Environ. Sci. Technol. 2005, 39, 17-23
Regional Comparisons of Coastal Sediment Contamination Detected by a Biomarker (P450 HRGS; EPA Method 4425) J A C K W . A N D E R S O N , * ,† S. IAN HARTWELL,‡ AND M. JAWED HAMEEDI‡ Columbia Analytical Services, P.O. Box 479, Kelso, Washington 98626, and NOAA, Center for Coastal Monitoring and Assessment, N/SCI1, 1305 East-West Highway, Silver Spring, Maryland 20910
Pollution investigations by the Center for Coastal Monitoring and Assessment of the National Oceanic and Atmospheric Administration (NOAA) have been conducted since 1984 and have incorporated extensive biological and chemical analyses. Since 1993, one of the biological tests utilized in these studies has been the biomarker P450HRGS, which is more recently described as EPA Method 4425. Extracts of sediments are applied to a human cell line with a reporter gene (firefly luciferase) at the CYP1A1 site. Light produced by the extracts is a function of the concentrations and potencies of those compounds with an affinity for Ah-receptor (certain polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and dioxins/ furans). These compounds are carcinogenic and can produce chronic toxicity, and those containing chlorine are persistent and bioaccumulated. Nineteen coastal regions and 1309 samples from the three U.S. coasts have been evaluated as part of the NOAA investigations. The stratified random sampling approach used by NOAA provides estimates of the areas (km2) of each region containing levels of the compounds above thresholds. From analysis of the database, sediments with concentrations at or below 11 µg benzo[a]pyrene equivalents (B[a]PEq)/g would not be expected to produce effects on the benthos. At 32 µg B[a]PEq/g and above there is the potential for impacts on the biota, and above 60 µg/g, the degradation of the benthic community has been observed. Several of the regional surveys found no samples at or above 60 µg B[a]PEq/g, but 60% of the samples from New York Harbor (280 km2) were above this level. Analyses of data from Puget Sound and Chesapeake Bay demonstrate an increase in samples above 32 µg B[a]PEq/g in more populated and industrial regions. Method 4425 serves as both a biomarker, simulating the response of an organism (with CYP1A) exposed to inducing compounds, and as a bioanalytical technique measuring the levels of these chemicals in the samples. A targeted investigation of the distribution of the three important classes of compounds identified by Method 4425 would be more cost-effective by first screening samples * Corresponding author present address: Pacific Northwest National Laboratory, Battelle Marine Sciences Laboratory, 1529 W. Sequim Bay Rd., Sequim, WA 98382; phone: (360)681-3631; fax: (360)681-4559; e-mail:
[email protected]. † Columbia Analytical Services. ‡ NOAA. 10.1021/es049283b CCC: $30.25 Published on Web 11/13/2004
2005 American Chemical Society
by this method before expending substantial funds in the detailed chemical analysis of all samples.
