Improving the design and conduct of aquatic toxicity studies with oils based on 20 years of CROSERF experience.

Laboratory toxicity testing is a key tool used in oil spill science, spill effects assessment, and mitigation strategy decisions to minimize environmental impacts. A major consideration in oil toxicity testing is how to replicate real-world spill conditions, oil types, weathering states, receptor organisms, and modifying environmental factors under laboratory conditions. Oils and petroleum-derived products are comprised of thousands of compounds with different physicochemical and toxicological properties, and this leads to challenges in conducting and interpreting oil toxicity studies. Experimental methods used to mix oils with aqueous test media have been shown to influence the aqueous-phase hydrocarbon composition and concentrations, hydrocarbon phase distribution (i.e., dissolved phase versus in oil droplets), and the stability of oil:water solutions which, in turn, influence the bioavailability and toxicity of the oil containing media. Studies have shown that differences in experimental methods can lead to divergent test results. Therefore, it is imperative to standardize the methods used to prepare oil:water solutions in order to improve the realism and comparability of laboratory tests. The CROSERF methodology, originally published in 2005, was developed as a standardized method to prepare oil:water solutions for testing and evaluating dispersants and dispersed oil. However, it was found equally applicable for use in testing oil-derived petroleum substances. The goals of the current effort were to: (1) build upon two decades of experience to update existing CROSERF guidance for conducting aquatic toxicity tests and (2) to improve the design of laboratory toxicity studies for use in hazard evaluation and development of quantitative effects models that can then be applied in spill assessment. Key experimental design considerations discussed include species selection (standard vs field collected), test substance (single compound vs whole oil), exposure regime (static vs flow-through) and duration, exposure metrics, toxicity endpoints, and quality assurance and control.

A Comparative Assessment of the Aquatic Toxicity of Corexit 9500 to Marine Organisms.

The use of chemical dispersants during oil spill responses has long been controversial. During the Deepwater Horizon (DWH) oil spill, 1.8 million gallons of dispersant, mainly Corexit 9500, were applied in offshore waters to mitigate the human health and coastal environmental impact of surface oil contamination. To evaluate the potential impact of the dispersant on marine life, 18 species, representing important ecological and commercial taxa, were tested using low-energy, dispersant-only water accommodated fractions (WAFs) of Corexit 9500 and standard acute toxicity test methods. All prepared WAFs were analytically characterized. Analyses included the two dispersant markers found in the dispersant and evaluated in samples collected during the DWH Response, dioctylsulfosuccinate sodium salt, and dipropylene glycol n-butyl ether (DPnB). The median lethal and effective concentrations (LC/EC50s) were calculated using a nominal exposure concentration (mg/L, based on the experimental loading rate of 50 mg/L) and measured DPnB (µg/L). Results ranged from 5.50 to > 50 mg/L dispersant and 492 to > 304,000 µg/L DPnB. Species sensitivity distributions of the data demonstrated that taxa were evenly distributed; however, algae and oysters were among the more sensitive organisms. The calculated 5% hazard concentration (HC5) for DPnB (1172 µg/L) was slightly higher than the USEPA chronic criteria of 1000 µg/L and substantially higher than all measured concentrations of DPnB measured in the Gulf of Mexico during the DWH oil spill response.

Interactive Effects of Mixtures of Phototoxic PAHs.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components in complex mixtures derived from petroleum based products. PAHs are unique in their ability to absorb UV light, resulting in significant increases in acute toxicity. The objective of this study was to determine if mixtures of the phototoxic PAHs fluoranthene, pyrene, and anthracene conform to the additive model of toxicity. Median lethal concentrations (LC50) were calculated for mysid shrimp (Americamysis bahia) and inland silverside (Menidia beryllina) exposed to individual, binary, and ternary mixtures of the selected PAHs. Mixtures were evaluated on a toxic unit basis to account for potency differences and toxicity data was analyzed using the concentration-addition and independent-action models. Data indicated that the model of additivity is sufficient in describing the toxicity of mixtures of phototoxic PAHs; therefore predictive models should consider an additivity model for assessing the toxicity of hydrocarbon mixtures.

The chronic toxicity of molybdate to marine organisms. I. Generating reliable effects data.

