Sensitivity of the Indo-Pacific coral Acropora millepora to aromatic hydrocarbons.

The risks posed by petroleum spills to coral reefs are poorly understood and quantifying acute toxicity thresholds for aromatic hydrocarbons to reef-building corals is required to assess their sensitivity relative to other taxa. In this study, we exposed Acropora millepora to toluene, naphthalene and 1-methylnaphthalene (1-MN) in a flow-through system and assessed survivorship and sublethal responses including growth, colour and the photosynthetic performance of symbionts. Median 50% lethal concentrations (LC50s) decreased over the 7-d exposure period, reaching asymptotic values of 22,921, 5,268, 1167 µg L-1 for toluene, naphthalene and 1-MN, respectively. Corresponding toxicokinetic parameters (εLC50) defining the time progression of toxicity were 0.830, 0.692, and 0.256 d-1, respectively. Latent effects after an additional 7-d recovery in uncontaminated seawater were not observed. Effect concentrations (EC50s) for 50% growth inhibition were 1.9- to 3.6-fold lower than the LC50s for each aromatic hydrocarbon. There were no observed effects of aromatic hydrocarbon exposure on colour score (a proxy for bleaching) or photosynthetic efficiency. Acute and chronic critical target lipid body burdens (CTLBBs) of 70.3 ± 16.3 and 13.6 ± 18.4 µmol g-1 octanol (± standard error) were calculated for survival and growth inhibition based on 7-d LC50 and EC10 values, respectively. These species-specific constants indicate adult A. millepora is more sensitive than other corals reported so far but is of average sensitivity in comparison with other aquatic taxa in the target lipid model database. These results advance our understanding of acute hazards of petroleum contaminants to key habitat-building tropical coral reef species.

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.

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.

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.

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.

Temporal chemical composition changes in water below a crude oil slick irradiated with natural sunlight.

Photooxidation can alter the environmental fate and effects of spilled oil. To better understand this process, oil slicks were generated on seawater mesocosms and exposed to sunlight for 8 days. The molecular composition of seawater under irradiated and non-irradiated oil slicks was characterized using ion mobility spectrometry-mass spectrometry and polyaromatic hydrocarbons analyses. Biomimetic extraction was performed to quantify neutral and ionized constituents. Results show that seawater underneath irradiated oil showed significantly higher amounts of hydrocarbons with oxygen- and sulfur-containing by-products peaking by day 4-6; however, concentrations of dissolved organic carbon were similar. Biomimetic extraction indicated toxic units in irradiated mesocosms increased, mainly due to ionized components, but remained <1, suggesting limited potential for ecotoxicity. Because the experimental design mimicked important aspects of natural conditions (freshly collected seawater, natural sunlight, and relevant oil thickness and concentrations), this study improves our understanding of the effects of photooxidation during a marine oil spill.

Oil Irradiation Experiments Document Changes in Oil Properties, Molecular Composition, and Dispersant Effectiveness Associated with Oil Photo-Oxidation.

While chemical dispersants are a powerful tool for treating spilled oil, their effectiveness can be limited by oil weathering processes such as evaporation and emulsification. It has been suggested that oil photo-oxidation could exacerbate these challenges. To address the role of oil photo-oxidation in dispersant effectiveness, outdoor mesocosm experiments with crude oil on seawater were performed. Changes in bulk oil properties and molecular composition were quantified to characterize oil photo-oxidation over 11 days. To test relative dispersant effectiveness, oil residues were evaluated using the Baffled Flask Test. The results show that oil irradiation led to oxygen incorporation, formation of oxygenated hydrocarbons, and higher oil viscosities. Oil irradiation was associated with decreased dispersant efficacy, with effectiveness falling from 80 to <50% in the Baffled Flask Test after more than 3 days of irradiation. Increasing photo-oxidation-induced viscosity seems to drive the decreasing dispersant effectiveness. Comparing the Baffled Flask Test results with field data from the Deepwater Horizon oil spill showed that laboratory dispersant tests underestimate the dispersion of photo-oxidized oil in the field. Overall, the results suggest that prompt dispersant application (within 2-4 days), as recommended by current oil spill response guidelines, is necessary for effective dispersion of spilled oil.

Toxicity of two representative petroleum hydrocarbons, toluene and phenanthrene, to five Atlantic coral species.

