Sediment samples for contaminant and benthic analyses were collected at the start of the experiment, 1 month after dosing, and 3 months after dosing. Near-nominal mean ERM quotients of 0.001, 0.075, and 0.818 were measured initially after dosing and remained fairly constant throughout the experiment. Measures of benthic condition, diversity, and richness were significantly reduced in both treatment groups relative to the control 1 month after dosing and persisted in TRT B at 3 months. The results demonstrate that benthic community effects can be observed at mean ERM quotients that are about an order of magnitude lower than levels that have been shown to be associated with significant toxicity in acute laboratory bioassays with single species (e.g., amphipods) in other studies.

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1.

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.

Effects of fluoxetine exposure on serotonin-related activity in the sheepshead minnow (Cyprinodon variegatus) using LC/MS/MS detection and quantitation.

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor and is among the top 100 drugs prescribed yearly in the United States and the United Kingdom. Tissue and water extraction methods were developed to detect and quantify FLX, norfluoxetine and the associated biological compounds serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophan (TRP) and melatonin (MEL) using LC/MS/MS. Acute mortality and sublethal physiological effects of FLX were assessed using standard static renewal toxicity tests in which juvenile sheepshead minnows (Cyprinodon variegatus) were exposed to FLX. Fluoxetine did not cause significant mortality at levels near currently reported environmental concentrations. Significant changes in neurotransmitter levels were observed within the serotonergic system in juvenile sheepshead minnows exposed at concentrations approximately one order of magnitude above those currently reported in the environment. Transformation activity ratios of a product to a precursor compound (5-HT/TRP, 5-HIAA/5-HT and MEL/5-HT) also exhibited significant changes with FLX treatment. Fluoxetine exposure did not only affect 5-HT but had additional effects both upstream and downstream of 5-HT within its synthesis and metabolic pathways. These sublethal changes within the serotonergic pathway may result in behavioral changes which could, in turn, have implications for the ecological response of populations to additional environmental stressors.

Effects of the antifouling compound, Irgarol 1051, on a simulated estuarine salt marsh ecosystem.

Toxicity effects of the antifouling compound, Irgarol 1051, were examined using a simulated estuarine salt marsh ecosystem. The 35 day mesocosm exposure incorporated tidal flux and contained seawater, sediments, marsh grass, and estuarine biota. Irgarol (10.0 microg/l) caused a significant reduction in phytoplankton biomass and primary productivity. HPLC pigment analysis indicated significant effects of irgarol on both phytoplankton and periphyton community composition, with decreased concentrations of pigments representative of diatom species. There was also a significant decrease in dissolved oxygen levels in the 10.0 microg/l irgarol treatment. Growth of the hard shell clam was significantly reduced in the 1.0 and 10.0 microg/l irgarol treatments. The effects observed occurred at irgarol concentrations greater than those typically measured in the environment. Prolonged exposure, the accumulation of irgarol in sediments, plant, or animal tissues, and the interaction of irgarol with other chemicals in the environment; however, could increase risk.

Coalescence of viscous liquid drops.

We report on studies of the early stage of coalescence of two liquid drops. The drops were high viscosity silicon oil immersed in a water-alcohol mixture of the same density in order to eliminate the effects of gravity. The viscosity was sufficiently large that measurements could be made under the conditions of Stokes flow. Measurements were made of the radius of the neck between the drops as a function of the time from the onset of coalescence, and the results compared with theoretical predictions.

The effects of the contemporary-use insecticide (fipronil) in an estuarine mesocosm.

To examine the effects of environmentally realistic fipronil concentrations on estuarine ecosystems, replicated mesocosms containing intact marsh plots and seawater were exposed to three treatments of fipronil (150, 355, and 5000 ng/L) and a Control. Juvenile fish (Cyprinidon variegatus), juvenile clams (Mercenaria mercenaria), oysters (Crassostrea virginica), and grass shrimp (Palaemonetes pugio) were added prior to fipronil in an effort to quantify survival, growth, and the persistence of toxicity during the planned 28-day exposure. Results indicated that there were no fipronil-associated effects on the clams, oysters, or fish. Shrimp were sensitive to the highest two concentrations (40% survival at 355 ng/L and 0% survival at 5000 ng/L). Additionally, the highest fipronil treatment (5000 ng/L) was toxic to shrimp for 6 weeks post dose. These results suggest that fipronil may impact shrimp populations at low concentrations and further use in coastal areas should be carefully assessed.

Toxicity and bioconcentration potential of the agricultural pesticide endosulfan in phytoplankton and zooplankton.

