A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

Acute toxicity of the UV filter oxybenzone to the coral Galaxea fascicularis.

Coral reefs are impacted by a variety of anthropogenic stressors including inputs of chemical contaminants. Although data is currently limited, sunscreens containing ultraviolet (UV) filters have recently been suggested as an emerging class of chemical contaminants. To provide further data on the toxicity of the UV filter oxybenzone (benzophenone-3 or BP-3) to corals, we conducted three independent acute toxicity tests exposing the colonial stony coral Galaxea fascicularis to BP-3 (0.31 to 10 mg/L nominal concentrations). Assessments included daily analytical verification of the exposure concentrations, calculation of the lethal concentration to result in 50% mortality (LC50) and numerous biological endpoints to further investigate the potential impact to both the coral and symbiont. LC50s for the three tests were similar and averaged 6.53 ± 0.47 mg/L nominal concentration BP-3 (4.45 mg/L measured dissolved BP-3). BP-3 did not initiate coral bleaching or show a significant loss of symbionts from the coral tissue in this species as reductions in measurements used for bleaching (i.e. visual color, color saturation and photosynthetic pigment concentrations) were only seen concurrently with tissue loss (i.e. at ≥2.5 mg/L nominal concentration BP-3). Polyp retraction, the most sensitive endpoint of this test, was seen to be a sub-lethal behavioral response to BP-3 exposure. Using the calculated LC50 with measured concentrations from a high-quality UV filter monitoring study in Hawaii, a preliminary, conservative risk quotient for BP-3 was calculated at 0.032. These results suggest that BP-3 likely does not pose an acute risk of mortality to G. fascicularis and additional testing is required to determine sublethal impacts of BP-3 under environmentally relevant concentrations and longer-term chronic exposures. This study highlights complications in conducting toxicity tests with organic UV filters including under-estimations of exposure concentrations and provides recommendations to improve these methods for better comparisons between studies.

A Critical Review of Organic Ultraviolet Filter Exposure, Hazard, and Risk to Corals.

There has been a rapid increase in public, political, and scientific interest regarding the impact of organic ultraviolet (UV) filters to coral reefs. Such filters are found in sunscreens and other consumer products and enter the aquatic environment via direct (i.e., recreational activities, effluents) or indirect (i.e., land runoff) pathways. This review summarizes the current state of the science regarding the concentration of organic UV filters in seawater and sediment near coral reef ecosystems and in coral tissues, toxicological data from early and adult life stages of coral species, and preliminary environmental risk characterizations. Up to 14 different organic UV filters in seawater near coral reefs have been reported across 12 studies, with the majority of concentrations in the nanograms per liter range. Nine papers report toxicological findings from no response to a variety of biological effects occurring in the micrograms per liter to milligrams per liter range, in part given the wide variations in experimental design and coral species and/or life stage used. This review presents key findings; scientific data gaps; flaws in assumptions, practice, and inference; and a number of recommendations for future studies to assess the environmental risk of organic UV filters to coral reef ecosystems. Environ Toxicol Chem 2021;40:967-988. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Nonlethal Biomarkers of Oxidative Stress in Oiled Sediment Exposed Southern Flounder (Paralichthys lethostigma): Utility for Field-Base Monitoring Exposure and Potential Recovery.

The Deepwater Horizon (DWH) blowout resulted in the deposition of toxic polycyclic aromatic hydrocarbons (PAHs), in the coastal sediments of the Gulf of Mexico. The immediate effects on an ecosystem from an oil spill are clearly recognizable, however the long-term chronic effects and recovery after a spill are still not well understood. Current methodologies for biomonitoring wild populations are invasive and mostly lethal. Here, two potential nonlethal biomonitoring tools for the assessment of PAH toxicity and induced biological alterations in the field, were identified using laboratory-validated methods. In this study, subadult southern flounder (Paralichthys lethostigma) were chronically exposed to DWH surrogate oiled sediments for 35 days; a subset of these exposed flounder were then provided a clean nonexposure period to ascertain the utility of selected biomarkers to monitor recovery post exposure. After chronic exposure, there was an increase in gene expression of cytochrome P450 1A but not glutathione S-transferase. There was also a notable imbalance of oxidants to antioxidants, measured as reduced glutathione, oxidized glutathione, and their ratio in the blood. Evidence of subsequent oxidative damage due to chronic exposure was found through lipid peroxidation and DNA damage assessments of liver, gill, and blood.

