Changes in Temperature Alter the Toxicity of Polycyclic Aromatic Compounds to American Lobster (Homarus americanus) Larvae.

Polycyclic aromatic compounds (PACs) present in the water column are considered to be one of the primary contaminant groups contributing to the toxicity of a crude oil spill. Because crude oil is a complex mixture composed of thousands of different compounds, oil spill models rely on quantitative structure-activity relationships like the target lipid model to predict the effects of crude oil exposure on aquatic life. These models rely on input provided by single species toxicity studies, which remain insufficient. Although the toxicity of select PACs has been well studied, there is little data available for many, including transformation products such as oxidized hydrocarbons. In addition, the effect of environmental influencing factors such as temperature on PAC toxicity is a wide data gap. In response to these needs, in the present study, Stage I lobster larvae were exposed to six different understudied PACs (naphthalene, fluorenone, methylnaphthalene, phenanthrene, dibenzothiophene, and fluoranthene) at three different relevant temperatures (10, 15, and 20 °C) all within the biological norms for the species during summer when larval releases occur. Lobster larvae were assessed for immobilization as a sublethal effect and mortality following 3, 6, 12, 24, and 48 h of exposure. Higher temperatures increased the rate at which immobilization and mortality were observed for each of the compounds tested and also altered the predicted critical target lipid body burden, incipient median lethal concentration, and elimination rate. Our results demonstrate that temperature has an important influence on PAC toxicity for this species and provides critical data for oil spill modeling. More studies are needed so oil spill models can be appropriately calibrated and to improve their predictive ability. Environ Toxicol Chem 2023;42:2389-2399. © 2023 SETAC.

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".

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.

The effect of chemical dispersion and temperature on the metabolic and cardiac responses to physically dispersed crude oil exposure in larval American lobster (Homarus americanus).

Despite their potential vulnerability to oil spills, little is known about the physiological effects of petroleum exposure and spill responses in cold-water marine animal larvae. We investigated the effects of physically dispersed (water-accommodated fraction, WAF) and chemically dispersed (chemically enhanced WAF, CEWAF; using Slickgone EW) conventional heavy crude oil on the routine metabolic rate and heart rate of stage I larval American lobster (Homarus americanus). We found no effects of 24-h exposure to sublethal concentrations of crude oil WAF or CEWAF at 12 °C. We then investigated the effect of sublethal concentrations of WAFs at three environmentally relevant temperatures (9, 12, 15 °C). The highest WAF concentration increased metabolic rate at 9 °C, whereas it decreased heart rate and increased mortality at 15 °C. Overall, metabolic and cardiac function of American lobster larvae is relatively resilient to conventional heavy crude oil and Slickgone EW exposure, but responses to WAF may be temperature-dependent.

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.

Calibration of an acute toxicity model for the marine crustacean, Artemia franciscana, nauplii to support oil spill effect assessments.

Oil spill risk and impact assessments rely on time-dependent toxicity models to predict the hazard of the constituents that comprise crude oils and petroleum substances. Dissolved aromatic compounds (ACs) are recognized as a primary driver of aquatic toxicity in surface spill exposure scenarios. However, limited time-dependent toxicity data are available for different classes of ACs to calibrate such models. This study examined the acute toxicity of 14 ACs and 3 binary AC mixtures on Artemia franciscana nauplii at 25 °C. Toxicity tests for 3 ACs were also conducted at 15 °C to evaluate the role of temperature on toxicity. The ACs investigated represented parent and alkylated homocyclic and nitrogen-, sulfur- and oxygen-containing heterocyclic structures with octanol-water partition coefficients (log Kow) ranging from 3.2 to 6.6. Passive dosing was used to expose and maintain concentrations in toxicity tests which were confirmed using fluorometry, and independently validated for 6 ACs using GC-MS analysis. Mortality was assessed at 6, 24, and 48 h to characterize the time course of toxicity. No mortality was observed for the most hydrophobic AC tested, 7,12-dimethylbenz[a]anthracene, due to apparent water solubility constraints. Empirical log LC50 s for the remaining ACs were fit to a linear regression with log Kow to derive a critical target lipid body burden (CTLBB) based on the target lipid model. The calculated 48 h CTLBB of 47.1 ± 8.1 µmol/g octanol indicates that Artemia nauplii exhibited comparable sensitivity to other crustaceans. A steep concentration-response was found across all compounds as evidenced by a narrow range (1.0-3.1) in the observed LC50 /LC10 ratio. Differences in toxicokinetics were noted, and no impacts of temperature-dependence of AC toxicity were found. Toxicity data obtained for individual ACs yielded acceptable predictions of observed binary AC mixture toxicity. Results from this study advance toxicity models used in oil spill assessments.