Introduction Environmental indicators have received an increasingly important role in communicating credible information about the status and trends in environmental conditions and sustainability of resources in coastal and marine waters. Indicators serve as valuable tools for linking resource management goals to specific performance measures that can be derived from the results of well-planned environmental assessment, monitoring, and research studies. Since its inception in 1984, the National Oceanic and Atmospheric Administration (NOAA) National Status and Trends (NS&T) Program has used environmental indicators in the form of benthic ecological indices and a broad suite of toxicity bioassays for assessing environmental quality in the coastal and estuarine areas of the United States. NS&T Program activities include region-specific assessments of coastal contamination and its associated adverse biological effects. To date, such assessments have been conducted in about 30 different estuaries and coastal waters. As a corollary to these studies, the program develops or applies environmental indicators (biomarkers as well as ecological indices), sediment quality guidelines, a quality assurance-quality control protocol, and historic trend analyses (based on sediment cores). One of the biological tests used in the coastal studies conducted by NOAA from 1994 through 2001 has been the screening of sediment samples for the presence of compounds, which have an affinity for the Ah-receptor (AhR) in the cells of wildlife and humans. These compounds include high molecular weight polycyclic aromatic hydrocarbons (PAHs), coplanar polychlorinated biphenyls (PCBs), and dioxins and furans (TCDDs/TCDFs). The carcinogenic potential of eight 4-6-ring PAHs (1, 2) and 12 PCBs and 17 TCDDs/TCDFs (3) have been assessed and expressed as toxic equivalency factors (TEFs). The screening test used on extracts of sediments that is the subject of this paper has been called the Reporter Gene system (RGS), the Human Reporter Gene System (HRGS), and most recently, EPA Method 4425 (4). Previous investigations of contaminated soils and sediments utilizing this assay have been described (5-8). Method 4425 has also been applied to extracts of water and tissue samples (9). In a recent study (10), the assay was used for both fish liver tissue and sediment analyses on samples from a contaminated site and a reference site in Puget Sound. Analyses by Method 4425 compared well with the CYP1A measured in the fish tissue and the levels of contaminants (PAHs, PCBs, dioxins/furans) in the sediments from the two locations. Structural differences in the gill DNA, measured by Fourier-transformed infrared spectra, of fish collected from the contaminated site (Duwamish Waterway) as compared to the reference site indicated DNA damage, and this approach may be a useful biomarker without sacrificing the fish. Method 4425 assay is rapid (60 samples can be tested in 16 h) and inexpensive as compared to chemical analyses of the three classes of organics detected by Method 4425. The method utilizes a cell line (101L) derived from the human hepatoma cell line (HepG2) in which a plasmid containing the human CYP1A1 promoter and 5′-flanking sequences has been stably integrated (11). The sequences consist of three xenobiotic responsive elements (XREs) fused to a reporter gene that produces firefly luciferase. The response (fold induction) of the Method 4425 assay to a mixture of the 17 VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Relative Order of Potency for Response of Method 4425 to Specific High Molecular Weight PAHsa polycyclic aromatic hydrocarbon
ratio to benzo[a]pyrene
benzo[k]fluoranthrene dibenz[a,h]anthracene benzo[b]fluoranthrene indeno[1,2,3-cd]pyrene benzo[a]pryene benzo[a]anthracene chrysene benzo[ghi]perylene
25 4 3 3 1 0.4 0.4 32 >60 (km2) (% >32) (km2 >32) 2 1 2 18 5 33 21 20 4 3 0 9 4 9.4
0 0 0 13 4 19 14 7 1 1 0 1 0 4.6
2 8 19 0 83 0
0 3 15 0 72 0
245.9 1351.1 271.4 2346.8 806.2 731.7 857.7 2265.0 3859.4 2201.3 23.2 989.2 989.2 1302.9
0.81 0.15 0.11 3.43 0.04 3.20 5.00 27.98 1.36 0.16 0.00 7.00 1.00 3.9
2.0 2.0 0.3 80.5 0.3 23.7 43.1 633.8 52.7 3.5 0.0 76.4 9.8 71.4
7.3 6.6 5.5 5.0 503.0 n/a
28.90 31.81 47.27 0.00 55.70 0.00
2.1 2.1 2.6 0.0 280.2 0.0
a Values listed represent Method 4425 responses (µg of B[a]PEq/g dry wt), percentages of samples exceeding estimated thresholds, and spatial extent of exceedences. At 32 µg B[a]PEq/g and above biological effects are possible, and at 60 µg/g they are probable. b Samples were selected from a larger group of samples and are therefore not representative of the random sampling approach used in other NOAA surveys. c The high contamination levels of NY Harbor sediments and the contribution of dioxins and furans separate this investigation from others. d The sampling plan for Biscayne Bay in 1999 was not a random approach but designed to identify sources e na ) not appropriate, as described in footnotes b-d.
