Analytical
Approach
Developing methods that accurately and rapidly measure eromatic components of oil spills in marine biota and habitats is a challenge for analytical chemists. A
J B aintaining the quality and function of coastal habitats is essential to sustaining marine species. In many urban estuaries, influxes of anthropogenic contaminants have degraded the quality of the water and sediment, impacting the health of marine biota. Among the most common sources of pollution are the thousands of gallons of petroleum products discharged or spilled into coastal waters each year. Petroleum contains a wide variety of chemical components (Table 1) the most
toxic of which are certain aromatic compounds that have the potential to damage fishery resources and affect the health of tiiose animals and humans that consume contaminated fish (i). Marine biota take up lipophilic aromatic compounds from water, sediment, and food. Fish and otiier marine vertebrates can efficiently metabolize aromatic compounds in their livers and then excrete the metabolites, primarily into bile {2,3). Thus, even if the animals have been heavily exposed to aro-
186 A Analytical Chemistry News & Features, March 1, 1998
matics, high levels of these compounds are unlikely to be transferred to consumers (Figure 1). In contrast to vertebrates, aromatic compounds can accumulate in marine invertebrates, because these animals do not efficiently metabolize aromatic compounds. Thus, parent aromatic compounds can be transferred up the food web to higher-level consumers (4). The challenge for analytical chemists is to develop methods that can accurately and rapidly measure both petroleumrelated aromatic compounds and their metabolites in marine biota and habitats. Following an oil spill, chemical analyses are needed to determine the extent of De-
Margaret M. Krahn John E. Stein National Oceanic & Atmospheric Administration
Assessing Exposure of Marine Biota and Habitats to Petroleum Compounds troleum contamination of sediment and biota and to prevent contaminated seafood from reaching the marketplace. Often, the very large numbers of samples requiring analysis lead to difficulties in setting analytical priorities and in completing the analyses in a timely manner. In the recent past, the majority of the analyses have been conducted by detailed methods (e.g., GC with detection by MS or flame ionization detection) that determine individual petroleum chemicals (5). This approach is timeconsuming, expensive, and in most cases probably unnecessary from a resource management perspective Alternatively, a plan for sampling and analysis following oil spills can be designed to provide the necessary information in a timely and cost-effective manner. First of all, resource managers, regulators, and scientists must collaborate to answer key questions that will determine the nature of the investigation needed. The types of samples to be collected and the chemical analytes to be measured can then be identified. Next, the best methods for obtaining quantitative information within a reasonable time must be selected, most likely using a tiered approach with an initial screening? for taojet compounds followed by detailed GC/MS analyses of a subset of samples to confirm analyte identifications and concentrations Finally a quality assurance plan must bf* written for evaluating the data This aDoroach allows data of known quality to be ranidlv disseminated to those who must make reoiilatorv d e n sions such as whethe t t 11
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following spills. In this Report, we will provide examples of the questions that must be answered before establishing a sampling and analysis
plan, discuss the use of a tiered approach to analysis, and review quality assurance procedures necessary to produce reliable data. Examples will be given of how these procedures were applied to a relatively small spill of high-density residual and industrial oil into the Columbia River in March 1984; the 11-million gallon Exxon Valdez crude oil spill in Prince William Sound, AK, in March 1989; the release of millions of barrels of Kuwaiti crude oil into the waters of the Arabian Gulf during the 1991 war; and the spill of 828,000 gallons of No. 2 fuel oil from the grounded barge North Cape ofo ffe eoast of Rhode Island in January 1996 Questions that need answers
What is the source of the spill? Is one purpose of the investigation to identify
the source of the petroleum? Does the spilled petroleum have any special chemical characteristics that will distinguish it from other potential sources and from background hydrocarbons? What are the toxicity and fate of the source? At what concentrations are petroleum components toxic to marine biota and humans? What is the primary emphasis of the investigation? Is it to determine the toxic effects of petroleum on marine fish, invertebrates, and terrestrial wildlife, the potential for transfer of contaminants up the marine food chain, or the risk to the human consumer from eating contaminated fishery products? What natural resources are at risk? Which marine species in the area are most susceptible to the toxic effects
T a b l e 1 . P e t r o l e u m c o m p o u n d s in c r u d e o i l . Class
Abundance
Bioavailability*
Persistence in tissues
Toxicity
Aliphatic hydrocarbons Alicyclic hydrocarbons (e.g., cycloalkanes, cycloalkenes) Aromatics (e.g., parent and alkyl aromatic hydrocarbons, N- and S-heterocycles) Polar compounds (e.g., acids, phenols, thiols, thiophenols) Elements (e.g., sulfur, vanadium. nickel, iron) Insoluble c o m ponents (e.g., asphaltenes, resins, tar)
High
High
Low
Low
High
High
Low
Low
High
High
Variable, depending on species
Variable
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
(a) Bioavailability is that portion of the total concentration of a chemical that is potentially available for biological uptake by aquatic organisms.