Occurrence and distribution of UV-filters and other anthropogenic contaminants in coastal surface water, sediment, and coral tissue from Hawaii.

The occurrence of UV-filters in the environment has raised concerns over potentially adverse impacts on corals. In this study, the concentrations of 13 UV-filters and 11 hormones were measured in surface seawater, sediment, and coral tissue from 19 sites in Oahu, Hawaii. At least eight UV-filters were detected in seawater, sediment, and coral tissue and total mass concentrations of all UV-filters were <750 ng L-1, <70 ng g-1 dry weight (dw), and <995 ng g-1 dw, respectively. Four UV-filters were detected in water, sediment, and coral tissue at detection frequencies of 63-100%, 56-91%, and 82-100%, respectively. These UV-filter concentrations generally varied as follows: water, homosalate (HMS) > octisalate (OS) > benzophenone-3 (BP-3, also known as oxybenzone) > octocrylene (OC); sediment, HMS > OS > OC > BP-3; coral, OS ≈ HMS > OC ≈ BP-3. BP-3 concentrations in surface seawater were <10 ng L-1 at 12 of 19 sites and highest at Waikiki beach (e.g., 10.9-136 ng L-1). While BP-3 levels were minimal in sediment (e.g., <1 ng g-1 dw at 18 of 19 sites), and ranged from 6.6 to 241 ng g-1 dw in coral tissue. No quantifiable levels of 2-ethylhexyl 4-methoxycinnamate (also known as octinoxate) were recorded in surface seawater or coral tissues, but 5-12.7 ng g-1 dw was measured for sediment at 5 of 19 sites. No hormones were detected in seawater or sediment, but 17α-ethinylestradiol was present in three corals from Kaneohe Bay. Surfactant degradation products were present in seawater, especially at Waikiki beach. These results demonstrate ubiquitous parts-per-trillion concentrations of UV-filters in surface seawater and is the first report of UV-filters in coral tissue from U.S.A. coastal waters. These data inform the range of environmentally-relevant concentrations for future risk assessments on the potential impacts of UV-filters on coral reefs in Oahu, Hawaii.

Characterization of dissolved and particulate phases of water accommodated fractions used to conduct aquatic toxicity testing in support of the Deepwater Horizon natural resource damage assessment.

In response to the Deepwater Horizon oil spill, the Natural Resource Trustees implemented a toxicity testing program that included 4 different Deepwater Horizon oils that ranged from fresh to weathered, and 3 different oil-in-water preparation methods (including one that used the chemical dispersant Corexit 9500) to prepare a total of 12 chemically unique water accommodated fractions (WAFs). We determined how the different WAF preparation methods, WAF concentrations, and oil types influenced the chemical composition and concentration of polycyclic aromatic hydrocarbons (PAHs) in the dissolved and particulate phases over time periods used in standard toxicity tests. In WAFs prepared with the same starting oil and oil-to-water ratio, the composition and concentration of the dissolved fractions were similar across all preparation methods. However, these similarities diverged when dilutions of the 3 WAF methods were compared. In WAFs containing oil droplets, we found that the dissolved phase was a small fraction of the total PAH concentration for the high-concentration stock WAFs; however, the dissolved phase became the dominant fraction when it was diluted to lower concentrations. Furthermore, decreases in concentration over time were mainly related to surfacing of the larger oil droplets. The initial mean diameters of the droplets were approximately 5 to 10 µm, with a few droplets larger than 30 µm. After 96 h, the mean droplet size decreased to 3 to 5 µm, with generally all droplets larger than 10 µm resurfacing. These data provide a detailed assessment of the concentration and form (dissolved vs particulate) of the PAHs in our WAF exposures, measurements that are important for determining the effects of oil on aquatic species. Environ Toxicol Chem 2017;36:1460-1472. © 2017 SETAC.

Characterization of oil and water accommodated fractions used to conduct aquatic toxicity testing in support of the Deepwater Horizon oil spill natural resource damage assessment.