the regions, eight of the 19 areas sampled (351 samples) contained no sediments with B[a]PEq values at or above 60 µg/g. At this high level of contamination, it has been observed that the structure of the benthic community was degraded (21). In a recent evaluation (268 total samples) of the benthos obtained in a 1998 survey of the Southern California Bight, it was found that benthic index values were all 20 or higher at stations where the B[a]PEq values were 60 µg/g or greater. Higher index values in this investigation infer decreasing health (abnormality) of the benthic community (23). Sixty percent of the New York Harbor sediments contained B[a]PEq values greater than 60 µg/g. Most of the NOAA studies covered approximately 10003000 km2, but some were less than 300 km2 (Table 2). Testing by Method 4425 was only conducted on a portion of the samples collected from Winyah Bay, Charleston Harbor, San Diego Bay, and the Southern California Bays, so the areas defining these samples were quite small (5-7.3 km2). In general, those strata containing samples with Method 4425 responses of 32 µg B[a]PEq/g and greater represented a relatively low percentage of the total area sampled (mean of 3.9%). For those regions sampled in the random stratified manner, only the upper region of the Chesapeake Bay, sampled in 1998, exhibited a high number of contaminated stations (28%), distributed over a wide area (634 km2). When all surveys listed in Table 2 are considered, the percentages of those samples over 32 µg B[a]PEq/g increased from 29% to 47%. The New York Harbor sediments were clearly in a class by themselves, with a mean of 290 µg B[a]PEq/g, and 56% of the area sampled contained samples over 32 µg B[a]PEq/g. Comparison with Chemical Analyses. Table 3 summarizes the statistical comparison between the Method 4425 results and the analyses of total PAHs (TPAH) in the sediment samples. NOAA contractors provided the detailed analyses of sediments from each of the sample locations for 44 PAH analytes or analyte groups, and the sum of those concentrations was plotted against the previously measured µg B[a]PEq/g values. The correlation coefficients (r 2 values) from
TABLE 3. Relationships between Measured B[a]P Equivalents and Total PAHs (TPAHs) in Sedimentsa
location
stations
Winyah Bay, SC, 1993 Charleston Harbor, SC, 1993 So. Calif. Bays, 1994 San Diego Bay, CA, 1994 Sabine Lake, TX, 1995 Galveston Bay, TX, 1996 Biscayne Bay, FL, 1996 Delaware River & Bay, 1997 N. Puget Sound, 1997 C. Puget Sound, 1998 S. Puget Sound, 1999 New York Harbor, 1998 Biscayne Bay, FL, 1999 N. Chesapeake Bay, MD C. Chesapeake Bay, DC S. Chesapeake Bay, VA San Francisco, CA, 2000 San Francisco, CA, 2001 St. Lucie Bay, FL, 2001 total mean standard deviation
9 20 29 30 65 75 121 81 100 100 100 119 31 63 69 78 99 99 21 1309
a
ratio B[a]PEq/ TPAH
correlation r2
16 18 22 16 10 7 4 17 2 8 11 24 23 17 8 2 6 4 9
0.86 0.87 0.68 0.71 0.84 0.68 0.53 0.81 0.70 0.73 0.87 0.87 0.69 0.62 0.68 0.64 0.64 0.63 0.43
11.7 7.2
0.71 0.12
Both values are in µg/g dry weight.