Analytical Chemistry News & Features, March 1, 1998
187 A
Analytical
Approach
from components of the oii? Arr eny oo these species atriskbecause of the particular life stage at the time of the spill (i.e., reproductive, larval, or juvenile stages may be more susceptible to toxic compounds)? Are threatened or endangered species in the path of the spill? What is the goal of the investigation? Is it to assess environmental damage or to determine the safety of seafood? For damage assessment studies, the primary emphasis of the investigation is on determining the exposure of natural resources and the toxic effects (injury) caused by the petroleum. Sediment and tissue samples (e.g., bile, liver, and muscle)fromfish and invertebrate species most at riskfromthe particular spill need to be collected. In a seafood safety investigation, the most important consideration is to eliminate any risk to the human
consumerfromeating contaminated fishery products and, secondarily, to maintain consumer confidence by preventing contaminated seafood productsfromreaching the marketplace. Although the samples collected for seafood safety investigations are most often the edible tissues of commercially important seafood species, other tissues and sediments may be collected to establish exposure of the animals. Sampling design considerations
After these questions have been answered, the numbers, types (e.g., sediment, tissue, or bilefrommarine species), and locations (e.g., intertidal or subtidal sediment, benthic biota, surface-feeding fish) of samples, as well as thefrequencyof sampling, can be determined and a sampling plan can be developed. Furthermore, a statistically de-
Chemical composition of the petroleum source
Aromatics in oil
Uptake
Uptake
Invertebrift/fe^Y ^ L Slow, inefficient metabolism 1
Vertebrates Fast, efficient metabolism Transfer to sediment
High molecular weight compounds
Reactive intermediates • Can bind to intracellular targets (e.g., DNA) and alter function
Metabolites • Water soluble • Excreted into bile • Eliminated from oraanism
Accumulation in tissues • Can cause acute effects to an organism, including death • Concern for safety of seafood caused by contamination of edible tissue
HPLC/fluorescence screening or GC/MS analysis for Bile metabolites
Aromatics in sediment
Aromatics in tissues
Figure 1 . Fate of petroleum-related aromatic compounds t a k e n up by vertebrates and invertebrates and analytical methods used to establish concentrations of these compounds in biota and sediments. 188 A
signed sampling plan can ensure sufficient sampling density in answering the questions posed and also in attaining the statistical power needed to distinguish significant differences among samples. Oversampling may be cost-effective, because it is often less expensive to collect extra samples when the equipment is on-site and to archive them than it would be to resample later. Furthermore, once a time point has passed (on the order of weeks to months), the opportunity for resampling is lost; that is, one will never again be able to collect "oneyear postspill" samples after that time has passed. The sampling plan must also contain information about how the samples will be tracked, transported, and stored, as well as steps that will be taken to prevent contamination (e.g., from other samples collected using the same sampling equipment) or loss of analytes during sampling and storage.
