Improving the design and conduct of aquatic toxicity studies with oils based on 20 years of CROSERF experience.

Laboratory toxicity testing is a key tool used in oil spill science, spill effects assessment, and mitigation strategy decisions to minimize environmental impacts. A major consideration in oil toxicity testing is how to replicate real-world spill conditions, oil types, weathering states, receptor organisms, and modifying environmental factors under laboratory conditions. Oils and petroleum-derived products are comprised of thousands of compounds with different physicochemical and toxicological properties, and this leads to challenges in conducting and interpreting oil toxicity studies. Experimental methods used to mix oils with aqueous test media have been shown to influence the aqueous-phase hydrocarbon composition and concentrations, hydrocarbon phase distribution (i.e., dissolved phase versus in oil droplets), and the stability of oil:water solutions which, in turn, influence the bioavailability and toxicity of the oil containing media. Studies have shown that differences in experimental methods can lead to divergent test results. Therefore, it is imperative to standardize the methods used to prepare oil:water solutions in order to improve the realism and comparability of laboratory tests. The CROSERF methodology, originally published in 2005, was developed as a standardized method to prepare oil:water solutions for testing and evaluating dispersants and dispersed oil. However, it was found equally applicable for use in testing oil-derived petroleum substances. The goals of the current effort were to: (1) build upon two decades of experience to update existing CROSERF guidance for conducting aquatic toxicity tests and (2) to improve the design of laboratory toxicity studies for use in hazard evaluation and development of quantitative effects models that can then be applied in spill assessment. Key experimental design considerations discussed include species selection (standard vs field collected), test substance (single compound vs whole oil), exposure regime (static vs flow-through) and duration, exposure metrics, toxicity endpoints, and quality assurance and control.

A Comparative Assessment of the Aquatic Toxicity of Corexit 9500 to Marine Organisms.

The use of chemical dispersants during oil spill responses has long been controversial. During the Deepwater Horizon (DWH) oil spill, 1.8 million gallons of dispersant, mainly Corexit 9500, were applied in offshore waters to mitigate the human health and coastal environmental impact of surface oil contamination. To evaluate the potential impact of the dispersant on marine life, 18 species, representing important ecological and commercial taxa, were tested using low-energy, dispersant-only water accommodated fractions (WAFs) of Corexit 9500 and standard acute toxicity test methods. All prepared WAFs were analytically characterized. Analyses included the two dispersant markers found in the dispersant and evaluated in samples collected during the DWH Response, dioctylsulfosuccinate sodium salt, and dipropylene glycol n-butyl ether (DPnB). The median lethal and effective concentrations (LC/EC50s) were calculated using a nominal exposure concentration (mg/L, based on the experimental loading rate of 50 mg/L) and measured DPnB (µg/L). Results ranged from 5.50 to > 50 mg/L dispersant and 492 to > 304,000 µg/L DPnB. Species sensitivity distributions of the data demonstrated that taxa were evenly distributed; however, algae and oysters were among the more sensitive organisms. The calculated 5% hazard concentration (HC5) for DPnB (1172 µg/L) was slightly higher than the USEPA chronic criteria of 1000 µg/L and substantially higher than all measured concentrations of DPnB measured in the Gulf of Mexico during the DWH oil spill response.

Chronic Toxicity of Unweathered and Weathered Macondo Oils to Mysid Shrimp (Americamysis bahia) and Inland Silversides (Menidia beryllina).

Chronic, 21-28-day toxicity tests of Macondo source (Massachusetts, or MASS) and weathered Slick A (CTC) and Slick B (Juniper) oils field collected during the 2010 Deepwater Horizon (DWH) Incident in the Gulf of Mexico (GOM) were conducted using standardized procedures. Standard species, Americamysis bahia and Menidia beryllina, were evaluated for changes in survival and growth during daily static-renewal tests. Both species demonstrated an increased sensitivity to low-energy water accommodated fractions (WAFs) of un-weathered MASS oil, with growth and survival decreasing as oil loading rate increased from 0.01 to 1.0 g/L. Survival and growth of mysid shrimp exposed to weathered oil (Slick A and Slick B) did not differ from that of test controls. In contrast, survival and growth of inland silversides declined relative to that of test controls at loading rates of 1 g/L for both weathered oils. Based on the concentration of total polycyclic aromatic hydrocarbons (TPAH42), no observed effect concentrations were lower for inland silverside survival (5.00-7.61 µg/L) and growth (<2.02 to <7.61 µg/L) in chronic exposures to Slick B and Slick A weathered oils compared with mysids (4.75-17.9 µg/L). Average TPAH concentrations in full strength WAFs followed the weathering trend, with 165 ± 17.2, 17.9 ± 0.480, and 4.75 ± 0.521 µg/L for MASS, Slick A, and Slick B oils, respectively. The effect (LOEC, IC25) and no-effect exposure concentrations (in TPAHs) from the standardized laboratory toxicity studies with un-weathered and weathered oils are discussed relative to the actual exposure concentrations in the GOM in 2010. The exposures evaluated in the laboratory toxicity tests represent the highest concentrations of total PAHs that were rarely observed in water column samples collected in the GOM during the release and post release periods of the DWH incident.