the regression analyses ranged from 0.43 for St. Lucie Bay to 0.87 for New York Harbor, with a mean r 2 and standard deviation for all regions (1309 samples) of 0.71 and 0.12. The two Florida investigations with the lowest correlations (St. Lucie and Biscayne Bay, 1996) contained relatively low levels of the measured PAHs, suggesting that the small amount of CYP1A1 induction observed was produced by other compounds. Also listed in Table 3, from the correlation curves, are the ratios of µg/g B[a]PEq to µg/g of total PAHs. The mean of these ratios was 12, with a standard deviation of 7. VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Study areas with ratios over 20 either contained low levels of PAHs (Southern California Bays, Biscayne Bay, 1999) or were found to contain other potent inducing compounds (dioxins/furans in New York Harbor). A consistent ratio for each study area would not necessarily be expected, since the Method 4425 assay with human cells responds to the concentrations and potencies of the specific compounds in the mixtures present on sediments. There are no doubt significant differences in the compositions of mixtures on sediments from the various coastal regions of the United States. The only investigation that included analyses of dioxins and furans in the sediments was New York Harbor, and all 12 coplanar PCBs (with affinity for the Ah-receptor) were not measured in any of these studies. Table 1 has demonstrated the wide differences in the potencies of several high molecular weight PAHs, and the potencies of coplanar PCBs have also been described (15). The most potent PAH and PCB were found to be benzo[k]fluoranthene and congener 81 (3,4,4′,5tetra CB), respectively. Significance and Use of Findings. The levels of B[a]P equivalents detected in the samples described above provide State and Federal resource managers with information on the relative concentrations of important organic chemicals in the sediments in their region that may be producing alterations in the benthic community from chronic toxicity or other adverse biological effects. In this regard, the application of this biomarker is just being realized. The Delaware Estuary Program recently included the P450HRGS results as one of the indicators of contaminated sediments in the estuary (24, 25) This method has been used in South Korea to evaluate the levels of CYP-inducing compounds (primarily PAHs) in sediment of Ulsan Bay (26). In Japan the method has been used for more than 3 years as an inexpensive analysis for dioxins (8), and it is being considered for approval as an official Japanese analytical method. Method 4425 can rapidly and inexpensively provide decision makers with a means of selecting those locations that no longer require attention and those which do. A significant cost saving could be gained if sediments were first screened by Method 4425 before moving forward on the extensive chemical and biological analyses that were included in the NOAA investigations described. This type of tiered approach has not been used in these investigations, primarily because of the usefulness of comprehensive databases for each survey region. As long as the funds are available, it can be argued that a complete data set is the best approach. When there is a need to investigate new regions or perhaps revisit areas to assess change and funds for a comprehensive evaluation are unavailable, screening by Method 4425 would be very useful. Since the chlorinated inducing compounds are persistent organic pollutants (POPs), which bioaccumulate, they may be transferred up the food web. There may be food-web magnification of these POPs of significant concern for wildlife and humans ingesting aquatic organisms from regions of high contamination. If the concern is only for the chlorinated inducing compounds, as in previous terrestrial investigations (8), Method 4425 can be applied after removal of PAHs from the extracts by silica gel and carbon columns (17). At those locations where there are particularly high levels of POPs, some type of remediation, such as dredging or capping of sediments, may be considered. The B[a]P equivalent data produced by Method 4425 are appropriate as the first step in identifying areas of contamination, so that subsequent chemical analyses of PAHs, PCBs, and dioxins and furans can be cost-effectively directed.
Acknowledgments We very much appreciate the extensive high-quality laboratory testing and evaluation provided by Jennifer M. Jones 22
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and Daniel L. McCoy in the production of findings presented in this paper. We wish to thank the NOAA contractors for the detailed analyses of PAHs used in comparisons to the 4425 data. NOAA data from some of the earlier investigations were provided by Ed Long, and we appreciate his aid. The time to compile and assess past data and prepare this manuscript was contributed by Columbia Analytical Services and the Pacific Northwest National Laboratory, operated for the Department of Energy by Battelle Memorial Institute.