Agricultural pesticide runoff in southeastern coastal regions of the United States is a critical issue. Bioconcentration of pesticides by phytoplankton and zooplankton at the base of the aquatic food web may increase the persistence of pesticides in aquatic ecosystems and cause effects at higher trophic levels. This study examined the toxicity of a widely used agricultural pesticide, endosulfan, to Pseudokirchneriella subcapitatum (freshwater green alga) and Daphnia magna (freshwater cladoceran). We then investigated the potential of both plankton species to sequester endosulfan from their surrounding media. We also assessed the degree to which endosulfan is accumulated by D. magna via food (endosulfan-contaminated P. subcapitatum). A 96-h growth rate EC50 of 427.80 microg/L endosulfan was determined for P. subcapitatum, whereas a 24-h immobilization EC50 of 366.33 microg/L endosulfan was determined for D. magna. The 5-h EC50s for filtration and ingestion in D. magna were 165.57 microg/L and 166.44 microg/L, respectively. An average bioconcentration factor (BCF) of 2,682 was determined for P. subcapitatum exposed to 100 microg/L endosulfan for 16 h. An average BCF of 3,278 was determined for D. magna in a 100 microg/L endosulfan water-only exposure. There was negligible uptake of endosulfan by D. magna feeding on contaminated algae in clean water (BCF approximately 0). Different proportions of parent isomers (endosulfan I and II) and the primary degradation product (endosulfan sulfate) were detected among treatments. Endosulfan was rapidly accumulated and concentrated from water by P. subcapitatum and D. magna neonates. Endosulfan contained in phytoplankton, however, was not bioaccumulated by zooplankton. These findings may prove useful in assessing ecosystem risk, because uptake from the water column appears to be the dominant route for bioconcentration of endosulfan by zooplankton.

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.

Bacterial symbiosis and paratransgenic control of vector-borne Chagas disease.

The triatomine vectors of Chagas disease are obligate haematophagous insects, feeding on vertebrate blood throughout their entire developmental cycle. As a result of obtaining their nutrition from a single food source, their diet is devoid of certain vitamins and nutrients. Consequently, these insects harbour populations of bacterial symbionts within their intestinal tract, which provide the required nutrients that are lacking from their diet. We have isolated and characterised symbiont cultures from various triatomine species and developed a method for genetically transforming them. We can then reintroduce them into their original host species, thereby producing stable paratransgenic insects in which we are able to express heterologous gene products. Using this methodology, we have generated paratransgenic Rhodnius prolixus that are refractory for infection with Trypanosoma cruzi. Two examples of potentially refractory genes are currently being expressed in paratransgenic insects. These include the insect immune peptide cecropin A and active single chain antibody fragments. We have also developed an approach that would allow introduction of genetically modified bacterial symbionts into natural populations of Chagas disease vectors. This approach utilises the coprophagic behaviour of these insects, which is the way in which the symbionts are transmitted among bug populations in nature. The production and ultimate release of transgenic or paratransgenic insects for public health applications is potentially very promising but also worthy of much careful consideration with respect to environmental, political, and human safety concerns.

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.

Characterization of VspB of Borrelia turicatae, a major outer membrane protein expressed in blood and tissues of mice.

Serotypes A and B of the relapsing fever spirochete Borrelia turicatae produce different disease manifestations in infected mice. Whereas serotype B causes more severe arthritis and reaches higher densities in the blood of mice than serotype A, serotype A invades the central nervous system earlier than serotype B during infection. These differences between serotypes A and B in mice are associated with the expression of different surface proteins, VspA and VspB, respectively, in the culture medium. To determine whether these proteins, in particular, VspB, are also expressed in vivo, scid mice infected with B. turicatae were studied. The expression of VspB by spirochetes in the blood was demonstrated in Coomassie blue-stained polyacrylamide gels and Western blots with a specific monoclonal antibody. Indirect immunofluorescence and immunoperoxidase studies confirmed the expression of VspB in the blood and also demonstrated VspB expression in the joints and heart. The gene for VspB was next identified and cloned by using partial amino acid sequencing, reverse transcriptase PCR, and a specific monoclonal antibody. The vspB gene encodes a protein of 216 amino acids that is 68% identical to VspA of B. turicatae and 44 to 56% identical to representative Vsp and OspC lipoproteins of other Borrelia spp. The processed VspB protein was distinguished from 26 other Vsp and OspC proteins by a high predicted isoelectric point at 9.39. The promoter region for vspB was similar to the promoter region for the vsp33 gene of Borrelia hermsii and for the ospC gene of Borrelia burgdorferi, two genes known to be environmentally regulated. These studies established that the virulence-associated VspB protein is expressed by spirochetes in the mouse and that VspB is a novel member of the Vsp-OspC family of proteins.

Immunologic and genetic analyses of VmpA of a neurotropic strain of Borrelia turicatae.

In mice infected with serotype A but not serotype B of the relapsing fever spirochete Borrelia turicatae, early invasion of the brain occurs. Serotypes A and B are further distinguished by the abundant surface protein they produce: VmpA and VmpB, respectively. Western blotting with monoclonal antibodies, one-dimensional peptide mapping, and partial amino acid sequencing demonstrated regions of the VmpA protein that differed from VmpB. Oligonucleotide primers based on the partial amino acid sequences of unique regions were used to amplify a portion of the VmpA gene (vmpA) by PCR, and the product was used as a probe in Southern blot and Northern blot analyses. These experiments showed that (i) expression of the vmpA sequence was determined at the level of transcription and (ii) the vmpA sequence was in two locations in serotype A and one location in serotype B. The vmpA gene at the expression-linked locus of serotype A was cloned and sequenced. An open reading frame would encode a polypeptide of 214 amino acids. The polypeptide expressed by Escherichia coli was bound by VmA-specific but not VmpB-specific antibody. Primer extension analysis identified a consensus sigma70-type promoter for vmpA at the expression locus. Phylogenetic analysis revealed that VmpA is homologous to small Vmp (Vsp) proteins of B. hermsii and to OspC proteins of B. burgdorferi. These findings indicate that a function of the Vsp-OspC family of proteins of Borrelia spp. may be differential localization in organs, including the brain, during infection.