Anal. Chem. 1999, 71, 4023-4029
Simultaneous Determination of PAHs, Hetero-PAHs (N, S, O), and Their Degradation Products in Creosote-Contaminated Soils. Method Development, Validation, and Application to Hazardous Waste Sites Susanne Meyer, Simone Cartellieri, and Hans Steinhart*
Institut fu¨r Biochemie und Lebensmittelchemie, Universita¨t Hamburg, D-20146 Hamburg, Germany
A simple and reproducible method which provides the simultaneous determination of PAHs and hetero-PAHs (N,S,O) and their metabolites in contaminated soils has been developed. Contaminants extracted from the soil sample were separated by polarity and acid-base characteristics using solid phase extraction (SPE) on silica gel and a strong basic anion exchange material. A subfraction containing PANHs and neutral metabolites was subsequently fractionated into neutral and basic compounds using a strong acidic cation exchange material. The identification and quantification was performed using different GC and HPLC methods. A method validation was carried out for 21 PAHs, 22 hetero-PAHs, and 19 metabolites in a five-level matrix calibration. Uncontaminated soil (AhA1-layer/compost mixture) was spiked with the chosen standard compounds. The method showed good linearity (coefficient of correlation) and high precision (coefficient of variation) for major and minor compounds over a wide range of concentration as well as high sensitivity (limit of detection). The method was successfully applied to different authentic tar oil contaminated sites. Aside from the typical tar oil PAHs, hetero-PAHs and metabolites predominantly with ketonic or quinonic structure were identified on a mg/kg concentration level. Soil contamination based on coal tar creosotesa product of high-temperature treatment of hard coalsis one of the most common problems on waste sites. Creosote is a complex mixture of several hundred compounds that can be assigned to four different chemical classes: polycyclic aromatic hydrocarbons (PAH, 85 wt %); hetero-PAHs containing nitrogen, sulfur, or oxygen (PANH, PASH, PAOH, 5-13 wt %); phenolic compounds (1-10 wt %); and monoaromatic hydrocarbons (MAH, 95%) were obtained from Aldrich (Steinheim, Germany), Fluka (Buchs, Switzerland), Merck (Darmstadt, Germany), and Promochem (Wesel, Germany). Fourteen of the 16 EPA-PAHs (except benzo[k]fluoranthene and indeno[1,2,3-cd]pyrene) and methylated and dimethylated derivatives of EPAPAHs as well as indene, biphenyl, and cyclopenta[def]phenanthrene were used as standard compounds during method development and validation. Hetero-PAHs and metabolites used are listed in Table 1. The solvents used were of HPLC grade (nhexane, n-heptane; Biomol, Hamburg, Germany) or of analyticalgrade (hydrochloric acid (30%), trifluoroacetic acid (100%), ammonia (25%); Merck). Dichloromethane, acetonitrile, and methanol (Merck, Darmstadt, Germany) were of synthetic-grade and were distilled before use. Sodium sulfate, potassium hydroxide, and
Figure 1. Analytical protocol for chemical class separation of PAHs, hetero-PAHs, and their metabolites.
potassium dihydrogenphosphate were of analytical-grade (Merck). The SPE materialss8 mL borosilicate glass SPE columns, PTFE frits, stainless steel brass taps with PTFE cones, and silica gel (average particle size 40 µm, particle size distribution 30-60 µm)s were purchased from Baker (Gross-Gerau, Germany). The strong basic anion exchange material, Chromabond SB, and 3-mL polypropylene cartridges containing 500 mg of strong acidic cation exchange material, Chromabond SA, were purchased from Macherey and Nagel (Du¨ren, Germany). Safety Considerations. All national laws concerning the handling of chemicals were observed during the whole work. Soil/Compost Mixture and Authentic Creosote-Contaminated Soils. To avoid varying recoveries due to inhomogeneities of native analytes in authentic contaminated soils, method development and validation was carried out using an uncontaminated AhA1 horizon of a parabrown soil from Northern Germany (1.1% organic carbon; 6.5% kaolin; density, 2.66 g/cm3; maximum water holding capacity, 35.9% (w/w); sieved at 2-mm mesh size) mixed with biocompost to increase the content of organic carbon (degree of maturity, V; maximum water holding capacity, 213% (w/w)) to result in a ratio of 9:1 (w/w), related to dry weight. Soil/compost humidity was adjusted to 55% of the maximum water capacity for method development and validation studies. Data from application of the method to the following creosote-contaminated soils are shown: Contamination A, Sandy soil from a wood-processing plant in Northern Germany (unsaturated zone); Contamination B, Creosote contaminated military existing material in Northern Germany (unsaturated zone). Spiking Procedure. The AhA1/compost mixture was spiked with PAHs, hetero-PAHs, and metabolites dissolved in dichlo-