The Deepwater Horizon blowout resulted in the release of millions of barrels of crude oil. As part of the Trustees' Natural Resource Damage Assessment, a testing program was implemented to evaluate the toxicity of Deepwater Horizon oil and oil/dispersant mixtures to aquatic organisms from the Gulf of Mexico. Because of the variety of exposures that likely occurred, the program included 4 Deepwater Horizon oils, which encompassed a range of weathering states, and 3 different oil-in-water mixing methods, for a total of 12 unique water accommodated fractions (WAFs). The present study reports on the chemical characteristics of these 4 Deepwater Horizon oils and 12 WAFs. In addition, to better understand exposure chemistry, an examination was conducted of the effects of WAF preparation parameters-including mixing energy, starting oil composition, and oil-to-water mixing ratios-on the chemical profiles and final concentrations of these 12 WAFs. The results showed that the more weathered the starting oil, the lower the concentrations of the oil constituents in the WAF, with a shift in composition to the less soluble compounds. In addition, higher mixing energies increased the presence of insoluble oil constituents. Finally, at low to mid oil-to-water mixing ratios, the concentration and composition of the WAFs changed with changing mixing ratios; this change was not observed at higher mixing ratios (i.e., >1 g oil/L). Ultimately, the present study provides a basic characterization of the oils and WAFs used in the testing program, which helps to support interpretation of the more than 500 Deepwater Horizon Natural Resource Damage Assessment toxicity testing results and to enable a comparison of these results with different tests and with the field. Environ Toxicol Chem 2017;36:1450-1459. © 2016 SETAC.

Investigating physiological, cellular and molecular effects in juvenile blue crab, Callinectus sapidus, exposed to field-collected sediments contaminated by oil from the Deepwater Horizon Incident.

Juvenile blue crabs, Callinectus sapidus, were exposed for 31 days to six different sediments collected within the Pass a Loutre State Wildlife Management Area approximately 6 months or 1.5 years post-capping of the Macondo-252 well-head following the Deepwater Horizon (DWH) Incident. Based on forensic analysis to fingerprint for DWH oil, these sediments differed in their levels of DWH oil contamination, and included one reference sediment collected from a location with no detectable DWH oil present. The concentration of 50 individual parent and alkylation group polycyclic aromatic hydrocarbons (PAHs), saturated hydrocarbons (37 total), and total extractable hydrocarbons were determined in each sediment, as were biologically relevant metals, grain size distribution, percent total organic carbon, and percent total solids. Total concentrations of 50 PAHs (TPAH50) of initial treatment sediments ranged from 187 µg kg(-1) (reference site) to 2,086,458 µg kg(-1) (the highest DWH oil contaminated site). Multiple biological endpoints were measured including mortality, growth, and ecdysis. Additionally, early biomarkers of biological stress were examined in the hemolymph and hepatopancreas of crabs, including DNA damage (Comet assay) and expression of genes encoding Cu-metallothionein (CuMT), glutathione-S-transferase (GST), and manganese superoxide dismutase (MnSOD). Over the 31 day exposure, there were no treatment related mortalities in juvenile blue crabs. The overall growth and molting of the crabs were not substantially different between the various sediment exposures over the exposure period. Additionally, none of the early biomarkers of biological stress were correlated with PAH concentrations. Overall, juvenile blue crabs did not appear to be negatively impacted during the 31 day exposure by DWH oil contaminated sediments collected at least 6 months post-capping of the Macondo-252 well-head.

Acute toxicity of current and alternative oil spill chemical dispersants to early life stage blue crabs (Callinectes sapidus).

The aim of this study was to examine the acute toxicity of five oil spill chemical dispersants on the ecologically and economically important coastal and estuarine species, blue crab Callinectes sapidus. Static, non-renewal 48 h acute toxicity tests were performed on stage-II blue crab zoea. The median lethal concentration (LC50) was calculated for each dispersant at 24 h and 48 h using nominal concentrations for each dispersant tested. The 48 h LC50 values from the most to the least toxic ranged from 10.1 mg L(-1) for Dispersit SPC 1000 to 76.5 mg L(-1) for Orca. For all dispersants, the swimming activity and mobility of larvae decreased with increasing dispersant concentration within 24h of exposure and reached relative immobility at concentrations below LC50 values. These results show that the dispersants examined in this study are only slightly toxic after 48 h exposure to the earliest life stage of blue crabs that might likely be exposed to dispersants in the environment, with the exception of Dispersit SPC 1000 that bordered between slightly and moderately toxic. Although the dispersants themselves appear to not cause substantial acute toxicity, sublethal and potentially delayed impacts, such as, reduced mobility or food source availability could indirectly remove larvae from the population and need to be further examined, as do larval responses in standard chronic toxicity tests. Furthermore, dispersants are not released into the environment in isolation and so the impact of dispersed-oil using these dispersant formulations also needs to be investigated to translate into real-world situations.