Bridging the lab to field divide: Advancing oil spill biological effects models requires revisiting aquatic toxicity testing.

Oil fate and exposure modeling addresses the complexities of oil composition, weathering, partitioning in the environment, and the distributions and behaviors of aquatic biota to estimate exposure histories, i.e., oil component concentrations and environmental conditions experienced over time. Several approaches with increasing levels of complexity (i.e., aquatic toxicity model tiers, corresponding to varying purposes and applications) have been and continue to be developed to predict adverse effects resulting from these exposures. At Tiers 1 and 2, toxicity-based screening thresholds for assumed representative oil component compositions are used to inform spill response and risk evaluations, requiring limited toxicity data, analytical oil characterizations, and computer resources. Concentration-response relationships are employed in Tier 3 to quantify effects of assumed oil component mixture compositions. Oil spill modeling capabilities presently allow predictions of spatial and temporal compositional changes during exposure, which support mixture-based modeling frameworks. Such approaches rely on summed effects of components using toxic units to enable more realistic analyses (Tier 4). This review provides guidance for toxicological studies to inform the development of, provide input to, and validate Tier 4 aquatic toxicity models for assessing oil spill effects on aquatic biota. Evaluation of organisms' exposure histories using a toxic unit model reflects the current state-of the-science and provides an improved approach for quantifying effects of oil constituents on aquatic organisms. Since the mixture compositions in toxicity tests are not representative of field exposures, modelers rely on studies using single compounds to build toxicity models accounting for the additive effects of dynamic mixture exposures that occur after spills. Single compound toxicity data are needed to quantify the influence of exposure duration and modifying environmental factors (e.g., temperature, light) on observed effects for advancing use of this framework. Well-characterized whole oil bioassay data should be used to validate and refine these models.

Adopting a toxic unit model paradigm in design, analysis and interpretation of oil toxicity testing.

The lack of a conceptual understanding and unifying quantitative framework to guide conduct and interpretation of laboratory oil toxicity tests, has led investigators to divergent conclusions that can confuse stakeholders and impede sound decision-making. While a plethora of oil toxicity studies are available and continue to be published, due to differences in experimental design, results between studies often cannot be compared. Furthermore, much resulting data fails to advance quantitative effect models that are critically needed for oil spill risk and impact assessments. This paper discusses the challenges posed when evaluating oil toxicity test data based on traditional, total concentration-based exposure metrics and offers solutions for improving the state of practice by adopting a unifying toxic unit (TU) model framework. Key advantages of a TU framework is that differences in test oil composition, sensitivity of the test organism/endpoint, and toxicity test design (i.e., type of test) can be taken into quantitative account in predicting aquatic toxicity. This paradigm shift is intended to bridge the utility of laboratory oil toxicity tests with improved assessment of effects in the field. To illustrate these advantages, results from literature studies are reassessed and contrasted with conclusions obtained based on past practice. Using instructive examples, model results are presented to explain how dissolved oil composition and concentrations and resulting TUs vary in WAFs prepared using variable loading or dilution test designs and the important role that unmeasured oil components contribute to predicted oil toxicity. Model results are used to highlight how the TU framework can serve as a valuable aid in designing and interpreting empirical toxicity tests and provide the data required to validate/refine predictive toxicity models. To further promote consistent exposure and hazard assessment of physically and chemically dispersed oil toxicity tests recommendations for advancing the TU framework are presented.

Intraspecific Variation in the Sublethal Effects of Physically and Chemically Dispersed Crude Oil on Early Life Stages of Atlantic Cod (Gadus morhua).

