Comprehensive Two-Dimensional Gas Chromatography with Peak Tracking for Screening of Constituent Biodegradation in Petroleum UVCB Substances.

Petroleum substances, as archetypical UVCBs (substances of unknown or variable composition, complex reaction products, or biological substances), pose a challenge for chemical risk assessment as they contain hundreds to thousands of individual constituents. It is particularly challenging to determine the biodegradability of petroleum substances since each constituent behaves differently. Testing the whole substance provides an average biodegradation, but it would be effectively impossible to obtain all constituents and test them individually. To overcome this challenge, comprehensive two-dimensional gas chromatography (GC × GC) in combination with advanced data-handling algorithms was applied to track and calculate degradation half-times (DT50s) of individual constituents in two dispersed middle distillate gas oils in seawater. By tracking >1000 peaks (representing ∼53-54% of the total mass across the entire chromatographic area), known biodegradation patterns of oil constituents were confirmed and extended to include many hundreds not currently investigated by traditional one-dimensional GC methods. Approximately 95% of the total tracked peak mass biodegraded after 64 days. By tracking the microbial community evolution, a correlation between the presence of functional microbial communities and the observed progression of DT50s between chemical classes was demonstrated. This approach could be used to screen the persistence of GC × GC-amenable constituents of petroleum substance UVCBs.

Mixture Effects on Biodegradation Kinetics of Hydrocarbons in Surface Water: Increasing Concentrations Inhibited Degradation whereas Multiple Substrates Did Not.

Most biodegradation tests are conducted using single chemicals at high concentrations, although these chemicals are present in the environment as mixtures at low concentrations. A partitioning-based platform was recently developed for biodegradation testing of composed mixtures of hydrophobic chemicals at ng/L to µg/L concentrations. We used this platform to study the concentration and mixture effect on biodegradation kinetics. Biodegradation tests were conducted in 20 mL vials using environmental water samples as inocula. Passive dosing was applied (1) to vary initial test concentrations of individual test compounds and (2) to vary the number of mixture components between 1 and 16. Automated solid-phase microextraction coupled to gas chromatography-mass spectrometry was used to measure substrate depletion relative to abiotic controls. The number of mixture components had no or only a limited effect on the biodegradation half times for three compounds when tested at environmentally relevant concentrations. In contrast, longer lag phases and half lives were observed for single compounds when tested at higher concentrations that approached aqueous solubility. The obtained results support that simultaneous testing of multiple chemicals at low concentrations can accelerate the generation of biodegradation kinetic data, which are more environmentally relevant compared with data from tests conducted with single chemicals at much higher concentrations.

The Rate of Crude Oil Biodegradation in the Sea.

Various groups have studied the rate of oil biodegradation in the sea over many years, but with no consensus on results. This can be attributed to many factors, but we show here that the principal confounding influence is the concentration of oil used in different experiments. Because of dilution, measured concentrations of dispersed oil in the sea are sub-parts-per-million within a day of dispersal, and at such concentrations the rate of biodegradation of detectable oil hydrocarbons has an apparent half-life of 7-14 days. This can be contrasted with the rate of degradation at the higher concentrations found in oil slicks or when stranded on a shoreline; there the apparent half-life varies from many months to many years.

Assessing Aromatic-Hydrocarbon Toxicity to Fish Early Life Stages Using Passive-Dosing Methods and Target-Lipid and Chemical-Activity Models.

Aromatic hydrocarbons (AH) are known to impair fish early life stages (ELS). However, poorly defined exposures often confound ELS-test interpretation. Passive dosing (PD) overcomes these challenges by delivering consistent, controlled exposures. The objectives of this study were to apply PD to obtain 5 d acute embryo lethality and developmental data and 30 d chronic embryo-larval survival and growth-effects data using zebrafish with different AHs; to analyze study and literature toxicity data using target-lipid (TLM) and chemical-activity (CA) models; and to extend PD to a mixture and test the assumption of AH additivity. PD maintained targeted exposures over a concentration range of 6 orders of magnitude. AH toxicity increased with log Kow up to pyrene (5.2). Pericardial edema was the most sensitive sublethal effect that often preceded embryo mortality, although some AHs did not produce developmental effects at concentrations causing mortality. Cumulative embryo-larval mortality was more sensitive than larval growth, with acute-to-chronic ratios of <10. More-hydrophobic AHs did not exhibit toxicity at aqueous saturation. The relationship and utility of the TLM-CA models for characterizing fish ELS toxicity is discussed. Application of these models indicated that concentration addition provided a conservative basis for predicting ELS effects for the mixture investigated.