Analytical Chemistry News & &eatures, March 1, 1,98
Before analyzing for petroleum-related chemicals in tissues or sediment samples collectedfromthe spill area, it is important to learn as much as possible about the nature of the spilled petroleum (7). Sizeexclusion HPLC with fluorescence detection provides a chromatogram that profiles the molecular weight (size) distribution of a particular petroleum product and often allows general identification of the petroleum. For example, the chromatograms of diesel fuel (Figure 2a), marine lubricating oil (Figure 2c) and Exxon Valdez ccude oii (Figure 2e) exhibit different chromatographic profiles (8) In contrast similarities exist between chromatograms of the crude oilsfromthe Exxon Valdez (Figure 2e) and Gulf snills (Figure 2e) In addition chromatogranhic profiles of diesel fuel (Figure 2a) and No 2 fuel oil from the
North Catop (Fieiire 2b) are similar because these fractions distill in the same temperahire range and contain comnnunds of similar molecular weights. Thus, the type of petroleum distillate and possible ,
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sources can be suggestedfromthe HPLC analysis, but detailed analyses are needed to provide a more definitive answer. The identities and proportions of the aromatic compounds in a petroleum source are usually determined by GC/MS to pro-
pounds from the parent crude from which they were distilled. For example, the No. 2 fuel oil from die North Cape contained substantial proportions of the (VCg alkyl dibenzothiophenes (the C2 homologues are shown in Figure 3), indicating that the source of die North Cape distillate was likely an Alaskan north slope crude oil. Thus, the proportions of marker compounds in a petroleum source, as well as the proportions of other aromatic, aliphatic, or metallic compounds provide a fingerprint that can be used to identify that source in environmental samples (7) Advanced statistical techniques such as principal-component and leastsquares methods can tiien be used to determine me contribution of each petroleumrelarpd source when multiple sources are
Figure 2. Chromatocirams from the size-exclusion HPLC/fluorescence analysis of petroleum fractions. (a) Diesel fuel, (b) No. 2 fuel oil spilled from the North Cape, (c) Marrne lubrrcattng oil. .d) Extract of sediment from Knight Island in Prince William Sound, AK. (e) Exxon Valdez crude oil. .f) Extract of sediment from the Abu Ali area of Saudi Arabia, (g) Weathered Persian Gulf crude. Fluorescence excitation was recorded at 260 nm and emission at 380 nm. The peak at about 4.5 min is the polystyrene internal standard. (Adapted from Ref. 8.)
vide a fingerprint of the source (Figure 3) (7-10). Because spilled oil is degraded in time by physical, chemical, and microbial processes, the aromatic-fraction fingerprint of the weathered oil will be dominated by those aromatic compounds that are most resistant to weathering. For example, weathered crude oil from the Exxon Valdez oil spill contained high proportions of degradation-resistant alkylated naphtha-
lenes and phenanthrenes typical of petroleum contamination, as well as the Cj-Cg alkylated dibenzothiophenes that have been identified as marker compounds for Alaskan north slope crude oils (11). These alkyl dibenzothiophenes, as well as the C4 homologue, were found in even greater proportions in the Kuwaiti crude oil (12). Furthermore, refined (distilled) petroleum fractions often retain marker com-
Aromatic compounds in sediment Following the Exxon Valdez zpill, hundrrds of sediment samples were collected to determine the distribution and concentration of the oil (8). Analyzing all these samples by the usual GC methods would have been excessively expensive and timeconsuming, so analytical priorities needed to be set. Therefore, the size-exclusion HPLC technique with fluorescence detection mentioned earlier was used as a firsttier approach to estimate concentrations of petroleum-related aromatic compounds in more than 400 sediment samples from a large number of sites in the spill Next concentrations of aromatic pounds were confirmed in a selected subset of the sediments by GC/MS The correlation with the screening method was excellerif nrhiiQ rpQiilts fmm Qrrppniticr analyses can be used to describe the and temporal distribution of the oil follow inp- a snill can aid in designintr the sam plincr nrot'ncol and evaluating site clean strategies, and can be used to increase the confidence in the results by providing suffi. cient data for adequate statistical analyses. *