Responses of the soft coral Xenia elongata following acute exposure to a chemical dispersant.

Limited toxicology data are available regarding oil dispersant exposure to coral species. Corexit® EC9500A (Corexit) is a commonly applied dispersant most well known for its use after the Deepwater Horizon spill in April, 2010. There is limited evidence that Corexit can cause a bleaching response in corals. The aims of the study were: (1) to determine the extent of bleaching after acute 24 h and 72 h exposures of sublethal concentrations (0-50 ppm) of Corexit to the pulsing soft coral Xenia elongata and (2) to investigate a percent symbiont loss calculation using zooxanthellae density. The percent symbiont loss calculation was compared to a traditional metric of normalizing zooxanthellae density to soluble protein content. Percent symbiont loss was an effective measure of coral stress in acute Corexit exposures, while protein normalized zooxanthellae density was more variable. The bleaching data suggest a positive relationship between dispersant concentration and percent symbiont loss, culminating in excessive tissue necrosis and coral mortality within 72 h in high concentration exposures (p < 0.001). Percent beaching ranged from 25% in 5 ppm exposures to 100% in 50 ppm exposures. Corexit also caused a significant decrease in pulse activity (p < 0.0001) and relative oxygen saturation (p < 0.001), possibly indicating a reduction in photosynthetic efficiency. This study and other similar research indicate that dispersant exposure is highly damaging to marine organisms, including ecologically important coral species.

Investigating the use of oil platform marine fouling invertebrates as monitors of oil exposure in the Northern Gulf of Mexico.

The concentration of 51 parent and alkylated PAHs was examined in oysters, Ostrea equestris, and corals, Tubastrea coccinea, collected from oil-rig structures off the coast of Louisiana during April and May 2011 to investigate their potential use as monitors for offshore contamination events. Corals and oysters collected from both sampling trips had lower PAH accumulation than most bivalves collected in previous studies near the shoreline of Louisiana and elsewhere in the Gulf of Mexico. In April, total PAH (TPAH) concentrations ranged from 8.73 to 15.17 ng g(-1) in corals and 2.52 to 22.04 ng g(-1) in oysters. In May, corals and oysters had elevated concentrations of TPAH ranging from 24.28 to 79.23 ng g(-1) and 7.18 to 95.55 ng g(-1), respectively. This increase could be a result of Mississippi River flooding that occurred during that time, as evidenced by the high perylene concentrations (3.92-41.49 ng g(-1)) measured in May oysters. Oysters and corals collected in May from MC21B, the closest rig to the Mississippi River Delta, had the highest TPAH concentrations observed among all locations and the only rig to have predominantly petrogenic source inputs. Overall, given the low baseline of PAHs demonstrated in this study and the rapid accumulation of diagnostic chemicals in response to a possible contamination event (i.e. sediment plume from May flooding), oil-rig invertebrates could make excellent monitoring tools to examine the exposure to and recovery from oil (and oil-spill response options) in the offshore Northern Gulf of Mexico. Pre-spill baseline data of chemical and biological biomarkers of contamination is key to better estimating the impacts and recovery of oil exposure. Therefore, this screen of PAH accumulation represents a crucial first step in determining baseline contaminant levels in order to utilize these unique resources as monitors for offshore oil exposure in the Northern Gulf of Mexico.

Species specific differences in the in vitro metabolism of the flame retardant mixture, Firemaster® BZ-54.

Firemaster(®) BZ-54 is a flame retardant additive and consists of a brominated benzoate (2-ethylhexyl 2,3,4,5-tetrabromobenzoate; TBB) and a brominated phthalate (bis (2-ethylhexyl) 2,3,4,5-tetrabromophthalate; TBPH). Previous research has shown that fathead minnows exposed in vivo to Firemaster(®) BZ-54 accumulate TBB and TBPH. This study examined the in vitro biotransformation potential of TBB and TBPH in hepatic subcellular fractions (i.e., S9, microsomes and cytosol) in the fathead minnow, common carp, mouse and snapping turtle. Metabolism was evaluated by measuring the loss of the parent TBB or TBPH and identifying potential metabolites in the sample extracts. Metabolic loss of TBPH was measured for all species, while TBB loss was observed for all species except for the snapping turtle. Several metabolites were observed in all of the incubations except for snapping turtle. Metabolites observed appeared to be derived from TBB, given their structures and lack of appearance in the snapping turtle incubations. One of these metabolites, 2,3,4,5-tetrabromomethylbenzoate has been identified for the first time in a biological system. When metabolized, TBB and TBPH loss was found in each subcellular fraction suggesting that the enzyme(s) involved are present in both soluble and membrane-bound forms. It can be concluded that a broad range of species are capable of metabolizing TBB and TBPH to various metabolites and further research should be carried out to ascertain the specific products formed from metabolism of TBB and TBPH.

