Evaluation of two direct immunoassays for rapid detection of petroleum products on marine birds

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Abstract

To reduce rehabilitation time and improve survival, a practical, objective test is needed to determine if marine birds are contaminated with oil before they enter captivity. The RaPID Assay® and EnviroGardTM immunoassays for detection of polycyclic aromatic hydrocarbons (PAHs) in soil were evaluated for their ability to detect petroleum on feather samples from 30 intermediate fuel oil contaminated and 30 uncontaminated common murres (Uria aalge). Sensitivity, specificity, positive and negative predictive value, precision, and time required to run each assay were determined. The RaPID Assay® was 96.7% sensitive and specific, while the EnviroGardTM assay was 93.3% sensitive, and 90.0% specific. Sensitivity decreased at higher dilutions for both assays. Intra-replicate variation was less than 20%. Our evaluation showed that these immunoassays are rapid and cost-effective methods for detecting oil-contamination on the plumage of seabirds, with the EnviroGardTM assay being more practical in most oil spill response situations due to ease of use and rapidity of results.

Introduction

Marine birds and mammals admitted to rehabilitation centers following oil spills are subject to prolonged time in captivity, stress from handling, and increased risk for disease due to overcrowding. For example, during the 1989 Exxon Valdez oil spill in Prince William Sound, Alaska, gastric ulcerations, aspergillosis, and keel and hock abrasions were observed in marine birds most likely as consequences of the physiological stress induced by captivity, as well as the facility limitations of the rehabilitation centers (Holcomb and White, 1990). Furthermore, tissues from presumed uncontaminated sea otters that died while being cared for at rehabilitation centers during the Exxon Valdez spill response displayed evidence of gastric erosions, hepatic lipidosis, renal tubular lipidosis and interstitial pulmonary emphysema (Lipscomb et al., 1993). In contrast, tissues collected from apparently healthy, wild sea otters from uncontaminated waters surrounding the Kuril Islands, Russia did not show evidence of such lesions (Lipscomb et al., 1993). In addition to morbidity and mortality in captivity, poor post-release survival following rehabilitation has been documented in multiple seabird species and sea otters (Sharp, 1996; Anderson et al., 1996; Estes, 1991). There is evidence that the degree of oiling and time in captivity were correlated with post-release survival of oiled and cleaned seabirds (Sharp, 1996).

In the field, determining whether or not a bird has been contaminated with oil can be difficult. Many marine bird species have dark plumage and a natural sheen that may mimic oil contamination. Because of the potential for hypothermia and physiological dysfunction even with a low degree of oil exposure, birds whose exposure status is in doubt are often considered exposed and admitted to rehabilitation centers for oil removal and medical care. Morbidity and mortality associated with prolonged captivity have led to concerns about subjecting non-exposed marine wildlife to what may be unnecessary rehabilitation (Estes, 1998).

To reduce time in captivity and improve survival, a rapid, objective test is needed to determine if seabirds are contaminated with oil before they undergo the rehabilitation process. Gas chromatography/mass spectroscopy (GC/MS), a laboratory analytical method for determination of concentrations of polycyclic aromatic hydrocarbons (PAHs), can provide accurate and quantitative estimates of petroleum product contamination (Wang and Fingas, 1997); however, GC/MS is not practical to use during an oil spill because it is time consuming, expensive, and most often only performed by trained personnel in a fully equipped laboratory (Mazet et al., 1997).

In recent years, commercially available direct competitive immunoassays have been developed to detect PAHs in soil samples contaminated with petroleum products. Immunoassays are portable, relatively easy to use, provide rapid results, and are far less expensive per sample than GC/MS analysis (Berry and Burton, 1997; Chuang et al., 1998). In 1997, the QuantixTM Portable Workstation direct immunoassay for PAH detection in soil was shown to be a sensitive, specific and cost-effective test for detection of PAH contamination on the fur of American mink (Mustela vison) experimentally exposed to known concentrations of oil (Mazet et al., 1997). The results suggested that a PAH direct immunoassay was a valuable tool to discriminate externally oil-contaminated from uncontaminated wildlife; however the QuantixTM assay is no longer commercially available. Therefore, the optimization of another PAH immunoassay for use before a bird is admitted to the rehabilitation center is needed. Such an assay would allow uncontaminated birds to be translocated and released before spending prolonged time in captivity, and allow rapid, objective documentation of contamination status.