Direct and indirect photodegradation in aquatic systems mitigates photosensitized toxicity in screening-level substance risk assessments of selected petrochemical structures.

Photochemical processes are typically not incorporated in screening-level substance risk assessments due to the complexity of modeling sunlight co-exposures and resulting interactions on environmental fate and effects. However, for many substances, sunlight exerts a profound influence on environmental degradation rates and ecotoxicities. Recent modeling advances provide an improved technical basis for estimating the effect of sunlight in modulating both substance exposure and toxicity in the aquatic environment. Screening model simulations were performed for 25 petrochemical structures with varied uses and environmental fate properties. Model predictions were evaluated by comparing the ratios of predicted exposure concentrations with and without light to the corresponding ratios of toxicity thresholds under the same conditions. The relative ratios of exposure and hazard in light vs. dark were then used to evaluate how inclusion of light modulates substance risk analysis. Results indicated that inclusion of light reduced PECs by factors ranging from 1.1- to 63-fold as a result of photodegradation, while reducing PNECs by factors ranging from 1- to 49-fold due to photoenhanced toxicity caused by photosensitization. Consequently, the presence of light altered risk quotients by factors that ranged from 0.1- to 17-fold, since the predicted increase in substance hazard was mitigated by the reduction in exposure. For many structures, indirect photodegradation decreases environmental exposures independently of the direct photolysis pathway which is associated with enhanced phototoxicity. For most of the scenarios and chemicals in the present work, photosensitization appears to be mitigated by direct and indirect degradation from sunlight exposure.

A Light Touch: Solar Photocatalysis Detoxifies Oil Sands Process-Affected Waters Prior to Significant Treatment of Naphthenic Acids.

Environmental reclamation of Canada's oil sands tailings ponds is among the single largest water treatment challenges globally. The toxicity of oil sands process-affected water (OSPW) has been associated with its dissolved organics, a complex mixture of naphthenic acid fraction components (NAFCs). Here, we evaluated solar treatment with buoyant photocatalysts (BPCs) as a passive advanced oxidation process (P-AOP) for OSPW remediation. Photocatalysis fully degraded naphthenic acids (NAs) and acid extractable organics (AEO) in 3 different OSPW samples. However, classical NAs and AEO, traditionally considered among the principal toxicants in OSPW, were not correlated with OSPW toxicity herein. Instead, nontarget petroleomic analysis revealed that low-polarity organosulfur compounds, composing <10% of the total AEO, apparently accounted for the majority of waters' toxicity to fish, as described by a model of tissue partitioning. These findings have implications for OSPW release, for which a less extensive but more selective treatment may be required than previously expected.

Recommendations for advancing test protocols examining the photo-induced toxicity of petroleum and polycyclic aromatic compounds.

Photo-induced toxicity of petroleum products and polycyclic aromatic compounds (PACs) is the enhanced toxicity caused by their interaction with ultraviolet radiation and occurs by two distinct mechanisms: photosensitization and photomodification. Laboratory approaches for designing, conducting, and reporting of photo-induced toxicity studies are reviewed and recommended to enhance the original Chemical Response to Oil Spills: Ecological Research Forum (CROSERF) protocols which did not address photo-induced toxicity. Guidance is provided on conducting photo-induced toxicity tests, including test species, endpoints, experimental design and dosing, light sources, irradiance measurement, chemical characterization, and data reporting. Because of distinct mechanisms, aspects of photosensitization (change in compound energy state) and photomodification (change in compound structure) are addressed separately, and practical applications in laboratory and field studies and advances in predictive modeling are discussed. One goal for developing standardized testing protocols is to support lab-to-field extrapolations, which in the case of petroleum substances often requires a modeling framework to account for differential physicochemical properties of the constituents. Recommendations are provided to promote greater standardization of laboratory studies on photo-induced toxicity, thus facilitating comparisons across studies and generating data needed to improve models used in oil spill science.

