Environ. Sci. Technol. 2000, 34, 2035-2041
Determination of Polycyclic Aromatic Hydrocarbons in Contaminated Water and Soil Samples by Immunological and Chromatographic Methods DIETMAR KNOPP,* MARTIN SEIFERT, V I R P I V A¨ A¨ N A¨ N E N , # A N D REINHARD NIESSNER Institute of Hydrochemistry and Chemical Balneology, Technical University Munich, Marchioninistrasse 17, D-81377 Mu ¨ nchen, Germany
An immunoassay was developed that can be used for the detection of polycyclic aromatic hydrocarbons (PAHs) in water, landfill leachate, and soil. As test format an indirect competitive microtiter plate ELISA (enzyme-linked immunosorbent assay) was applied. While groundwater samples from a former manufactured gas plant site could be analyzed directly, soil and landfill leachate had to be extracted and required at least a 100-fold dilution prior to immunochemical measurement. PAHs could be recovered from fortified reference soils as well as aged field samples with high yield using 1-h ultrasonication with acetonitrile. Extraction efficiency was comparable to Soxhlet extraction and ultrasonication with tetrahydrofurane. Recovery was lower with agitation but would still be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions such as identifying “hot spots”, site mapping, monitoring of remediation processes, and selecting site samples for laboratory analysis. Classification of ELISA data showed that it was possible to estimate the PAH contamination in soils with about 5% false positive and 5% false negative results that may have arisen from heterogeneity of samples, cross-reactivity of compounds with a similar structure, humic acids, or unknown interferences.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitious environmental pollutants of natural or anthropogenic origin. They are formed due to incomplete combustion of various materials particularly fossil fuels. Sources of PAHs are power stations, domestic and industrial heating systems, combustion engines (diesel and petrol), and refuse burning. PAHs present in the atmosphere are distributed between gas and particle phases. They are transported over long distances and can be found in wet and dry deposition. Contamination of soils can differ widely depending on local emissions. Jones et al. reported an increase in soil PAHs starting from about 300 ppb at the end of the former century to approximately 2 ppm 100 years later, observed at a semirural location in * Corresponding author phone: ++49 89 7095 7994; fax: ++49 89 7095 7999; e-mail:
[email protected]. # Present address: Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland. 10.1021/es991215f CCC: $19.00 Published on Web 04/14/2000
2000 American Chemical Society
southeast England that may be representative for agricultural soils in other industrialized countries or regions (1). Manufactured gas plant (MGP) sites and cokery sites are known for significant PAH-release into surrounding soil (2, 3). Such sites can be found in every large town in Germany. Many of these plants were shut down and now await development as old neglected deposits. In Germany, the number of total MGP sites is estimated to be 1000. The International Agency for Research on Cancer (IARC) stated that there is sufficient evidence that some of these compounds such as 4-ringed PAHs chrysene and benzo[a,h]anthracene and the 6-ringed PAH indeno[1,2,3-cd]pyrene are carcinogenic to experimental animals. Research on their toxic properties has focused on genotoxicity. Many nongenotoxic PAHs, however, have been shown to be epigenetically toxic (4). Little is known about the human cancer risk of the widespread distribution of low levels of PAHs in food products and polluted air. Although hundreds of PAHs exist in the environment, the U.S. Environmental Protection Agency (EPA) has identified 16 unsubstituted PAHs as priority pollutants (naphthalene, acenaphthene, acenaphthylene, phenanthrene, anthracene, fluorene, benzo[a]anthracene, chrysene, fluoranthene, pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene) which are monitored routinely for regulatory purposes. The German Drinking Water Act recommends the analytical determination of only six compounds (fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene). In the new European Drinking Water Guideline (98/83/EC) special attention is given to benzo[a]pyrene by setting a threshold limit of 10 ng/L for this single compound. For the analysis of environmental samples, purification and enriching followed by a powerful chromatographic separation and identification is required. Most utilized is HPLC with UV (254 nm) or fluorescent detection. Rapid, more simple, and cost-effective methods would facilitate analytical measurements. A modern approachsimmunochemical techniques (immunoassays)soften can satisfy these criteria and has become a common analytical tool in clinical laboratory