Complex mixture toxicology: Evaluation of toxicity to freshwater aquatic receptors from biodegradation metabolites in groundwater at a crude oil release site, recent analogous results from other authors, and implications for risk management.

Aquatic toxicity posed by the complex mixture of biodegradation metabolites and related oxygen-containing organic compounds (OCOCs) in groundwater at typical petroleum release sites is of concern to regulatory agencies; several are using results from laboratory studies in older literature that are not appropriate analogs for risk management. Recent field studies from typical sites and natural groundwater should be utilized. In this study, OCOCs downgradient of the biodegrading crude oil release at the USGS Bemidji site were tested for freshwater aquatic toxicity using unaltered whole groundwater samples. This type of testing is optimal because the entire mixture of OCOCs present is tested directly and assessment is not affected by analytical limitations. Ceriodaphnia dubia and Pimephales promelas were tested for toxicity using USEPA Methods 1002 and 1000, which estimate chronic toxicity. OCOCs in representative samples up to the maximum concentration tested of 1710 ug/L Total Petroleum Hydrocarbons (TPH) (nC10 to nC40; without silica gel cleanup) did not result in effects relative to the lab control for C. dubia survival, or for P. promelas survival or growth; and did not result in effects above background for C. dubia reproduction. This is consistent with findings using the same testing methods and species on samples from 14 biodegrading fuel release sites: OCOCs did not cause increased toxicity relative to background at a maximum tested concentration of 1800 ug/L TPH (nC10 to nC28). Based on their toxicity testing using the same species and USEPA methods on groundwater from a biodegrading diesel release site, Washington Department of Ecology recently set a freshwater screening level for OCOCs at 3000 ug/L TPH ("Weathered DRO"). These studies indicate that, in the absence of dissolved hydrocarbons, OCOCs in groundwater from typical biodegrading fuel or crude oil releases are not toxic to C. dubia or P. promelas at typical concentrations.

Orbitrap ESI-MS evaluation of solvent extractable organics from a crude oil release site.

The concentrations of oxygen-containing organic compounds (OCOC), measured as dissolved organic carbon (DOC), in groundwater exceeds those of dissolved hydrocarbons, measured as total petroleum hydrocarbons (TPH), at a crude oil release site. Orbitrap mass spectrometry was used to characterize OCOC in samples of the oil, water from upgradient of the release, source area, and downgradient wells, and a local lake. Chemical characterization factors included carbon number, oxygen number, formulae similarity, double bond equivalents (DBE) and radiocarbon dating. Oil samples were dominated by formulae with less than 30 carbons, four or fewer oxygens, and a DBE of less than four. In water samples, formulae were identified with more than 30 carbons, more than 10 oxygens, and a DBE exceeding 30. These characteristics are consistent with DOC found in unimpacted water. Between 65% and 92% of the formulae found in samples collected within the elevated OCOC plume were also found in the upgradient or surface water samples. Evidence suggests that many of the OCOC are not petroleum degradation intermediates, but microbial products generated as a result of de novo synthesis by organisms growing on carbon supplied by the oil. Implications of these results for understanding the fate and managing the risk of hydrocarbons in the subsurface are discussed.

Human and Aquatic Toxicity Potential of Petroleum Biodegradation Metabolite Mixtures in Groundwater from Fuel Release Sites.

The potential toxicity to human and aquatic receptors of petroleum fuel biodegradation metabolites (oxygen-containing organic compounds [OCOCs]) in groundwater has been investigated as part of a multi-year research program. Whole mixtures collected from locations upgradient and downgradient of multiple fuel release sites were tested using: 1) in vitro screening assays for human genotoxicity (the gamma-H2AX assay) and estrogenic effects (estrogen receptor transcriptional activation assay), and 2) chronic aquatic toxicity tests in 3 species (Ceriodaphnia dubia, Raphidocelis subcapitata, and Pimephales promelas). In vitro screening assay results demonstrated that the mixtures did not cause genotoxic or estrogenic effects. No OCOC-related aquatic toxicity was observed and when aquatic toxicity did occur, upgradient samples typically had the same response as samples downgradient of the release, indicating that background water quality was impacting the results. This information provides additional support for previous work that focused on the individual compounds and, taken together, indicates that OCOCs from petroleum degradation at fuel release sites are unlikely to cause toxicity to human or freshwater receptors at the concentrations present. Environ Toxicol Chem 2020;39:1634-1645. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Identification of ester metabolites from petroleum hydrocarbon biodegradation in groundwater using GC×GC-TOFMS.

In an effort to understand the nature and toxicity of petroleum hydrocarbon degradation metabolites, 2-dimensional gas chromatography linked to a time-of-flight mass spectrometer (GC×GC-TOFMS) was used to conduct nontargeted analysis of the extracts of 61 groundwater samples collected from 10 fuel release sites. An unexpected result was the tentative identification of 197 unique esters. Although esters are known to be part of specific hydrocarbon degradative pathways, they are not commonly considered or evaluated in field studies of petroleum biodegradation. In addition to describing the compounds identified, the present study discusses the role for nontargeted analysis in environmental studies. Overall, the low toxicological profile of the identified esters, along with the limited potential for exposure, renders them unlikely to pose any significant health risk.