Methods for assessing the bioaccumulation of hydrocarbons and related substances in terrestrial organisms: A critical review.

This study investigates and reviews methods for the assessment of the terrestrial bioaccumulation potential of hydrocarbons and related organic substances. The study concludes that the unitless biomagnification factor (BMF) and/or the trophic magnification factor (TMF) are appropriate, practical, and thermodynamically meaningful metrics for identifying bioaccumulative substances in terrestrial food chains. The study shows that various methods, including physical-chemical properties like the KOA and KOW , in vitro biotransformation assays, quantitative structure-activity relationships, in vivo pharmacokinetic and dietary bioaccumulation tests, and field-based trophic magnification studies, can inform on whether a substance has the potential to biomagnify in a terrestrial food chain as defined by a unitless BMF exceeding 1. The study further illustrates how these methods can be arranged in a four-tier evaluation scheme for the purpose of screening assessments that aim to minimize effort and costs and expediate bioaccumulation assessment of the vast numbers of organic substances in commerce, identifies knowledge gaps, and provides recommendations for further research to improve bioaccumulation assessment. Integr Environ Assess Manag 2023;19:1433-1456. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

A Review of Mechanistic Models for Predicting Adverse Effects in Sediment Toxicity Testing.

Since recognizing the importance of bioavailability for understanding the toxicity of chemicals in sediments, mechanistic modeling has advanced over the last 40 years by building better tools for estimating exposure and making predictions of probable adverse effects. Our review provides an up-to-date survey of the status of mechanistic modeling in contaminated sediment toxicity assessments. Relative to exposure, advances have been most substantial for non-ionic organic contaminants (NOCs) and divalent cationic metals, with several equilibrium partitioning-based (Eq-P) models having been developed. This has included the use of Abraham equations to estimate partition coefficients for environmental media. As a result of the complexity of their partitioning behavior, progress has been less substantial for ionic/polar organic contaminants. When the EqP-based estimates of exposure and bioavailability are combined with water-only effects measurements, predictions of sediment toxicity can be successfully made for NOCs and selected metals. Both species sensitivity distributions and toxicokinetic and toxicodynamic models are increasingly being applied to better predict contaminated sediment toxicity. Furthermore, for some classes of contaminants, such as polycyclic aromatic hydrocarbons, adverse effects can be modeled as mixtures, making the models useful in real-world applications, where contaminants seldomly occur individually. Despite the impressive advances in the development and application of mechanistic models to predict sediment toxicity, several critical research needs remain to be addressed. These needs and others represent the next frontier in the continuing development and application of mechanistic models for informing environmental scientists, managers, and decisions makers of the risks associated with contaminated sediments. Environ Toxicol Chem 2023;00:1-17. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Petroleum refinery effluent contribution to chemical mixture toxic pressure in the environment.