The use of ephyrae of a scyphozoan jellyfish, Aurelia aurita, in the aquatic toxicological assessment of Macondo oils from the Deepwater Horizon incident.

Ephyrae of the scyphozoan jellyfish, Aurelia aurita, were evaluated in 96-hr acute toxicity tests for lethal response to Macondo crude oils from the Deepwater Horizon (DWH) incident in the Gulf of Mexico (GOM), Corexit 9500, and oil-dispersant mixtures. Water accommodated fractions (WAFs) of weathered and unweathered Macondo crude oils were not acutely toxic to ephyrae (LC50s > 100% WAF). The total PAHs (TPAHs), measured as the sum of 46 PAHs, averaged 21.1and 152 µg TPAH/L for WAFs of weathered and unweathered oil, respectively. Mortality was significantly (p = <0.0001) higher in the three highest exposure concentrations (184-736 µg TPAH/L) of chemically dispersed WAFs (CEWAF) compared to controls. Dispersant only tests resulted in a mean LC50 of 32.3 µL/L, which is in the range of previously published LC50s for marine zooplankton. Changes in appearance and muscle contractions were observed in organisms exposed to CEWAF dilutions of 12.5 and 25%, as early as 24 h post-exposure. Based on the results of these tests, crude oil alone did not cause significant acute toxicity; however, the presence of chemical dispersant resulted in substantial mortality and physical and behavioral abnormalities either due to an increase in hydrocarbons or droplet exposure.

Factors affecting toxicity test endpoints in sensitive life stages of native Gulf of Mexico species.

Indigenous species are less commonly used in laboratory aquatic toxicity tests compared with standard test species due to (1) limited availability lack of requisite information necessary for their acclimation and maintenance under laboratory conditions and (2) lack of information on their sensitivity and the reproducibility of toxicity test results. As part of the Natural Resource Damage Assessment aquatic toxicity program in response to the Deepwater Horizon Oil incident (2010), sensitive life stages of native Gulf of Mexico species were evaluated in laboratory toxicity tests to determine the potential effects of the spill. Fish (n = 5) and invertebrates (n = 2) selected for this program include the following: the Florida pompano (Trachinotus carolinus), red drum (Sciaenops ocellatus), spotted sea trout (Cynoscion nebulosus), cobia (Rachycentron canadum), red porgy (Pagrus pagrus), blue crab (Callinectes sapidus), and the common moon jellyfish (Aurelia aurita). Initially in the program, to establish part of the background information, acute tests with reference toxicants (CdCl2, KCl, CuSO4) were performed with each species to establish data on intraspecies variability and test precision as well as identify other factors that may affect toxicity results. Median lethal concentration (LC50) values were calculated for each acute toxicity test with average LC50 values ranging from 248 to 862 mg/L for fish exposures to potassium chloride. Variability between test results was determined for each species by calculating the coefficient of variation (%CV) based on LC50 values. CVs ranged from 11.2 % for pompano (96-h LC50 value) to 74.8 % for red porgy 24-h tests. Cadmium chloride acute toxicity tests with the jellyfish A. aurita had the lowest overall CV of 3.6 %. By understanding acute toxicity to these native organisms from a compound with known toxicity ranges and the variability in test results, acute tests with nonstandard species can be better interpreted and used appropriately when determining risk.

Acute aquatic toxicity studies of Gulf of Mexico water samples collected following the Deepwater Horizon incident (May 12, 2010 to December 11, 2010).

The potential for the Deepwater Horizon MC-252 oil incident to affect ecosystems in the Gulf of Mexico (GOM) was evaluated using Americamysis bahia, Menidia beryllina and Vibrio fischeri (Microtox® assay). Organisms were exposed to GOM water samples collected in May-December 2010. Samples were collected where oil was visibly present on the water surface or the presence of hydrocarbons at depth was indicated by fluorescence data or reduced dissolved oxygen. Toxicity tests were conducted using water-accommodated fractions (WAFs), and oil-in-water dispersions (OWDs). Water samples collected from May to June 2010 were used for screening tests, with OWD samples slightly more acutely toxic than WAFs. Water samples collected in July through December 2010 were subjected to definitive acute testing with both species. In A. bahia tests, total PAH concentrations for OWD exposures ranged from non-detect to 23.0 µg L(-1), while WAF exposures ranged from non-detect to 1.88 µg L(-1). Mortality was >20% in five OWD exposures with A. bahia and three of the WAF definitive tests. Total PAH concentrations were lower for M. beryllina tests, ranging from non-detect to 0.64 µg L(-1) and non-detect to 0.17 µg L(-1) for OWD and WAF exposures, respectively. Only tests from two water samples in both the WAFs and OWDs exhibited >20% mortality to M. beryllina. Microtox® assays showed stimulatory and inhibitory responses with no relationship with PAH exposure concentrations. Most mortality in A. bahia and M. beryllina occurred in water samples collected before the well was capped in July 2010 with a clear decline in mortality associated with a decline in total PAH water concentrations.