Accumulation and DNA damage in fathead minnows (Pimephales promelas) exposed to 2 brominated flame-retardant mixtures, Firemaster 550 and Firemaster BZ-54.

Firemaster 550 and Firemaster BZ-54 are two brominated formulations that are in use as replacements for polybrominated diphenyl ether (PBDE) flame retardants. Two major components of these mixtures are 2,3,4,5-tetrabromo-ethylhexylbenzoate (TBB) and 2,3,4,5-tetrabromo-bis(2-ethylhexyl) phthalate (TBPH). Both have been measured in environmental matrices; however, scant toxicological information exists. The present study aimed to determine if these brominated flame-retardant formulations are bioavailable and adversely affect DNA integrity in fish. Fathead minnows (Pimephales promelas) were orally exposed to either FM 550, FM BZ54, or the nonbrominated form of TBPH, di-(2-ethylhexyl) phthalate (DEHP) for 56 d and depurated (e.g., fed clean food) for 22 d. At several time points, liver and blood cells were collected and assessed for DNA damage. Homogenized fish tissues were extracted and analyzed on day 0 and day 56 to determine the residue of TBB and TBPH and the appearance of any metabolites using gas chromatography-electron-capture negative ion mass spectrometry (GC/ECNI-MS). Significant increases (p < 0.05) in DNA strand breaks from liver cells (but not blood cells) were observed during the exposure period compared with controls, although during depuration these levels returned to control. Both parent compounds, TBB and TBPH, were detected in tissues at approximately 1% of daily dosage along with brominated metabolites. The present study provides evidence for accumulation, metabolism, and genotoxicity of these new formulation flame retardants in fish and highlights the potential adverse effects of TBB- and TBPH-formulated fire retardants to aquatic species.

Shipboard trials of an ozone-based ballast water treatment system.

Legislation introduced by the United Nations International Maritime Organization (IMO) has focused primarily on standards defining successful treatments designed to remove invasive species entrained in ballast water. An earlier shipboard study found that ozone introduced into salt water ballast resulted in the formation of bromine compounds, measured as total residual oxidants (TRO) that were toxic to both bacteria and plankton. However, the diffuser system employed to deliver ozone to the ballast water tanks resulted in patchiness in TRO distribution and toxicity to entrained organisms. In this follow-up study, the shipboard diffuser system was replaced by a single Venturi-type injection system designed to deliver a more homogeneous biocide distribution. Within-tank variability in TRO levels and associated toxicity to zooplankton, phytoplankton and marine bacteria was measured via a matrix of tubes deployed to sample different locations in treated and untreated (control) tanks. Three trials were conducted aboard the oil tanker S/T Prince William Sound in the Strait of Juan de Fuca off Port Angeles, Washington State, USA, between June and December 2007. Mortalities of plankton and bacteria and oxidant concentrations were recorded for treated and untreated ballast water up to 3days following treatment, and residual toxicity beyond this period was measured by bioassay of standard test organisms. Results indicated uniform compliance with current IMO standards, but only partial compliance with other existing and pending ballast water legislation.

Alterations in dimethylsulfoniopropionate (DMSP) levels in the coral Montastraea franksi in response to copper exposure.