practice. Immunoassays are selective and sensitive methods that use biological binding proteins (antibodies) for target analyte detection. Rapidity, ease of use, low cost, and the ability to process large numbers of samples at the same time make this technology of interest to the environmental analytical laboratory. In recent years, an increasing research in the immunochemical determination of environmental pollutants could be noticed. The vast majority of these methods was applied for pesticides. The spectrum will be extended more and more also for other trace contaminants such as polycyclic aromatic compounds, dioxins, polychlorinated biphenyls, and other chemicals (5-8). Initially, commercial immunochemical test kits for the determination of total PAH content in soil were offered by BAKER, Millipore, EnSys (all tube-kits), Quantix (microtiter plate-kit), and SDI (immunofiltration device). Application of these kits for water analysis was only reported by Millipore (LOD 1 µg/L), EnSys (LOD 14.5 µg/L), SDI (30 µg/L), and Quantix (LOD 50 µg/L). A few studies became known which used these test kits for the determination of PAHs in water and soil samples (9-11). Additional immunochemical assays for PAHs were developed at the lab scale and applied for the determination, including immunoextraction, of these compounds in different environmental sample types (12-16). The present paper will VOL. 34, NO. 10, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Scheme of the former manufactured gas plant site. demonstrate the utility of a PAH-enzyme immunoassay (PAHELISA) that was developed at our group for field screening and monitoring of PAH contamination in water and soil samples.
Experimental Section Groundwater Samples. Description of the Sampling Site (Figure 1). Water samples were pumped out from remediation wells (P1 to P5) which were screened in the upper quarternary aquifer downstream of the contaminated area of a former MGP. Gas production for household heating took place at this site until the late 1960s. Thirty years later house-building was started in that area, and, consequently, an increased release of PAHs from contaminated soil into the underlaying aquifer was measured. For in situ remediation of this location it was decided to exploit natural PAH wash-out. For that, the contaminated groundwater was continuously removed, purified with multilayer filters and activated charcoal (at the groundwater cleaning station), and then returned to the aquifer (pump-and treat technology). Remediation process was controlled by the PAH analysis of groundwater samples which were taken from additional downstream wells that were located nearby (A0 to A4) or distant (B1 to B3) from the contaminated area. PAH measurements in groundwater samples from upstream wells (U1, U2) enabled determination of background levels. In general, samples could be characterized as clear, odorless, and colorless; however, samples from protection wells in some cases were yellow-colored, slightly cloudy, and smelled of coal tar. The immunological determination of PAHs was carried out directly in these samples without any pretreatment. For the HPLC analysis a sample cleanup was necessary: To 500 mL of filtered (GFC 6, Schleicher und Schu ¨ ll, Dassel, Germany) water sample were added 10 g of NaCl and 10 mL of cyclohexane. After stirring for 1 h with a magnetic stirrer a volume of 8 mL of the organic phase was separated and evaporated under nitrogen. The residue was taken up with 400 µL of triphenylene in acetonitrile (0.5 ng/µL) as internal standard. Landfill Leachate. Seepage water samples from two different waste deposits were analyzed which were located at different sites: a municipal waste deposit (near Augsburg) and a special waste deposit (near Nu ¨ rnberg). The water sample from the latter was a dark fluid covered by an oily layer (about 10% of the total volume) and with a strong smell of coal tar. This layer was sucked off from the water, and a 10-µL aliquot was diluted 1:1000 with acetonitrile and further analyzed separately for the PAH content with HPLC and ELISA. A 500-mL sample from the municipal waste deposit 2036
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was extracted with cyclohexane as described for the groundwater samples, concentrated in N2 flow nearly to dryness, and redissolved in 8 mL acetonitrile. Five hundred milliliters of seepage water from the special waste deposit was extracted with some higher volume of cyclohexane (25 mL). After separation 20 mL of the organic phase was removed and centrifugated at 5000 g/min for 5 min, and then 10 mL of the cyclohexane was concentrated in N2 flow nearly to dryness and redissolved in 10 mL of acetonitrile. HPLC and ELISA measurements were done as outlined below. Soil Samples. Sample Collection. For fortification experiments five standard soils (air-dried, grain size
