Recommendations for the advancement of oil-in-water media and source oil characterization in aquatic toxicity test studies.

During toxicity testing, chemical analyses of oil and exposure media samples are needed to allow comparison of results between different tests as well as to assist with identification of the drivers and mechanisms for the toxic effects observed. However, to maximize the ability to compare results between different laboratories and biota, it has long been recognized that guidelines for standard protocols were needed. In 2005, the Chemical Response to Oil Spills: Ecological Effects Research Forum (CROSERF) protocol was developed with existing common analytical methods that described a standard method for reproducible preparation of exposure media as well as recommended specific analytical methods and analyte lists for comparative toxicity testing. At the time, the primary purpose for the data collected was to inform oil spill response and contingency planning. Since then, with improvements in both analytical equipment and methods, the use of toxicity data has expanded to include their integration into fate and effect models that aim to extend the applicability of lab-based study results to make predictions for field system-level impacts. This paper focuses on providing a summary of current chemical analyses for characterization of oil and exposure media used during aquatic toxicity testing and makes recommendations for the minimum analyses needed to allow for interpretation and modeling purposes.

Setting the stage to advance oil toxicity testing: Overview of knowledge gaps, and recommendations.

The Chemical Response to Oil Spills: Ecological Effects Research Forum created a standardized protocol for comparing the in vivo toxicity of physically dispersed oil to chemically dispersed oil to support science-based decision making on the use of dispersants in the early 2000s. Since then, the protocol has been frequently modified to incorporate advances in technology; enable the study of unconventional and heavier oils; and provide data for use in a more diverse manner to cover the growing needs of the oil spill science community. Unfortunately, for many of these lab-based oil toxicity studies consideration was not given to the influence of modifications to the protocol on media chemistry, resulting toxicity and limitations for the use of resulting data in other contexts (e.g., risk assessments, models). To address these issues, a working group of international oil spill experts from academia, industry, government, and private organizations was convened under the Multi-Partner Research Initiative of Canada's Oceans Protection Plan to review publications using the CROSERF protocol since its inception to support their goal of coming to consensus on the key elements required within a "modernized CROSERF protocol".

Advances to the CROSERF protocol to improve oil spill response decision making.

The Chemical Response to Oil Spills: Ecological Effects Research Forum (CROSERF) created a standardized protocol for comparing the toxicity of physically dispersed oil versus chemically dispersed oil to address environmental concerns related to the proposed use of dispersants in the early 2000s. Since then, many revisions have been made to the original protocol to diversify the intended use of the data generated, incorporate emerging technologies, and to examine a wider range of oil types including non-conventional oils and fuels. Under the Multi-Partner Research Initiative (MPRI) for oil spill research under Canada's Oceans Protection Plan (OPP), a network of 45 participants from seven countries representing government, industry, non-profit, private, and academic sectors was established to identify the current state of the science and formulate a series of recommendations to modernize the oil toxicity testing framework. The participants formed a series of working groups, targeting specific aspects of oil toxicity testing, including: experimental conduct; media preparation; phototoxicity; analytical chemistry; reporting and communicating results; interpreting toxicity data; and appropriate integration of toxicity data to improve oil spill effects models. The network participants reached a consensus that a modernized protocol to assess the aquatic toxicity of oil should be sufficiently flexible to address a broad range of research questions in a 'fit-for-purpose' manner, where methods and approaches are driven by the need to generate scientifically-defensible data to address specific study objectives. Considering the many needs and varied objectives of aquatic toxicity tests currently being conducted to support and inform oil spill response decision making, it was also concluded that the development of a one size fits all approach would not be feasible.

Recommendations for advancing media preparation methods used to assess aquatic hazards of oils and spill response agents.