Literature Cited (1) U.S. Environmental Protection Agency. Provisional Guidance for Quantitative Risk Assessment of Polycyclic Aromatic Hydrocarbons; EPA/600/R-93/089; Environmental Criteria and Assessment Office, Office of Research and Development: Washington, DC, 1993. (2) Delistraty, D. Toxic equivalency factor approach for risk assessment of polycyclic aromatic hydrocarbons. Environ. Toxicol. Chem. 1997, 64, 81. (3) Van den Berg, M.; Birnbaum, L.; Bosveld, A. T.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J. P.; Hanberg, A.; Hasegawa, R.; Kennedy, S. W.; Kubiak, T.; Larsen, J. C.; van Leeuwen, F. X.; Liem, A. K.; Nolt, C.; Peterson, R. E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health Perspect. 1998, 106, 775. (4) U.S. Environmental Protection Agency. Method 4425: Screening Extracts of Environmental Samples for Planar Organic Compounds (PAHS, PCBS, PCDDS/PCDFS) by a Reporter Gene on a Human Cell Line; EPA Office of Solid Waste, SW 846 Methods, Update IVB; U.S. Government Printing Office: Washington, DC, 2000. (5) Anderson, J. W.; Rossi, S. S.; Tukey, R. H.; Tien Vu, Quattrochi, L. C. A Biomarker, 450 RGS, for assessing the potential toxicity of organic compounds in environmental samples. Environ. Toxicol. Chem. 1995, 14 (7), 1159-1169. (6) Anderson, J. W.; Zeng, E.; Jones, J. M. Correlation between the response of a human cell line (P450RGS) and the distribution of sediment PAHs and PCBs on the Palos Verdes Shelf, California. Environ. Toxicol. Chem. 1999, 18 (7), 1506-1510. (7) Anderson, J. W.; Jones, J. M.; McCoy, D. L. Cost-effective dioxin site characterization using a P450 human reporter gene system (HRGS; EPA 4425). Organohalogen Compd. 2000, 45, 208-211. (8) Anderson, J. W.; Jones, J. M.; McCoy, D. L.; Fujita, A.; Yamamoto, T.; Iijima, S. Comparison between a rapid biological screening Method (EPA 4425) for TCDDs/TCDFs and chemical analytical methods. Organohalogen Compd. 2003, 60, 271-274. (9) Anderson, J. W.; Jones, J. M.; Steinert, S.; Sanders, B.; Means, J.; McMillin, D.; Vu, T.; Tukey, R. Correlation of CYP1A1 induction, as measured by the P450 RGS biomarker assay, with high molecular weight PAHs in mussels deployed at various sites in San Diego Bay in 1993 and 1995. Mar. Environ. Res. 1999, 48, 389-405. (10) Malins, D. C.; Stegeman, J. J.; Anderson, J. W.; Johnson, P. M.; Gold, J.; Anderson, K. M. Structural changes in gill DNA reveal the effects of contaminants on Puget Sound fish. Environ. Health Perspect. 2004, 112 (5), 511-515. (11) Postlind, H.; Vu, T.; Tukey, R. H.; Quattrochi, L. C. Response of human CYP1-luciferase plasmids to 2,3,7,8-tetrachlorodibenzop-dioxin and polycylcic aromatic hydrocarbons. Toxicol. Appl. Pharmacol. 1993, 118, 255-262. (12) Tillitt, D. E.; Ankley, G. T.; Verbrugge, D. A.; Giesy, J. P.; Ludwig, J. P.; Kubiak, T. J. H411E rat hepatoma cell bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents in colonial fisheating waterbird eggs from the Great Lakes. Arch. Environ. Contam. Toxicol. 1991, 21, 91-101. (13) Garrison, P. A.; Tullis, K.; Aarts, J. M.; Brouwer, A.; Geisy, J. P.; Denison, M. S. Species-specific recombinant cell line as biossay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxinlike chemicals. Fundam. Appl. Toxicol. 1996, 30, 194-203. (14) Murk, A. J.; Legler, J.; Denison, M. S.; Giesy, J. P.; van de Guchte, C.; Brouwer, A. Chemical-activated luciferase gene expression (CALUX): A novel in vitro bioassay for Ah receptor active compounds in sediments and pore water. Fundam. Appl. Toxicol. 1996, 33, 149-160. (15) Jones, J. M.; Anderson, J. W. Relative potencies of PAHs and PCBs based on the response of human cells. Environ. Toxicol. Pharmacol. 1999, 7, 19-26.