A scientific research program was initiated by the International Molybdenum Association (IMOA) which addressed identified gaps in the environmental toxicity data for the molybdate ion (MoO(4)(2-)). These gaps were previously identified during the preparation of EU-REACH-dossiers for different molybdenum compounds (European Union regulation on Registration, Evaluation, Authorization and Restriction of Chemical substances; EC, 2006). Evaluation of the open literature identified few reliable marine ecotoxicological data that could be used for deriving a Predicted No-Effect Concentration (PNEC) for the marine environment. Rather than calculating a PNEC(marine) using the assessment factor methodology on a combined freshwater/marine dataset, IMOA decided to generate sufficient reliable marine chronic data to permit derivation of a PNEC by means of the more scientifically robust species sensitivity distribution (SSD) approach (also called the statistical extrapolation approach). Nine test species were chronically exposed to molybdate (added as sodium molybdate dihydrate, Na(2)MoO(4)·2H(2)O) according to published standard testing guidelines that are acceptable for a broad range of regulatory purposes. The selected test organisms were representative for typical marine trophic levels: micro-algae/diatom (Phaeodactylum tricornutum, Dunaliella tertiolecta), macro-alga (Ceramium tenuicorne), mysids (Americamysis bahia), copepod (Acartia tonsa), fish (Cyprinodon variegatus), echinoderms (Dendraster exentricus, Strongylocentrotus purpuratus) and molluscs (Mytilus edulis, Crassostrea gigas). Available NOEC/EC(10) levels ranged between 4.4 mg Mo/L (blue mussel M. edulis) and 1174 mg Mo/L (oyster C. gigas). Using all available reliable marine chronic effects data that are currently available, a HC(5,50%) (median hazardous concentration affecting 5% of the species) of 5.74(mg Mo)/L was derived with the statistical extrapolation approach, a value that can be used for national and international regulatory purposes.

The chronic toxicity of molybdate to freshwater organisms. I. Generating reliable effects data.

The European Union regulation on Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH) (EC, 2006) requires the characterization of the chronic toxicity of many chemicals in the aquatic environment, including molybdate (MoO(4)(2-)). Our literature review on the ecotoxicity of molybdate revealed that a limited amount of reliable chronic no observed effect concentrations (NOECs) for the derivation of a predicted no-effect concentration (PNEC) existed. This paper presents the results of additional ecotoxicity experiments that were conducted in order to fulfill the requirements for the derivation of a PNEC by means of the scientifically most robust species sensitivity distribution (SSD) approach (also called the statistical extrapolation approach). Ten test species were chronically exposed to molybdate (added as sodium molybdate dihydrate, Na(2)MoO(4)·2H(2)O) according to internationally accepted standard testing guidelines or equivalent. The 10% effective concentrations (EC10, expressed as measured dissolved molybdenum) for the most sensitive endpoint per species were 62.8-105.6 (mg Mo)/L for Daphnia magna (21day-reproduction), 78.2 (mg Mo)/L for Ceriodaphnia dubia (7day-reproduction), 61.2-366.2 (mg Mo)/L for the green alga Pseudokirchneriella subcapitata (72h-growth rate), 193.6 (mg Mo)/L for the rotifer Brachionus calyciflorus (48h-population growth rate), 121.4 (mg Mo)/L for the midge Chironomus riparius (14day-growth), 211.3 (mg Mo)/L for the snail Lymnaea stagnalis (28day-growth rate), 115.9 (mg Mo)/L for the frog Xenopus laevis (4day-larval development), 241.5 (mg Mo)/L for the higher plant Lemna minor (7day-growth rate), 39.3 (mg Mo)/L for the fathead minnow Pimephales promelas (34day-dry weight/biomass), and 43.2 (mg Mo)/L for the rainbow trout Oncorhynchus mykiss (78day-biomass). These effect concentrations are in line with the few reliable data currently available in the open literature. The data presented in this study can serve as a basis for the derivation of a PNEC(aquatic) that can be used for national and international regulatory purposes and for setting water quality criteria. Using all reliable data that are currently available, a HC(5,50%) (median hazardous concentration affecting 5% of the species) of 38.2 (mg Mo)/L was derived with the statistical extrapolation approach.

Aqueous solubility and Daphnia magna chronic toxicity of di(2-ethylhexyl) adipate.