Laboratory preparation of aqueous test media is a critical step in developing toxicity information needed for oil spill response decision-making. Multiple methods have been used to prepare physically and chemically dispersed oils which influence test outcome, interpretation, and utility for hazard assessment and modeling. This paper aims to review media preparation strategies, highlight advantages and limitations, provide recommendations for improvement, and promote the standardization of methods to better inform assessment and modeling. A benefit of media preparation methods for oil that rely on low to moderate mixing energy coupled with a variable dilution design is that the dissolved oil composition of the water accommodation fraction (WAF) stock is consistent across diluted treatments.  Further, analyses that support exposure confirmation maybe reduced and reflect dissolved oil exposures that are bioavailable and amenable to toxicity modeling.  Variable loading tests provide a range of dissolved oil compositions that require analytical verification at each oil loading. Regardless of test design, a preliminary study is recommended to optimize WAF mixing and settling times to achieve equilibrium between oil and test media. Variable dilution tests involving chemical dispersants (CEWAF) or high energy mixing (HEWAF) can increase dissolved oil exposures in treatment dilutions due to droplet dissolution when compared to WAFs. In contrast, HEWAF/CEWAFs generated using variable oil loadings are expected to provide dissolved oil exposures more comparable to WAFs. Preparation methods that provide droplet oil exposures should be environmentally relevant and informed by oil droplet concentrations, compositions, sizes, and exposure durations characteristic of field spill scenarios. Oil droplet generators and passive dosing techniques offer advantages for delivering controlled constant or dynamic dissolved exposures and larger volumes of test media for toxicity testing. Adoption of proposed guidance for improving media preparation methods will provide greater comparability and utility of toxicity testing in oil spill response and assessment.

Transcriptome Profiling in Larval Fathead Minnow Exposed to Commercial Naphthenic Acids and Extracts from Fresh and Aged Oil Sands Process-Affected Water.

Surface mining and extraction of oil sands results in the generation of and need for storage of large volumes of oil sands process-affected water (OSPW). More structurally complex than classical naphthenic acids (NAs), naphthenic acid fraction components (NAFCs) are key toxic constituents of OSPW, and changes in the NAFC profile in OSPW over time have been linked to mitigation of OSPW toxicity. Molecular studies targeting individual genes have indicated that NAFC toxicity is likely mediated via oxidative stress, altered cell cycles, ontogenetic differentiation, endocrine disruption, and immunotoxicity. However, the individual-gene approach results in a limited picture of molecular responses. This study shows that NAFCs, from aged or fresh OSPW, have a unique effect on the larval fathead minnow transcriptome and provides initial data to construct adverse outcome pathways for skeletal deformities. All three types of processed NAs (fresh, aged, and commercial) affected the immunome of developing fish. These gene networks included immunity, inflammatory response, B-cell response, platelet adhesion, and T-helper lymphocyte activity. Larvae exposed to both NAFCs and commercial NA developed cardiovascular and bone deformities, and transcriptomic networks reflected these developmental abnormalities. Gene networks found only in NAFC-exposed fish suggest NAFCs may alter fish cardiovascular health through altered calcium ion regulation. This study improves understanding regarding the molecular perturbations underlying developmental deformities following exposure to NAFCs.

Understanding the chronic impacts of oil refinery wastewater requires consideration of sediment contributions to toxicity.

Previous studies at an oil refinery in Saint John, New Brunswick, Canada, found a diminished fish community downstream of the effluent outfall that appeared to be associated with periodic low dissolved oxygen concentrations due to episodic discharges of contaminated transport vessel ballast water. This study was initiated after the ballast water was removed from the effluent to further investigate the potential causes of residual effects in the study stream, Little River. We used field caging of fish, laboratory bioassays, and chemical analysis of effluents and sediments from the field site to determine if the effluent or contaminated sediments were affecting the recovery of the fish community in Little River. The field studies suggested that exposed, caged fish were affected, displaying >40 % increases in liver sizes and increased liver detoxification enzyme activity (cytochrome P450 1A, CYP1A); however, similar responses were absent in laboratory exposures that used effluent only. Adding sediments collected from the vicinity of the refinery's outfall to the laboratory bioassays reproduced some of the field responses. Chemical analyses showed high concentrations of PAHs in sediments but low concentrations in the effluent, suggesting that the PAHs in the sediment were contributing more to the impacts than the effluent. Application of effects-based monitoring is suggested as beneficial to identify impacts to fisheries where refinery effluents of this type are involved.