Symbiotic corals routinely experience hyperoxic conditions within their tissues due to the photosynthesis of the endosymbiotic dinoflagellate microalgae (Symbiodinium spp.). Symbiodinium spp. produce high intracellular levels of the osmolyte dimethylsulfoniopropionate (DMSP). It has recently been discovered in marine algae that DMSP and its enzymatic breakdown products also play a significant role in the scavenging of cellular reactive oxygen species (ROS). To examine this potential for DMSP in corals, we exposed the hard coral Montastraea franksi to 1, 10 and 50 microg L(-1) (ppb) concentrations of the oxidative stressor, copper. Levels of total (DMSP(t), all coral tissue) were higher than particulate DMSP(p) (algal component only), demonstrating partitioning of DMSP between algal symbionts and coral host. Significant changes in levels of DMSP(t) and DMSP(p) occurred in M. franksi after 48 h, demonstrating a response to copper and indicating a potential antioxidant role for DMSP. DMSP(t) and DMSP(p) levels decreased with copper dose, although at the highest copper dose DMSP(p) levels increased, whereas DMSP(t) levels did not. This observed differential response to copper between DMSP(t) and DMSP(p) demonstrates that physiological changes may be overlooked if conclusions are based upon DMSP(t) levels alone, which is a common measure used in coral studies. Decreases in chlorophyll a and algal cell numbers in response to elevated copper were also observed. These indices are important physiological indicators and are often used as indices to normalize DMSP levels. Our data suggest that the use of these common indices for normalizing DMSP may not always be appropriate.

Characterizing the in vitro hepatic biotransformation of the flame retardant BDE 99 by common carp.

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardant chemicals known to biomagnify in aquatic foodwebs. However, significant biotransformation of some congeners via reductive dehalogenation has been observed during in vivo and in vitro laboratory exposures, particularly in fish models. Little information is available on the enzyme systems responsible for catalyzing this metabolic pathway in fish. This study was undertaken to characterize the biotransformation of one primary BDE congener, 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), using in vitro techniques. Hepatic sub-cellular fractions were first prepared from individual adult common carp (Cyprinus carpio) to examine metabolism in both microsomal and cytosolic sub-cellular fractions. Debromination rates (i.e. BDE-99 biotransformation to BDE-47) were generally higher in the microsomal fraction than in the cytosolic fraction, and some intra-species variability was observed. Further experiments were conducted to determine the biotransformation kinetics and the influence of specific co-factors, inhibitors and competitive substrates on metabolism using pooled carp liver microsomes. The apparent K(m) and V(max) values were 19.4microM and 1120pmolesh(-1)mgprotein(-1), respectively. Iodoacetate (IaC) and the two thyroid hormones, reverse triodothyronine (rT3) and thyroxine (T4), significantly inhibited the debromination of BDE-99 in microsomal sub-cellular fractions with IC(50) values of 2.2microM, 0.83microM, and >1.0microM, respectively. These results support our hypothesis that deiodinase enzymes may be catalyzing the metabolism of PBDEs in fish liver tissues. Further studies are needed to evaluate metabolic activity in other species and tissues that contain these enzymes.

Lack of biological effects of water accommodated fractions of chemically- and physically-dispersed oil on molecular, physiological, and behavioral traits of juvenile snapping turtles following embryonic exposure.

Snapping turtle (Chelydra serpentina) eggs were exposed to two concentrations of chemically- or physically-dispersed water accommodated fractions of weathered Arabian light crude oil (Low=0.5 and High=10 g oil/L water). Solutions were passed through nest substrate to simulate alterations in composition during percolation to egg depth. Hatchlings were raised for 13 months during which numerous endpoints were measured. Prior to percolation, total PAH ("tPAH"; the sum of 52 PAHs measured) in physically-dispersed oil fractions were similar (High, 43; Low 67 mg/L). Following percolation, tPAH was also similar in physically-dispersed fractions (High, 14; Low 24 mg/L). Addition of dispersant increased tPAH prior to percolation in the High treatment (302 mg/L) relative to Low (13 mg/L), but percolation resulted in nearly equal concentrations in both treatments (High, 30; Low, 22 mg/L) due to physical trapping of dispersed oil by the nest substrate. In both chemically- and physically-dispersed fractions, percolation reduced low molecular weight (MW) compounds such that embryos were exposed to primarily mid- to high MW compounds. Total PAH in eggs differed 15-fold between the chemically-dispersed High and physically-dispersed High treatments (560 and 36 microg/kg respectively), the former characterized by higher MW compounds than the latter. While eggs accumulated up to 560 microg/kg tPAH, we observed no effects on hatching success or hatchling/juvenile traits (DNA integrity, survival, growth, metabolism, energy storage, or behavior), our results demonstrate that PAH profiles are altered during percolation, suggesting that experiments with subsurface organisms should be designed to account for compositional changes that occur as the solutions percolate through the substrate.