The offshore oil industry in Atlantic Canada necessitates a greater understanding of the potential impacts of oil exposure and spill response measures on cold-water marine species. We used a standardized scoring index to characterize sublethal developmental impacts of physically and chemically dispersed crude oil in early life stages of Atlantic cod (Gadus morhua) and assessed intraspecific variation in the response among cod families. Cod (origin: Scotian Shelf, Canada) were laboratory-crossed to produce embryos from five specific families, which were subsequently exposed prehatch to gradient dilutions of a water-accommodated fraction (WAF) and a chemically enhanced WAF (CEWAF; prepared with Corexit 9500A) for 24 h. Postexposure, live embryos were transferred into filtered seawater and monitored to hatch; then, all live fish had sublethal endpoints assessed using the blue-sac disease (BSD) severity index. In both WAF and CEWAF groups, increasing exposure concentrations (measured as total petroleum hydrocarbons) resulted in an increased incidence of BSD symptoms (impaired swimming ability, increased degree of spinal curvature, yolk-sac edemas) in cod across all families. This positive concentration-dependent increase in BSD was similar between physically (WAF) versus chemically (CEWAF) dispersed oil exposures, indicating that dispersant addition does not exacerbate the effect of crude oil on BSD incidence in cod. Sensitivity varied between families, with some families having less BSD than others with increasing exposure concentrations. To our knowledge, our study is the first to demonstrate the occurrence in fishes of intraspecific variation among families in sublethal responses to oil and dispersant exposure. Our results suggest that sublethal effects of crude oil exposure will not be uniformly observed across cod populations and that sensitivity depends on genetic background. Environ Toxicol Chem 2022;41:1967-1976. © 2022 SETAC.

Newly Hatched Stage I American Lobster (Homarus americanus) Survival Following Exposure to Physically and Chemically Dispersed Crude Oil.

Standard model species are commonly used in toxicity tests due to their biological and technical advantages but studying native species increases the specificity and relevance of results generated for the potential risk assessment to an ecosystem. Accounting for intraspecies variability and other factors, such as chemical and physical characterization of test medium, is necessary to develop a reproducible bioassay for toxicity testing with native species. In this study, larval stage I American lobster (Homarus americanus), a commercially important and native species of Atlantic Canada, was used as the test species. Toxicity tests were first conducted by exposing lobster larvae to a reference toxicant of copper sulphate (CuSO4) and then to physically and chemically (using Corexit 9500A) dispersed oil (WAF and CEWAF, respectively). The effect on larval survival was estimated by calculating the 24-h median effect concentration (24-h EC50), and there was no difference between WAF or CEWAF exposure when the results are reported on a total petroleum hydrocarbon (TPH) basis. The 24-h EC50s ranged from 2.54 to 9.73 mg TPH/L when all trials (n = 19) are considered together. The HC5 (hazardous concentration for 5 per cent of the population) value was 2.52 mg TPH/L and similar to the EC50 value when all trials were pooled. To evaluate the reproducibility of the lobster toxicity tests, inter-trial variability was determined, and the resultant coefficients of variation (%CV) were compared to those reported for two standard test species, mysid shrimp (Americamysis bahia) and inland silverside (Menidia beryillina). This comparison showed that the %CV for the lobster toxicity tests were lower than those for the standard species tests indicating that the described larval lobster toxicity bioassay produces reliable and repeatable results.

Assessing the Toxicity of Individual Aromatic Compounds and Mixtures to American Lobster (Homarus americanus) Larvae Using a Passive Dosing System.

Aquatic exposures to aromatic compounds (ACs) may be important contributors to biological effects of oil spills. The present study examined the acute toxicity of 11 ACs and 3 binary AC mixtures on stage 1 American lobster larvae using a passive dosing test design. The ACs investigated covered a range of classes and log octanol-water partition coefficient values (KOW ; 2.5-5.5). Silicone O-rings were used to partition ACs into seawater and maintain stable exposures. Exposed lobster larvae were assessed for mobility and survival at 3, 6, 12, 24, 36, and 48 h. Fluorometry and gas chromatography-mass spectrometry measurements confirmed well-defined substance exposures. Expressing lethality in terms of chemical activities yielded values between 0.01 and 0.1, consistent with a baseline mode of action. Analysis of time-dependent median lethal/effect concentration (L/EC50) values were used to determine incipient values. An expected linear relationship between the incipient log L/EC50 and log KOW was fit to the empirical toxicity data to derive critical target lipid body burdens for immobilization and lethality endpoints. These values indicate that American lobster larvae fall on the sensitive end of the acute species sensitivity distribution. We used AC toxicity data to successfully predict toxicity of binary mixtures assuming additive toxicity. The observed time-dependent toxicity was inversely related to log KOW and occurred more quickly than reported previously. The results contribute to improving models for predicting oil spill impacts on American lobster larvae populations. Environ Toxicol Chem 2021;40:1379-1388. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.