romethane comparable to a 1% tar oil contamination. The spiked soil was mixed thoroughly in a brown glass vessel. The solvent was evaporated using a gentle stream of nitrogen, and the soil was stored about 16 h in the dark to ensure partial sorption. Optimized Sample Preparation. Twenty grams of contaminated soil was mixed with 1 mL of 1 M hydrochloric acid. Subsequently, the soil was dried by grinding with 20 g of sodium sulfate and extracted for 7.5 h with a mixture of dichloromethane (210 mL) and n-heptane (10 mL) using a Soxhlet apparatus. The dichloromethane was removed by concentrating the resulting extract to about 5 mL by rotary evaporation (40 °C, 600 mbar). The SPE columns were prepared by filling first 0.7 g of Chromabond SB and then 2.0 g of silica gel (dried at 180 °C, 25 h, and deactivated with 10% water (w/w) before use) between three PTFE frits. After equilibration with 12 mL of n-hexane, the Soxhlet extracts were transferred to the columns. The fractions were eluted and collected as shown in Figure 1. PAHs, PASHs, and PAOHs were eluted with 3 mL of n-hexane, 12 mL of n-hexane/ dichloromethane (85:15; v/v), and 2 mL of dichloromethane (fraction 1). Subsequently, PANHs and neutral metabolites were eluted with 1 mL od dichloromethane, 6 mL of methanol, and 3 mL of 0.05 N hydrochloric acid in methanol (fraction 2) followed by a fraction containing acidic metabolites eluted with 6 mL of 0.05 N hydrochloric acid in methanol (fraction 3). Fraction 2 (10 mL) was directly applied to the Chromabond SA cartridge, which was equilibrated with 5 mL of methanol. The neutral PANHs and neutral metabolites were not retained; they eluted immediately with the volume of fraction 2 followed by 5 mL of methanol (fraction 2a). The following fraction containing basic PANHs was eluted with 5 mL of 1 N ammonia in methanol (fraction 2b). Before Analytical Chemistry, Vol. 71, No. 18, September 15, 1999
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gas chromatographic determinations, fractions were diluted and internal standards were added as follows: 9-chloroanthracene and indeno[1,2,3-cd]fluoranthene for fraction 1, 2-chlorophenothiazin for fraction 2a and 3, and indole for fraction 2b, respectively. Validation. Detector linearity was determined by linear regression analyses of five-level (in some cases four level, see Table 1) calibration curves for each analyte (measurement of each level in triplicate). After achieving coefficients of regression r ) 0.99, linear regression equations and coefficients of variation (CV) were calculated. For the matrix calibration, spiked AhA1/compostsoil samples (5 points or 4, respectively, each point in triplicate) were extracted and prepared via the optimized method and analyzed by GC or HPLC, respectively. Coefficients of regression, coefficients of variation, and the linear-regression equations were calculated in addition to average recoveries for each analyte at each spiking level. Method limits of detection (LOD) were calculated from the chromatograms of the lowest matrix calibration level. LODs were calculated as the quantity of analyte required to give a response of 3 × the baseline noise at the expected retention time of the analyte in the chromatogram of nonfortified soil extract. Gas Chromatography-Flame Ionization Detection (GCFID). Quantitative determination of PAHs, PAOHs, and PASHs (fraction 1) during method development and validation was performed with a Carlo Erba HRGC 5160 gas chromatograph equipped with a flame ionization detector and the Chromstar evaluation software (SCPA, Stuhr-Brinkum, Germany). A DB-5 column (30 m × 0.32-mm i.d., 0.25-µm film thickness) (J&W Scientific, Folsom, USA) was used with a temperature program starting at 60 °C (4 min isothermal) and increasing to 300 °C at 6 °C/min (10 min isothermal). Split injection (split ratio, 1:4) was performed at an injector temperature of 300 °C and detector temperature of 320 °C. High-Performance Liquid Chromatography-Diode Array Detection (HPLC-DAD). The HPLC system consisted of a L-6200 low-pressure gradient system, a T-6300 column thermostat, and a AS-4000 autosampler (all Merck/Hitachi, Darmstadt, Germany). For detection a Millipore/Waters (Eschborn, Germany) 994 diode array detector and the evaluation software DART (Ifas, Hamburg, Germany) were connected. For the quantitative determination of fractions 2a and 2b during method development and validation a 150 mm × 4 mm i.d. CC 150-4 Nucleosil 100-5 