A water solubility of 5.5 (+/-0.22) microg/L for di(2-ethylhexyl) adipate (DEHA) was measured using the slow-stir method. This value is consistent with computer estimations and over two orders of magnitude lower than that previously determined using the shake-flask method. We performed a 21-day chronic Daphnia magna limit test at an average exposure of 4.4 microg/L in laboratory diluent water to avoid insoluble test material and avoid physical entrapment. One hundred percent of the DEHA-treated organisms survived compared to 90% survival in both the controls and solvent controls. Mean neonate reproduction was 152, 137, and 148 and mean dry weight per surviving female was 0.804, 0.779, and 0.742 mg in the DEHA treatment, control, and solvent control, respectively. No adverse effects were observed.

Gene expression in caged juvenile Coho Salmon (Oncorhynchys kisutch) exposed to the waters of Prince William Sound, Alaska.

The 1989 Exxon Valdez oil spill (EVOS) resulted in the release of 258,000 barrels of crude oil into the waters of Prince William Sound (PWS), Alaska. The current study, conducted in 2004, sought to use juvenile Coho salmon (Oncorhynchus kisutch) caged in situ to determine whether biomarker induction differed at sites where the adjacent shoreline contained buried residues from the 1989 oil spill compared to sites that were never oiled. Juvenile Coho salmon were caged at five sites; three oiled during the 1989 EVOS and two that were not oiled. Tissue samples were collected from organisms caged at each site as well as a control group housed onboard the research vessel. Analysis of CYP1A, superoxide dismutase (SOD), and glutathione peroxidase (GPO) gene expression was conducted using real time reverse transcriptase polymerase chain reaction (rtRT-PCR). Statistically significant levels of CYP1A expression were observed at some sites indicating increased hydrocarbon exposure. No patterns were observed regarding sites that were originally oiled or not oiled by the 1989 EVOS, indicating that sources of PAHs other than EVOS oil occur in PWS.

MTBE ambient water quality criteria development: a public/private partnership.

A public/private partnership was established in 1997, under the administrative oversight of the American Petroleum Institute (API), to develop aquatic toxicity data sufficient to calculate ambient water quality criteria for methyl tertiary-butyl ether (MTBE), a gasoline oxygenate. The MTBE Water Quality Criteria Work Group consisted of representatives from private companies, trade associations, and USEPA. Funding was provided by the private entities, while aquatic biological/toxicological expertise was provided by industry and USEPA scientists. This public/private partnership constituted a nonadversarial, cost-effective, and efficient process for generating the toxicity data necessary for deriving freshwater and marine ambient water quality criteria. Existing aquatic toxicity data were evaluated for acceptability, consistent with USEPA guidance, and nineteen freshwater and marine tests were conducted by commercial laboratories as part of this effort to satisfy the federal criteria database requirements. Definitive test data were developed and reported under the oversight of industry study monitors and Good Laboratory Practice standards auditors, and with USEPA scientists participating in advisory and critical review roles. Calculated, preliminary freshwater criteria for acute (Criterion Maximum Concentration) and chronic (Criterion Continuous Concentration) exposure effect protection are 151 and 51 mg MTBE/L, respectively. Calculated, preliminary marine criteria for acute and chronic exposure effect protection are 53 and 18 mg MTBE/L, respectively. These criteria values may be used for surface water quality management purposes, and they indicate that ambient MTBE concentrations documented in U. S. surface waters to date do not constitute a risk to aquatic organisms.

The cost of hepatitis A infections in American adolescents and adults in 1997.

The incidence of hepatitis A has declined in the United States for several decades, leading to an increased number of susceptible adolescents and adults. Because clinical severity increases with age, hepatitis A infections in older individuals cause greater morbidity, mortality, and treatment costs. Because the economic burden of hepatitis A has not been well described, we estimated its costs, from the societal perspective, for patients infected at distinct ages. A case-series study described treatment patterns, work loss, and symptom duration. Disease outcomes were estimated by an expert panel. Economic losses were calculated by applying normative data reflecting treatment charges, employee compensation, and the value of housekeeping services. In the case-series study, mean treatment charges were $740 for nonhospitalized patients versus $6,914 for hospitalized patients (P <.001). Symptom duration (67.8 vs. 34.4 days, P <.001) and work loss (33.2 vs. 15.5 days, P <.01) were also greater for those hospitalized. Nationally, we estimate 63,363 symptomatic hepatitis A infections occurred among adolescents and adults during 1997, resulting in 8,403 hospital admissions and 255 deaths. Nearly 2.5 million days of symptomatic illness and 829,000 work-loss days resulted, and 7,466 years of life were lost. Under base-case assumptions, annual hepatitis A costs were estimated at $488.8 million. In sensitivity analyses, this estimate varied from $332.4 to $579.9 million. These costs may be reduced by regionally targeted vaccination of children, as recommended by the Advisory Committee on Immunization Practices, although the cost effectiveness of this policy has not yet been established.

