Carys L. Mitchelmore

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.

Life Histories, Salinity Zones, and Sublethal Contributions of Contaminants to Pelagic Fish Declines Illustrated with a Case Study of San Francisco Estuary, California, USA

Human effects on estuaries are often associated with major decreases in abundance of aquatic species. However, remediation priorities are difficult to identify when declines result from multiple stressors with interacting sublethal effects. The San Francisco Estuary offers a useful case study of the potential role of contaminants in declines of organisms because the waters of its delta chronically violate legal water quality standards; however, direct effects of contaminants on fish species are rarely observed. Lack of direct lethality in the field has prevented consensus that contaminants may be one of the major drivers of coincident but unexplained declines of fishes with differing life histories and habitats (anadromous, brackish, and freshwater). Our review of available evidence indicates that examining the effects of contaminants and other stressors on specific life stages in different seasons and salinity zones of the estuary is critical to identifying how several interacting stressors could contribute to a general syndrome of declines. Moreover, warming water temperatures of the magnitude projected by climate models increase metabolic rates of ectotherms, and can hasten elimination of some contaminants. However, for other pollutants, concurrent increases in respiratory rate or food intake result in higher doses per unit time without changes in the contaminant concentrations in the water. Food limitation and energetic costs of osmoregulating under altered salinities further limit the amount of energy available to fish; this energy must be redirected from growth and reproduction toward pollutant avoidance, enzymatic detoxification, or elimination. Because all of these processes require energy, bioenergetics methods are promising for evaluating effects of sublethal contaminants in the presence of other stressors, and for informing remediation. Predictive models that evaluate the direct and indirect effects of contaminants will be possible when data become available on energetic costs of exposure to contaminants given simultaneous exposure to non-contaminant stressors.

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.

Bromination of Marine Dissolved Organic Matter following Full Scale Electrochemical Ballast Water Disinfection

An extensively diverse array of brominated disinfection byproducts (DBPs) were generated following electrochemical disinfection of natural coastal/estuarine water, which is one of the main treatment methods currently under consideration for ballast water treatment. Ultra-high-resolution mass spectrometry revealed 462 distinct brominated DBPs at a relative abundance in the mass spectra of more than 1%. A brominated DBP with a relative abundance of almost 22% was identified as 2,2,4-tribromo-5-hydroxy-4-cyclopentene-1,3-dione, which is an analogue to several previously described 2,2,4-trihalo-5-hydroxy-4-cyclopentene-1,3-diones in drinking water. Several other brominated molecular formulas matched those of other known brominated DBPs, such as dibromomethane, which could be generated by decarboxylation of dibromoacetic acid during ionization, dibromophenol, dibromopropanoic acid, dibromobutanoic acid, bromohydroxybenzoic acid, bromophenylacetic acid, bromooxopentenoic acid, and dibromopentenedioic acid. Via comparison to previously described chlorine-containing analogues, bromophenylacetic acid, dibromooxopentenoic acid, and dibromopentenedioic acid were also identified. A novel compound at a 4% relative abundance was identified as tribromoethenesulfonate. This compound has not been previously described as a DBP, and its core structure of tribromoethene has been demonstrated to show toxicological implications. Here we show that electrochemical disinfection, suggested as a candidate for successful ballast water treatment, caused considerable production of some previously characterized DBPs in addition to novel brominated DBPs, although several hundred compounds remain structurally uncharacterized. Our results clearly demonstrate that electrochemical and potentially direct chlorination of ballast water in estuarine and marine systems should be approached with caution and the concentrations, fate, and toxicity of DBP need to be further characterized.

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.

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.

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.

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.

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.