Coral reefs are keystone coastal ecosystems that can be exposed to petroleum hydrocarbons from multiple sources, and when selecting spill response methods to limit environmental damages, corals represent one of the highest valued resources for protection. Because previous research to characterize the sensitivity of coral species to petroleum hydrocarbon exposures is limited, a continuous-flow passive dosing system and toxicity testing protocol was designed to evaluate the acute effects of two representative petroleum compounds, toluene and phenanthrene, on five coral species: Acropora cervicornis, Porites astreoides, Siderastera siderea, Stephanocoenia intersepta, and Solenastrea bournoni. Using analytically confirmed exposures, sublethal and lethal endpoints were calculated for each species, and used as model inputs to determine critical target lipid body burdens (CTLBBs) for characterizing species sensitivity. Further, quantification of the time-dependent toxicity of single hydrocarbon exposures is described to provide model inputs for improved simulation of spill impacts to corals in coastal tropical environments.

Miniaturised marine tests as indicators of aromatic hydrocarbon toxicity: Potential applicability to oil spill assessment.

Assessing oil spill toxicity in real time is challenging due to dynamic field exposures and lack of simple, rapid, and sensitive tests. We investigated the relative sensitivity of two commercially available marine toxicity tests to aromatic hydrocarbons using the target lipid model (TLM). State of the art passive dosing in sealed vials was used to assess the sensitivity of brine shrimp (Artemia franciscana) and rotifer (Brachionus plicatilis). Organisms were exposed to toluene, 1-methylnaphthalene and phenanthrene for 24 h. Toxicity results were analysed using the TLM to estimate the critical target lipid body burden and support comparison to empirical data for 79 other aquatic organisms. Our findings demonstrate the applicability of passive dosing to test small volumes and indicate that the two rapid cyst-based assays are insensitive in detecting hydrocarbon exposures compared to other aquatic species. Our results highlight the limitations of applying these tests for oil pollution monitoring and decision-making.

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.

Assessing toxicity of hydrophobic aliphatic and monoaromatic hydrocarbons at the solubility limit using novel dosing methods.

Reliable delineation of aquatic toxicity cut-offs for poorly soluble hydrocarbons is lacking. In this study, vapor and passive dosing methods were applied in limit tests with algae and daphnids to evaluate the presence or absence of chronic effects at exposures corresponding to the water solubility for representative hydrocarbons from five structural classes: branched alkanes, mono, di, and polynaphthenic (cyclic) alkanes and monoaromatic naphthenic hydrocarbons (MANHs). Algal growth rate and daphnid immobilization, growth and reproduction served as the chronic endpoints investigated. Results indicated that the dosing methods applied were effective for maintaining mean measured exposure concentrations within a factor of two or higher of the measured water solubility of the substances investigated. Chronic effects were not observed for hydrocarbons with an aqueous solubility below approximately 5 µg/L. This solubility cut-off corresponds to structures consisting of 13-14 carbons for branched and cyclic alkanes and 16-18 carbons for MANHs. These data support reliable hazard and risk evaluation of hydrocarbon classes that comprise petroleum substances and the methods described have broad applicability for establishing empirical solubility cut-offs for other classes of hydrophobic substances. Future work is needed to understand the role of biotransformation on the observed presence or absence of toxicity in chronic tests.

The sensitivity of the deepsea species northern shrimp (Pandalus borealis) and the cold-water coral (Lophelia pertusa) to oil-associated aromatic compounds, dispersant, and Alaskan North Slope crude oil.

This study investigated the sensitivity of two deepsea species using mortality of northern shrimp (Pandalus borealis) and polyp activity of stony coral (Lophelia pertusa) to dispersant, Corexit 9500 and aromatic hydrocarbons (toluene, 2-methylnaphthalene, phenanthrene) in 96-h tests. Resulting hydrocarbon toxicity data were fit to the Target Lipid Model to generate predictive models and determine species sensitivity. Toxicity of chemically enhanced water accommodated fractions of Alaskan North Slope crude oil (ANS-oil) was also investigated with shrimp using nominal loading, total petroleum hydrocarbons and biomimetic extraction (BE) as oil exposure metrics. Coral were more sensitive to dispersant than shrimp while similar sensitivity was observed for hydrocarbons. Study and literature findings indicate deepsea species exhibit acute sensitivities to dispersant, hydrocarbons and oil that are comparable to pelagic species. Results support use of passive sampling methods to quantify dissolved oil for interpreting oil toxicity tests and suggest models for predicting time-dependence of toxicity warrant re-evaluation.

The treatment of biodegradation in models of sub-surface oil spills: A review and sensitivity study.