Petroleum refinery effluents (PRE) are wastewaters from industries associated with oil refining. Within Europe, PREs are regulated through local discharge permits and receive substantial treatment before emission. After treatment, PREs can still contain low levels of various pollutants potentially toxic to organisms. Earlier work, including whole-effluent toxicity assessments, has shown that the toxicity of permitted PREs is often limited. However, the extent to which PREs contribute to chemical pollution already present in the receiving environment is unknown. Therefore, our study aimed to assess the contribution of PREs to mixture toxic pressure in the environment, using the multi-substance potentially affected fraction of species (msPAF) as an indicator. Based on measured chemical concentrations, compiled species sensitivity distributions (SSD) and a mechanistic solubility model, msPAF levels were estimated for undiluted effluents at discharge points and diluted effluents downstream in receiving waters. Median msPAF-chronic and msPAF-acute levels of PREs at discharge points were 74% (P50) and 40% (P95), respectively. The calculated msPAF levels were reduced substantially to <5% downstream for most effluents (82%), indicating low to negligible toxicity of PREs in receiving environments beyond the initial mixing zone. Regardless of differences in endpoints and locations, hydrocarbons (mainly total petroleum hydrocarbons) and inorganics (mainly ammonia) explained at least 85% of the mixture toxic pressure. The msPAF levels of PREs were on average 2.5-4.5 orders of magnitude lower than msPAF levels derived from background pollution levels, suggesting that PREs were minor contributors to the toxic pressure in the environment. This study presents a generic methodology for quantifying the potential toxic pressure of PREs in the environment, identifying hotspots where more effective wastewater treatment could be needed. We explicitly discuss the uncertainties for further refinement and development of the method.

Predicting Primary Biodegradation of Petroleum Hydrocarbons in Aquatic Systems: Integrating System and Molecular Structure Parameters using a Novel Machine-Learning Framework.

Quantitative structure-property relationship (QSPR) models for predicting primary biodegradation of petroleum hydrocarbons have been previously developed. These models use experimental data generated under widely varied conditions, the effects of which are not captured adequately within model formalisms. As a result, they exhibit variable predictive performance and are unable to incorporate the role of study design and test conditions on the assessment of environmental persistence. To address these limitations, a novel machine-learning System-Integrated Model (HC-BioSIM) is presented, which integrates chemical structure and test system variability, leading to improved prediction of primary disappearance time (DT50) values for petroleum hydrocarbons in fresh and marine water. An expanded, highly curated database of 728 experimental DT50 values (181 unique hydrocarbon structures compiled from 13 primary sources) was used to develop and validate a supervised model tree machine-learning model. Using relatively few parameters (6 system and 25 structural parameters), the model demonstrated significant improvement in predictive performance (root mean square error = 0.26, R2 = 0.67) over existing QSPR models. The model also demonstrated improved accuracy of persistence (P) categorization (i.e., "Not P/P/vP"), with an accuracy of 96.8%, and false-positive and -negative categorization rates of 0.4% and 2.7%, respectively. This significant improvement in DT50 prediction, and subsequent persistence categorization, validates the need for models that integrate experimental design and environmental system parameters into biodegradation and persistence assessment. Environ Toxicol Chem 2022;41:1359-1369. © 2022 ExxonMobil Biomedical Sciences, Inc. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Modeling Time-Dependent Aquatic Toxicity of Hydrocarbons: Role of Organism Weight, Temperature, and Substance Hydrophobicity.

Oil spill exposures are highly dynamic and are not comparable to laboratory exposures used in standard toxicity tests. Toxicokinetic-toxicodynamic (TKTD) models allow translation of effects observed in the laboratory to the field. To improve TKTD model calibration, new and previously published data from 148 tests were analyzed to estimate rates characterizing the time course of toxicity for 10 fish and 42 invertebrate species across 37 hydrocarbons. A key parameter in the TKTD model is the first-order rate that incorporates passive elimination, biotransformation, and damage repair processes. The results indicated that temperature (4-26 °C), organism size (0.0001-10 g), and substance log octanol-water partition coefficient (2-6) had limited influence on this parameter, which exhibited a 5th to 95th percentile range of 0.2-2.5 day-1 (median 0.7 day-1 ). A species sensitivity distribution approach is proposed to quantify the variability of this parameter across taxa, with further studies needed for aliphatic hydrocarbons and plant species. Study findings allow existing oil spill models to be refined to improve effect predictions. Environ Toxicol Chem 2022;41:3070-3083. © 2022 ExxonMobil Biomedical Science Inc. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Moving persistence assessments into the 21st century: A role for weight-of-evidence and overall persistence.