The methodologies of the RaPID Assay® and the EnviroGardTM PAH immunoassays distributed by Strategic Diagnostics (Newark, DE, USA) are similar to the QuantixTM assay. The RaPID Assay® is a magnetic particle immunoassay for semi-quantitative screening for PAHs in soil consistent with Environmental Protection Agency Office of Solid Waste SW-486 Method 4035 (United States Environmental Protection Agency, 1997). The RaPID Assay® has a range of PAH detection in soil of 200 ppb to 5 ppm, and results have been shown to correlate well with those of EPA Method 8310 using field contaminated and spiked soil samples (Web, J., personal communication). The assay is semi-quantitative in nature due to comparison of the absorbance response of the sample to a calibration curve of phenanthrene. The EnviroGardTM PAH assay uses polyclonal antibody coated test tubes to detect PAHs in soil, including the 16 compounds listed under EPA SW-846 methods 8270, 8310 and 8100. This assay allows for semi-quantitative screening for total PAH by comparison of sample results to 1, 10, or 100 ppm calibration controls, selected so that the levels of total PAH in a soil sample will be detected with a maximum 5% false-negative rate. The RaPID Assay® is intended to be more quantitative and cost effective in terms of per sample cost than the EnviroGardTM PAH assay, but requires more equipment and a higher level of operator training. Both tests are commercially available and can be shipped overnight in the event of an oil spill.

Therefore, the main objective of this study was to determine the sensitivity and specificity of the RaPID Assay® and the EnviroGardTM PAH assay to detect PAHs on feather samples from known oil contaminated and uncontaminated common murres (Uria aalge). Additionally, positive and negative predictive values of test results were assessed at differing presumed prevalences of oil contamination in the population, agreement between the two assays was evaluated, and intra-replicate variation for each assay was assessed. Lastly, the mean time needed to run each assay, adjusted for the number of samples run, was determined to evaluate efficiency and ease of use in an oil spill response situation. The overall goal of the study was to determine which of these assays had the most potential to be accurate and useful in an actual oil spill event.

Section snippets

Sample collection

During the first four days immediately following an intermediate grade fuel oil (IFO 180) spill in Humboldt Bay, CA, USA on September 6, 1999, feather samples were opportunistically taken from 30 dead, externally oil-contaminated common murres processed at the Marine Wildlife Care Center, Humboldt State University. Murres were considered externally contaminated if oil was visible on the plumage. True random sampling of murres was not possible due to personnel and time limitations resulting from

Results

The frequency distributions of mean PAH concentrations detected by the RaPID Assay® for sample extracts diluted 1:50 and 1:1000 are presented in Fig. 1, Fig. 2, respectively. At both dilutions, the distribution of PAH concentrations from oil-contaminated samples was spread across the range of the assay, indicating differing relative degrees of contamination among the samples. The RaPID Assay® using the 1:50 sample extract dilution misclassified one uncontaminated and one contaminated feather

Discussion

Assay performance results (Table 2) indicated that the RaPID Assay® is a sensitive and specific test at both dilution levels, and that the EnviroGardTM assay is sensitive and specific at the 1 ppm action level for detection of PAH on feathers samples contaminated with IFO 180. Increased sample extract dilution resulted in lowered sensitivity and increased specificity for both assays. Overall, the RaPID Assay® at the 1:50 dilution level had the best balance of sensitivity and specificity (96.7%

Acknowledgements

The authors thank John Webb of Strategic Diagnostics for technical support; Melissa Miller and David Jessup from the California Department of Fish and Game's Marine Wildlife Veterinary Care and Research Center in Santa Cruz, CA, USA for collection of uncontaminated murre samples; and the spill response personnel from Point Reyes Bird Observatory for their assistance in collection of oil-contaminated samples. All samples were collected in conjunction with California's Oiled Wildlife Care Network.

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