Uptake and partitioning of copper and cadmium in the coral Pocillopora damicornis.

Coral-reef ecosystems are increasingly being impacted by a wide variety of anthropogenic inputs, including heavy metals, which could be contributing to coral reef stress and bleaching episodes. Fragments of Pocillopora damicornis were exposed in the laboratory to cadmium (Cd) or copper (Cu) chlorides (0, 5, 50 microg l(-1)) for 14 days and analyzed for metal content in the whole association, algal or animal fractions. Various physiological and biochemical parameters were also measured, such as, algal cell counts, mitotic index, chlorophyll content and levels of the antioxidant glutathione (GSH). Cd and Cu accumulation were observed at all time points and doses; there was no evidence of differential metal partitioning between the algal or animal fractions. No changes in algal cell density, mitotic index or chlorophyll content from the controls were observed in any of the metal treatments. GSH levels were significantly higher in the 5 microg l(-1) Cd (Day 4) and Cu (Days 4 and 14) treatments compared with controls at the same time point. Although no evidence of a bleaching response occurred, corals in both 50 microg l(-1) metal exposures sloughed off tissues and did not survive the duration of the exposure period. Our results demonstrate the accumulation of Cd and Cu in P. damicornis and mortality in the absence of a bleaching response.

Induction of CYP1A and DNA damage in the fathead minnow (Pimephales promelas) following exposure to biosolids.

Biosolids (treated sewage sludge) are increasingly disposed of on land. Thus particle-sorbed and dissolved constituents have the potential to enter nearby watersheds. Although organic contaminants are known to be present in biosolids these are not currently regulated and little data exist on their potential toxicity to aquatic organisms. We exposed Pimephales promelas to two concentrations of biosolids (0.5 and 2.5 g l(-1)) for 28-days (static-renewal) and characterized contaminants present and the extent of CYP1A and DNA damage induction at various time points. Many organic contaminants were detected in the biosolids, with polycyclic aromatic hydrocarbons (PAHs) being the dominant class. Substantial levels of polybrominated diphenyl ethers (PBDEs) and nonylphenols (NPs) were also present. Significant induction of hepatic CYP1A protein compared with controls (P<0.05) was observed in both low (0.5 g l(-1)) and high (2.5 g l(-1)) exposed fish from Day 7. CYP1A levels peaked at Day 21 with 21-fold and 8-fold inductions over controls in high and low dose fish respectively. Induction of DNA damage in hepatocytes (single strand breaks as measured using the COMET assay) was observed in both exposures compared with controls on Days 14 and 28 (P<0.05). A significant correlation was found between CYP1A induction and DNA damage (Pearson correlation index, P<0.05). It is plausible that activation of PAHs may be responsible for the induction of CYP1A and resulting increase in DNA damage. Our data show the potential for detrimental effects in the event of exposure of aquatic organisms to biosolids and the need for further investigations of possible impacts due to constituents not covered by current guidelines.

Debromination of polybrominated diphenyl ether-99 (BDE-99) in carp (Cyprinus carpio) microflora and microsomes.

Based on previous findings in dietary studies with carp (Cyprinus carpio), we investigated the mechanism of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) debromination to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) using liver and intestinal components. In vitro aerobic and anaerobic experiments tested the ability of carp intestinal microflora to debrominate BDE-99. No debromination of BDE-99 to BDE-47 was observed in microfloral samples; therefore, carp enzymatic pathways were assessed for debromination ability. After sixty-min incubation, intestine and liver microsomes exhibited 83+/-34% and 106+/-18% conversions, respectively, of BDE-99 to BDE-47; with no significant (p>0.05) difference between organ debromination capabilities. Microsomal incubations with BDE-99, enzyme cofactors and competing substrates assessed the potential mechanisms of debromination. The presence of NADPH in the microsomal assay did not significantly (p>0.05) affect BDE-99 debromination, which suggest that cytochrome P450 enzymes are not the main debrominating pathway for BDE-99. Co-incubation of BDE-99 spiked microsomes with reverse thyronine (rT3) significantly (p<0.05) decreased the debromination capacity of intestinal microsomes indicating the potential of catalytic mediation via thyroid hormone deiodinases. The significant findings of this study are that intestinal microflora are not responsible for BDE-99 debromination, however, it is an endogenous process which occurs with approximately equal activity in intestine and liver microsomes and it can be inhibited by rT3.