C18-PAH column and a 8 mm × 4 mm i.d. CC 8/4 Nucleosil 120-5 C18 guard column (Macherey and Nagel, Du¨ren, Germany) were used. The separation of the neutral PANHs and neutral metabolites (fraction 2a) was performed at 35 °C with a flow rate of 1 mL/min and a ternary gradient consisting of acetonitrile (A), 0.1% trifluoroacetic acid in bidistilled water (T), and methanol (M) (Table 2a) and with the detector operating simultaneously at 228, 251, 258, 265, 272, and 288 nm. The separation of the basic PANHs (fraction 2b) was performed at 35 °C with a flow rate of 1 mL/min and a ternary gradient consisting of acetonitrile (A), phosphate buffer (0.68 g of potassium dihydrogenphosphate and 40 mL of 0.1 N potassium hydroxide per liter of bidistilled water, adjusted to pH 7.5) (P), and methanol (M) (Table 2b). The detector was operating simultaneously at 225, 250, 277, and 281 nm. For the quantitative determination of the acidic metabolites (fraction 3) during method development and validation, the system 4026 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999
Table 2. HPLC Gradients for Separation of PANH and Metabolites Fractions 2a and 2b Fraction 2a: Neutral PANH and Metabolites time (min) A (%) T (%) M (%) 0 6 12 17 22 30 42 50 55
time (min) 0 1 12 23 36 42
10 10 10 5 0 0 0 0 0
73 70 60 53 48 40 25 0 0
Fraction 2b: Basic PANH A (%) P (%) 20 20 40 53 100 100
60 60 60 47 0 0
17 20 30 42 52 60 75 100 100
M (%) 20 20 0 0 0 0
was fitted with a 250 mm × 4 mm i.d. ET 250-4 Nucleosil 100-5 C18-HD column and a 11 mm × 4 mm i.d. KS 11/4 Nucleosil 120-5 C18 guard column (Macherey and Nagel). The separation was performed at 35 °C with a flow rate of 1 mL/min and a binary gradient consisting of 0.1% trifluoroacetic acid (T) and methanol (M). The gradient elution program started with 35% T and 65% M which was held for 2 min and then changed linearly to 25% T and 75% M between 2 and 25 min. The detection was performed simultaneously at 238, 252, and 261 nm. Gas Chromatography-Mass Spectrometric Detection (GCMSD). The quantitative determination of PAHs, hetero-PAHs and neutral metabolites in authentic contaminated soils was performed on a Hewlett-Packard (Palo Alto, CA) model 5890 gas chromatograph connected to a HP 5971 mass-selective detector equipped with the HP evaluation unit HP G1043C Rev C.02.00 MS ChemStation and a HP 7673 autosampler. A DB-5 column (30 m × 0.25mm i.d., 0.25 µm film thickness) (J&W Scientific, Folsom, CA) was used. The injection port and MSD interface temperatures were 300 °C. Injections were performed in the splitless mode with the inlet liner purged 1.0 min after injection (split ratio, 1:10). The electron ionization energy was 70 eV, the MSD was operated in the single ion monitoring mode with changing target ions. For fraction 1, the initial oven temperature was 60 °C (5-min isothermal), and then it was programmed at 3.0 °C/min to 240 °C and at 6 °C/min to 300 °C (8-min isothermal). For fraction 2a, the initial oven temperature was 80 °C (1-min isothermal), and then it was programmed at 2.5 °C/min to 240 °C and at 8.0 °C/min to 300 °C (10-min isothermal). For fraction 2b, the initial temperature was 80 °C (1-min isothermal), and then it was programmed at 5 °C/ min to 170 °C (5-min isothermal) and at 6.5 °C/min to 300 °C (11-min isothermal). RESULTS AND DISCUSSION Method Development. An analytical procedure was developed to successfully isolate and separate PAHs and hetero-PAHs and their metabolites from creosote-contaminated soils with their wide range of polarities and different acid-base characteristics.
Table 3. Analytical Characteristics of the Validated Method: PAHs (Five-Level Calibration, Each Level in Triplicate) std calibration curve
matrix calibration curve
group of compds
no. of PAHs
ra
CVb (%)
r
CV (%)
recovery mean (%)
PAHs (GC-FID) two-ring PAHs three-ring PAHs four-ring PAHs five-ring PAHs six-ring PAHs
6 6 5 3 1
0.9981-0.9991 0.9981-0.9996 0.9955-0.9998 0.9973-0.9993 0.9993
2.15-3.12 1.36-3.13 0.99-4.83 1.85-3.76 1.83
0.9801-0.9980 0.9981-0.9993 0.9965-0.9989 0.9953-0.9988 0.9982
3.22-10.24 1.93-3.15 1.94-4.27 2.52-4.93 3.08
30.7-84.2 78.4-94.1 91.0-103.5 88.3-96.7 97.9
a
r, coefficient of correlation. b CV, coefficient of variation.
Soxhlet extraction with dichloromethane was the method of choice because of its simple handling and the excellent extraction characteristics of dichloromethane for nonpolar as well as for polar substances, which is well-documented in the literature.15-17 The extraction time of 7.5 h proved to be sufficient, as only trace amounts of the analytes were released by a subsequent saponification18,19 of the Soxhlet residue (the saponified part was