Biodegradation is important for the fate of oil spilled in marine environments, yet parameterization of biodegradation varies across oil spill models, which usually apply constant first-order decay rates to multiple pseudo-components describing an oil. To understand the influence of model parameterization on the fate of subsurface oil droplets, we reviewed existing algorithms and rates and conducted a model sensitivity study. Droplets were simulated from a blowout at 2000 m depth and were either treated with sub-surface dispersant injection (2% dispersant to oil ratio) or untreated. The most important factor affecting oil fate was the size of the droplets, with biodegradation contributing substantially to the fate of droplets ≤0.5 mm. Oil types, which were similar, had limited influence on simulated oil fate. Model results suggest that knowledge of droplet sizes and improved estimation of pseudo-component biodegradation rates and lag times would enhance prediction of the fate and transport of subsurface oil.

Modeling the toxicity of dissolved crude oil exposures to characterize the sensitivity of cod (Gadus morhua) larvae and role of individual and unresolved hydrocarbons.

Toxicity of weathered oil was investigated using Atlantic cod (Gadus morhua) larvae. A novel exposure system was applied to differentiate effects associated with dissolved and droplet oil with and without dispersant. After a 4-day exposure and subsequent 4-day recovery period, survival and growth were determined. Analytical data characterizing test oil composition included polyaromatic hydrocarbons (PAH) based on GC/MS and unresolved hydrocarbon classes obtained by two-dimensional chromatography coupled with flame ionization detection was used as input to an oil solubility model to calculate toxic units (TUs) of dissolved PAHs and whole oil, respectively. Critical target lipid body burdens derived from modeling characterizing the sensitivity of effect endpoints investigated were consistent across treatments and within the range previously reported for pelagic species. Individually measured PAHs captured only 3-11% of the TUs associated with the whole oil highlighting the limitations of traditional total PAH exposure metrics for expressing oil toxicity data.

Passive dosing yields dissolved aqueous exposures of crude oil comparable to the CROSERF (Chemical Response to Oil Spill: Ecological Effects Research Forum) water accommodated fraction method.

The Chemical Response to Oil Spill: Ecological Effects Research Forum's water accommodated fraction procedure was compared with 2 alternative techniques in which crude oil was passively dosed from silicone tubing or O-rings. Fresh Macondo oil (MC252) was dosed at 30 mg/L using each approach to investigate oil dissolution kinetics, which was monitored by fluorometry as estimated oil equivalents (EOEs). Subsequent experiments with each dosing method were then conducted at multiple oil loadings. Following equilibration, test media were analytically characterized for polyaromatic hydrocarbons (PAHs) using gas chromatography (GC)-mass spectrometry and dissolved oil using biomimetic solid-phase microextraction (SPME). The results showed that equilibrium was achieved within 72 h for all methods. Measured PAH concentrations were compared with oil solubility model predictions of dissolved exposures. The concentration and composition of measured and predicted dissolved PAHs varied with oil loading and were consistent between dosing methods. Two-dimensional GC compositional data for this oil were then used to calculate dissolved toxic units for predicting MC252 oil acute toxicity across the expected range of species sensitivities. Predicted toxic units were nonlinear with loading and correlated to both EOE and biomimetic SPME. Passive dosing methods provide a practical strategy to deliver and maintain dissolved oil concentrations while avoiding the complicating role that droplets can introduce in exposure characterization and test interpretation. Environ Toxicol Chem 2018;37:2810-2819. © 2018 SETAC.

The sensitivity of a deep-sea fish species (Anoplopoma fimbria) to oil-associated aromatic compounds, dispersant, and Alaskan North Slope crude oil.

A predominant concern following oil spills is toxicity to aquatic organisms. However, few data are available on effects in deep-sea cold water fishes. The present study had 3 major objectives. The first was to investigate the relative sensitivity of the deep-sea species Anoplopoma fimbria (sablefish) to acute effects of 3 aromatic compounds (toluene, 2-methylnaphthalene, and phenanthrene), dispersant alone, and chemically enhanced water accommodated fractions (CEWAFs) of Alaskan North Slope crude oil. The second was to determine the critical target lipid body burden (CTLBB) for sablefish by fitting aromatic hydrocarbon toxicity data to the target lipid model (TLM), which then allowed expression of CEWAF exposures in terms of dissolved oil toxic units. The final aim was to apply a passive sampling method that targets bioavailable, dissolved hydrocarbons as an alternative analytical technique for improved CEWAF exposure assessment. The results indicate that sablefish exhibit sensitivity to Corexit 9500 (96-h median lethal concentration [LC50] = 72.2 mg/L) within the range reported for other fish species. However, the acute CTLBB of 39.4 ± 2.1 µmol/goctanol lies at the lower end of the sensitivity range established for aquatic species. The utility of both toxic units and passive sampling measurements for describing observed toxicity of dispersed oil is discussed. The present study is novel in that a new test species is investigated to address the uncertainty regarding the sensitivity of deep-sea fishes, while also employing modeling and measurements to improve exposure characterization in oil toxicity tests. Environ Toxicol Chem 2018;37:2210-2221. © 2018 SETAC.