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gested by comparing chromatograms of sediment extracts with those of potential petroleum sources (Figure 2). For example, the chromatographic profile of sediment extract from northeast Knight Ieland (Prince Wilu Ham Sound, AK Figure 2d) was nearly su-
Analytical Chemistry News & Features, March 1, 1998 189 A
Analytical
Approach From 1989-91, more than 4000 fish of several species were collected from approximately 50 sites along the path of the Exxon Valdez zil spill for analysis (15,16). It was critically important to determine if the fish had been exposed to petroleum-related aromatic compounds, because of the potential for major economic impact that fishery closures would have on the commercial fishing industry. For example, immediately before the Pacific halibut fishery was scheduled to open in May 1989, concentrations of bile metabolites in this offshore bottom-dwelling species were found to be similar to those from fish sampled from a pristine site Moreover GC/MS analyses of selected samples of edible tissue from these halibut showed no detectable concentrations of aromatic compounds Thus the findings from the
analyses of both bile and edible catprl t h a t tViACA Viallhnt werc* n o t pynncpH tr»
Figure 3. Selected ion mass chromatograms for (left) C 2 -dibenzothiophenes and (right) C 2 -phenanthrenes provide a partial fingerprint of the spilled North Cape oil in extracts of sediment; lobster tail tissue from a lobster in the spill area and from lobster from a reference area.
perimposable with that of Exxon Valdez crude oil (Figure 2e), but it was different from those of other sources of contamination (other parts of Figure 2) that might be found in Alaskan sediments (8). Similarly, ,he chromatograph of sediment from the Abu Ali area in the Persian Gulf (Figure 2f) was nearly superimposable with that of weathered Persian Gulf oil (Figure 2g) (12)) Additional information about the contaminant source can be gained by comparing the identities and proportions of the aromatic compounds determined by GC/MS in sediments with similar characteristics of the probable sources. For example, the fingerprint of Alaskan north slope crude oil, which has high proportions of alkylated naphthalenes, phenanthrenes, and dibenzothiophenes, was found in Herring Bay sediment, suggesting Exxon Valdez crude as the contaminant source (8)).n addition a fingerprint similar to that of No. 2 fuel oil spilled from the North Cate was found in sediments collected from the spill area (Fiffure 3) However it is generally necessary to conduct statistical analyses of thefingerprintdata to identify and allocate 190 A
the sources of the aromatic compounds present (9). Metabolites of aromatic compounds
The reversed-phase HPLC method with fluorescence detection was used to rapidly estimate concentrations of metabolites in bile resulting from the uptake and transformation of aromatic compounds byfishand other vertebrates exposed to environmental contaminants (13). The first opportunity for field testing this method occurred following the spill of a relatively small amount of high-density residual and industrial oil into the Columbia River (14) The exposure of the fish to spilled oil was established by showing that the concentrations of metabolites in the bile of white sturgeon captured downstream from the spill were significantly higher than those of sturgeon captured upriver This study as well as previous laboratory investigation provided the screening method in assessing the exposure offish and marine mammals to oilfromthe Exxon Valdez znili l(31 (Figure 41
Analytical Chemistry News & Features, March 1, 1998
annreciable concentrations of crude oil tbns allowed thefishingseason to open as scheduled. Contrasting results were found when bile samples from nearshore bottom-dwelling species (e.g., rock sole and yellowfin sole) or pelagic species (e.g., salmon) were analyzed to address the concerns of native Alaskans who feared that their subsistence seafood might be contaminated (15,16). Elevated concentrations of metabolites were found in the bile of fish from sites in the path of the spill (Figure 4), and concentrations were elevated in bottom-dwelling fish from some sites for more than two years
afterward (11 15 16). These results suggest continuing petroleum contamination of nearshore subtidal sediments in the path of the spill, as well as continuing exposure of the fish. However, GC/MS analyses of the edible flesh showed relatively low concentrations of aromatic compounds, even in the fish that had the highest concentrations of metabolites of aromatic compounds in bile (the majority of the samples had concentrations of summed aromatic compounds