(16) Anderson, J. W.; Jones, J. M.; Hameedi, J.; Long, E.; Tukey, R. Comparative analysis of sediment extracts from NOAA’s Bioeffects studies by the biomarker, P450 RGS. Mar. Environ. Res. 1999, 48, 407-425. (17) U.S. Environmental Protection Agency. EPA Solid Waste (SW) 846 Methods, Update IVA; U.S. EPA Office of Solid Waste: Washington, DC, 1998; http://www.epa.gov/epaoswer/hazmat/ test/main.htm. (18) American Public Heath Association. P450 reporter gene response to dioxin-like organic compounds. Method 8070. In Standard Methods for the Examination of Water and Wastewater, 20th ed.; American Public Health Association: Washington, DC, 1998; pp 8-36-8-37. (19) American Society for Testing and Materials. Standard guide E 1853M-98 for measuring the presence of planar organic compounds which induce CYP1A, reporter gene test systems. In Biological Effects; Environmental Fate; Biotechnology; Pesticides; Section 11: Water and Environmental Technology; Annual Book of ASTM Standards, Vol. 11.05; American Society for Testing and Materials: West Conshohocken, PA, 1999. (20) Long, E. R.; Dutch, M.; Aasen, S.; Welch, K.; Hameedi, M. J. Chemical Contamination, Acute Toxicity in Laboratory Tests, and Benthic Impacts in Sediments of Puget Sound: A Summary of Results on the Joint 1997-1999 Ecology/NOAA Survey; Washington State Department of Ecology Publication No. 0303-049 and NOAA Technical Memorandum NOS NCCOS CCMA 163; 2003. (21) Fairey, R.; Bretz, C.; Lamerdin, S.; Hunt, J.; Anderson, B.; Tudor, S.; Wilson, C. J.; LaCaro, F.; Stephenson, M.; Puckett, M.; Long, E. R. Chemistry, toxicity, and benthic community conditions in
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sediments of the San Diego Bay Region; Final Report by State Water Resources Control Board, National Oceanic and Atmospheric Administration, California Department of Fish and Game-Marine Pollution Studies Laboratory, Moss Landing Marine Laboratories: Sacramento, CA, 1996. McCoy, D. L.; Jones, J. M.; Anderson, J. W.; Harmon, M.; Hartwell, I.; Hameedi, J. Distribution of CYP1A1-inducing chemicals in sediments of the Delaware River/Bay System. Environ. Toxicol. Chem. 2002, 21 (8), 1618-1627. Ranasinghe, J. A.; Montagne, D. E.; Smith, R. W.; Mikel, T. K.; Weisberg, S. B.; Cadien, D. B.; Velarde, R. G.; Dalkey, A. Southern California Bight 1998 Regional Monitoring Program: Benthic Macrofauna; Technical Report 382, Southern California Coastal Water Research Project, Westminster, CA, 2003. Delaware Estuary Program. Delaware Estuary: Environmental Indicators; Delaware Estuary Program: West Trenton, NJ, 2001. Hameedi, M. J. Environmental indicators as performance measures for improving estuarine environmental quality. In Estuarine Indicators; Bortone, S. A., Ed.; CRC Press: Boca Raton, FL, 2005; pp 453-467. Koh, C.-H.; Kim, G. B.; Maruya, K. A.; Anderson, J. W.; Jones, J. M.; Kang, S.-G. Induction of P450 reporter gene system bioassay by polycyclic aromatic hydrocarbons in Ulsan Bay (South Korea) sediments. Environ. Pollut. 2001, 111, 437-445.
Received for review May 13, 2004. Revised manuscript received September 24, 2004. Accepted September 28, 2004. ES049283B
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