A re-evaluation of PETROTOX for predicting acute and chronic toxicity of petroleum substances.

The PETROTOX model was developed to perform aquatic hazard assessment of petroleum substances based on substance composition. The model relies on the hydrocarbon block method, which is widely used for conducting petroleum substance risk assessments providing further justification for evaluating model performance. Previous work described this model and provided a preliminary calibration and validation using acute toxicity data for limited petroleum substance. The objective of the present study was to re-evaluate PETROTOX using expanded data covering both acute and chronic toxicity endpoints on invertebrates, algae, and fish for a wider range of petroleum substances. The results indicated that recalibration of 2 model parameters was required, namely, the algal critical target lipid body burden and the log octanol-water partition coefficient (KOW ) limit, used to account for reduced bioavailability of hydrophobic constituents. Acute predictions from the updated model were compared with observed toxicity data and found to generally be within a factor of 3 for algae and invertebrates but overestimated fish toxicity. Chronic predictions were generally within a factor of 5 of empirical data. Furthermore, PETROTOX predicted acute and chronic hazard classifications that were consistent or conservative in 93 and 84% of comparisons, respectively. The PETROTOX model is considered suitable for the purpose of characterizing petroleum substance hazard in substance classification and risk assessments. Environ Toxicol Chem 2017;36:2245-2252. © 2017 SETAC.

Investigating the role of dissolved and droplet oil in aquatic toxicity using dispersed and passive dosing systems.

Characterization of the aquatic toxicity of oil is needed to support hazard assessment and inform spill response. Natural processes and mitigation strategies involving dispersant use can result in exposures to both dissolved and droplet oil that are not typically differentiated when oil exposures are characterized in toxicity tests. Thus, the impact of droplets on aquatic toxicity is largely uncharacterized. To improve the understanding of the role of droplets, acute toxicity tests with Daphnia magna and Americamysis bahia were performed with Endicott crude oil in low-energy mixing systems with and without Corexit 9500 dispersant. Exposures were also prepared by placing crude oil in silicone tubing and passively dosing test media to provide dissolved oil exposures without droplets. A framework is described for characterizing dissolved phase exposures using both mechanistic modeling and passive sampling measurements. The approach is then illustrated by application to data from the present study. Expression of toxicity in terms of toxic units calculated from modeled dissolved oil concentrations or passive sampling measurements showed similar dose responses between exposure systems and organisms, despite the gradient in droplet oil. These results indicate that droplets do not appreciably contribute to toxicity for the 2 species investigated and further support hazard evaluation of dispersed oil on the basis of dissolved exposure metrics. Environ Toxicol Chem 2017;36:1020-1028. © 2016 SETAC.

Guidance for improving comparability and relevance of oil toxicity tests.

The complex nature and limited aqueous solubility of petroleum substances pose challenges for consistently characterizing exposures in aquatic life hazard assessments. This paper reviews important considerations for the design, conduct and interpretation of laboratory toxicity tests with physically and chemically dispersed oils based on an understanding of the behavior and toxicity of the hydrocarbons that comprise these substances. Guiding principles are provided that emphasize the critical need to understand and, when possible, characterize dissolved hydrocarbon exposures that dictate observed toxicity in these tests. These principles provide a consistent framework for interpreting toxicity studies performed using different substances and test methods by allowing varying dissolved exposures to be expressed in terms of a common metric based on toxic units (TUs). The use of passive sampling methods is also advocated since such analyses provide an analytical surrogate for TUs. The proposed guidance is translated into a series of questions that can be used in evaluating existing data and in guiding design of future studies. Application of these questions to a number of recent publications indicates such considerations are often ignored, thus perpetuating the difficulty of interpreting and comparing results between studies and limiting data use in objective hazard assessment. Greater attention to these principles will increase the comparability and utility of oil toxicity data in decision-making.