Chronic toxicity and carcinogenic evaluation of diisononyl phthalate in rats.

Groups of 110 Fischer 344 rats/sex were fed diisononyl phthalate (DINP) at dietary levels of 0, 0.03, 0.3, and 0.6 wt% for periods up to 2 years. Interim sacrifices of 10 predesignated rats/sex/dose were at 6, 12, and 18 months with surviving animals sacrificed at 24 months. At study termination, survival was in excess of 60% for every group. At the mid or high dose, the following biological effects were noted: slight decreases in food consumption and body weight; slight increase in mortality; a dose-related increase in relative organ weights of liver and kidney; and some slight effects on urinalysis, hematologic, and clinical chemistry parameters. No peroxisome induction was observed in livers of treated rats compared with controls. No clear treatment-related nonneoplastic or neoplastic lesions were found. However, mononuclear cell leukemia (MNCL) and changes known to be associated with an increased incidence of MNCL were seen in the mid-dose and high-dose groups. A literature review suggests that MNCL is a common finding in aging F344 rats and that this increased incidence in rats treated with DINP is not relevant to man. A clear no-observed-effect level was demonstrated for all biological end points at a dietary level of 0. 03 wt% or approximately 17 mg/kg/day of DINP.

An evaluation of the acute toxic properties of liquids derived from oil sands.

The acute toxicity of three materials derived from Athabasca Oil Sands--(1) bitumen plus naphtha, (2) untreated naphtha (0-250 degrees C) and (3) synthetic crude oil (0-500 degrees C)--was assessed in a battery of tests. In acute oral studies, all three test materials exhibited a low order of toxicity (LD50 greater than 5.0 g kg-1). The acute dermal LD50 was also low (greater than 3 g kg-1) for each test material. All three materials were judged to be 'slight' ocular irritants. Acute inhalation studies (6-h exposures at the maximum attainable concentrations) produced varied responses. Bitumen plus naphtha administered at a concentration of 1.46 mg l-1 did not cause mortality in exposed rats or mice. Lung discoloration was the only necropsy finding of note. Untreated naphtha administered at a concentration of 10.6 mg l-1 was lethal to essentially all of the mice; but only two rats died. Necropsy findings included elevated weights in the liver and kidneys of the exposed mice, elevated lung weights in male rats and elevated liver weights in female rats. Synthetic crude oil administered at a concentration of (4 mg l-1) was lethal to 5/10 mice, but none of the rats (0/10) died. Severe hair loss was noted in the surviving mice, and slight alopecia was also observed in rats. Both species exhibited elevated liver weight, and elevated lung weight was noted in female rats.

Evaluation of the dermal carcinogenic potential of tar sands bitumen-derived liquids.

The carcinogenic potential of Athabasca tar sands and six experimental liquids derived from crude bitumen was evaluated utilizing the mouse epidermal carcinogenesis model. Tar sands, bitumen, and untreated naphtha produced few, if any, tumors. Three thermally and catalytically cracked liquids, light (nominal boiling range: 149-316 degrees C) and heavy (nominal boiling range: greater than 316 degrees C) gas oils and gas oil blend (boiling range: greater than 316 degrees C), produced a significant number of epidermal neoplasms. A synthetic crude oil, prepared by blending naphtha and light and heavy gas oils, was moderately carcinogenic; however, the activity of this sample fell within the range of values obtained in studies of crude petroleum samples. Since the bitumen-derived streams do not differ substantially in carcinogenic potency from petroleum-derived materials of comparable boiling range and process history, industrial hygiene practices which limit exposures to levels comparable to those observed in the petroleum-refining industry should provide similar measures of protection.