Assessing the persistence of chemicals in the environment is a key element in existing regulatory frameworks to protect human health and ecosystems. Persistence in the environment depends on many fate processes, including abiotic and biotic transformations and physical partitioning, which depend on substances' physicochemical properties and environmental conditions. A main challenge in persistence assessment is that existing frameworks rely on simplistic and reductionist evaluation schemes that may lead substances to be falsely assessed as persistent or the other way around-to be falsely assessed as nonpersistent. Those evaluation schemes typically assess persistence against degradation half-lives determined in single-compartment simulation tests or against degradation levels measured in stringent screening tests. Most of the available test methods, however, do not apply to all types of substances, especially substances that are poorly soluble, complex in composition, highly sorptive, or volatile. In addition, the currently applied half-life criteria are derived mainly from a few legacy persistent organic pollutants, which do not represent the large diversity of substances entering the environment. Persistence assessment would undoubtedly benefit from the development of more flexible and holistic evaluation schemes including new concepts and methods. A weight-of-evidence (WoE) approach incorporating multiple influencing factors is needed to account for chemical fate and transformation in the whole environment so as to assess overall persistence. The present paper's aim is to begin to develop an integrated assessment framework that combines multimedia approaches to organize and interpret data using a clear WoE approach to allow for a more consistent, transparent, and thorough assessment of persistence. Integr Environ Assess Manag 2022;18:868-887. © 2021 ExxonMobil Biomedical Sciences, Inc. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Inter-laboratory comparison of water solubility methods applied to difficult-to-test substances.

Water solubility is perhaps the single most important physical-chemical property determining the environmental fate and effects of organic compounds. Its determination is particularly challenging for compounds with extremely low solubility, frequently referred to as "difficult-to-test" substances and having solubility's generally less than 0.1 mg/L. The existing regulatory water solubility test for these compounds is the column elution method. Its applicability, however, is limited, to non-volatile solid or crystalline hydrophobic organic compounds. There currently exists no test guideline for measuring the water solubility of very hydrophobic liquid, and potentially volatile, difficult-to-test compounds. This paper describes a "slow-stir" water solubility methodology along with results of a ring trial across five laboratories evaluating the method's performance. The slow-stir method was applied to n-hexylcyclohexane, a volatile, liquid hydrophobic hydrocarbon. In order to benchmark the inter-laboratory variability associated with the proposed slow-stir method, the five laboratories separately determined the solubility of dodecahydrotriphenylene, a hydrophobic solid compound using the existing column elution guideline. Results across the participating laboratories indicated comparable reproducibility with relative standard deviations (RSD) of 20% or less reported for each test compound - solubility method pair. The inter-laboratory RSD was 16% for n-hexylcyclohexane (mean 14 µg/L, n = 5) using the slow-stir method. For dodecahydrotriphenylene, the inter-laboratory RSD was 20% (mean 2.6 µg/L, n = 4) using the existing column elution method. This study outlines approaches that should be followed and the experimental parameters that have been deemed important for an expanded ring trial of the slow-stir water solubility method.

Application of the Target Lipid Model to Assess Toxicity of Heterocyclic Aromatic Compounds to Aquatic Organisms.

Heterocyclic aromatic compounds can be found in crude oil and coal and often co-exist in environmental samples with their homocyclic aromatic counterparts. The target lipid model (TLM) is a modeling framework that relates aquatic toxicity to the octanol-water partition coefficient (KOW ) that has been calibrated and validated for hydrocarbons. A systematic analysis of the applicability of the TLM to heterocyclic aromatic compounds has not been performed. The objective of the present study was to compile reliable toxicity data for heterocycles and determine whether observed toxicity could be successfully described by the TLM. Results indicated that the TLM could be applied to this compound class by adopting an empirically derived coefficient that accounts for partitioning between water and lipid. This coefficient was larger than previously reported for aromatic hydrocarbons, indicating that these heterocyclic compounds exhibit higher affinity to target lipid and toxicity. A mechanistic evaluation confirmed that the hydrogen bonding accepting moieties of the heteroatoms helped explain differences in partitioning behavior. Given the TLM chemical class coefficient reported in the present study, heterocyclic aromatics can now be explicitly incorporated in TLM-based risk assessments of petroleum substances, other products, or environmental media containing these compounds. Environ Toxicol Chem 2021;40:3000-3009. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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.

Characterization of raw and ozonated oil sands process water utilizing atmospheric pressure gas chromatography time-of-flight mass spectrometry combined with solid phase microextractionun.

This work describes a novel application of atmospheric pressure gas chromatography time-of-flight mass spectrometry (APGC-TOF-MS) combined with solid-phase microextraction (SPME) for the simultaneous analysis of hydrocarbons and naphthenic acids (NAs) species in raw and ozone-treated oil sands process water (OSPW). SPME method using polydimethylsiloxane (PDMS)-coated fibers was validated using gas chromatography with flame ionization detector (GC-FID) to ensure the SPME extractions were operated appropriately. The ionization pathways of the hydrocarbon species in OSPW in the APGC source were verified by analyzing a mixture of eight polyaromatic hydrocarbons which were ionized primarily via charge transfer to produce [M+] while NAs in OSPW were found to be ionized through protonation to generate [MH+] in the wet APGC source. SPME/APGC-TOF-MS analysis demonstrated a different composition profile in OSPW #1, with 74.5% of hydrocarbon species, 23.4% of O2-NAs, and 2.1% of the oxidized NA species at extraction pH 2.0 compared with that obtained by UPLC-TOF-MS analysis (36.9% of O2-NAs, 26.8% of O3-NAs, 24.9% of O4-NAs, 9.1% of O5-NAs, 2.3% of O6-NAs). Moreover, the peak areas of the total NAs and the total peak areas of NAs + hydrocarbons measured by SPME/APGC-TOF-MS correlated excellently with the total NA concentration determined by UPLC-TOF-MS (R2 = 0.90) and the concentrations of the total acid-extractable organics determined by SPME/GC-FID (R2 = 0.98), respectively. APGC-TOF-MS integrated with the SPME techniques could extend the range of target compounds and be a promising alternative to evaluate and characterize NAs and hydrocarbon in different water types.

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.

Acritical review and weight of evidence approach for assessing the bioaccumulation of phenanthrene in aquatic environments.

Bioaccumulation (B) assessment is challenging because there are various B-metrics from laboratory and field studies, multiple criteria and thresholds for classifying bioaccumulative (B), very bioaccumulative (vB), and not bioaccumulative (nB) chemicals, as well as inherent variability and uncertainty in the data. These challenges can be met using a weight of evidence (WoE) approach. The Bioaccumulation Assessment Tool (BAT) provides a transparent WoE assessment framework that follows Organisation for Economic Co-operation and Development (OECD) principles for performing a WoE analysis. The BAT guides an evaluator through the process of data collection, generation, evaluation, and integration of various lines of evidence (LoE) (i.e., B-metrics) to inform decision-making. Phenanthrene (PHE) is a naturally occurring chemical for which extensive B and toxicokinetics data are available. A B assessment for PHE using the BAT is described that includes a critical evaluation of 74 measured in vivo LoE for fish and invertebrate species from laboratory and field studies. The number of LoE are reasonably well balanced across taxa (i.e., fish and invertebrates) and the different B-metrics. Additionally, in silico and in vitro biotransformation rate estimates and corresponding model-predicted B-metrics are included as corroborating evidence. Application of the BAT provides a consistent, coherent, and scientifically defensible WoE evaluation to conclude that PHE is not bioaccumulative (nB) because the overwhelming majority of the bioconcentration, bioaccumulation, and biomagnification metrics for both fish and invertebrates are below regulatory thresholds. An analysis of the relevant data using fugacity ratios is also provided, showing that PHE does not biomagnify in aquatic food webs. The critical review identifies recommendations to increase the consistency of B assessments, such as improved standardization of B testing guidelines, data reporting requirements for invertebrate studies, and consideration of temperature and salinity effects on certain B-metrics. Integr Environ Assess Manag 2021;17:911-925. © 2021 Concawe. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).