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TECHNICAL REPORTS
Organic Compounds in the Environment

Validation of Procedures to Quantify Nonextractable Polycyclic Aromatic Hydrocarbon Residues in Soil

^a HortResearch, East Street, Private Bag 3123, Hamilton, New Zealand
^b Dep. of Environmental Science, Institute of Environmental and Natural Sciences, Lancaster Univ., Lancaster, LA1 4YQ, United Kingdom

^* Corresponding author (gnorthcott{at}hortresearch.co.nz)

Received for publication August 2, 2001.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
This study was conducted to optimize butanol solvent shake extraction, dichloromethane soxtec extraction, and methanolic saponification extraction for the selective extraction of aged polycyclic aromatic hydrocarbons from soil. Extraction kinetics for these methods was established to determine the optimal time necessary to achieve exhaustive compound extraction. This resulted in times of 12, 6, and 5 h, respectively, for butanol, dichloromethane, and saponification, to extract polycyclic aromatic hydrocarbons from previously spiked, then aged soil. Increasing the soil mass to butanol volume ratio reduced the proportion of polycyclic aromatic hydrocarbon extracted by butanol, highlighting the importance of determining and maintaining a constant soil to solvent ratio for comparative purposes. Drying soil samples before dichloromethane soxtec extraction reduced by 30 to 76% the amount of polycyclic aromatic hydrocarbons extracted. The effect of sample drying is discussed with relevance to enhancing the formation of nonextractable compounds in soil and compound losses previously assumed by volatilization. The optimized extraction procedures provided low variability with relative standard deviations 5.2% for analysis of multiple replicates. The results obtained by the optimized procedures provided equivalent or improved reproducibility to those obtained by other methods reported in the literature.

Abbreviations: B[a]P, benzo[a]pyrene • BuOH, n-butanol • DCM, dichloromethane • dpm, disintegrations per minute • LSC, liquid scintillation counting • MSE, methanolic saponification extraction • PAH, polycyclic aromatic hydrocarbon • Phe, phenanthrene • Pyr, pyrene • UGXR, Ultima Gold XR liquid scintillation fluid

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
THERE ARE SEVERAL REASONS for the continued interest in the fate of organic contaminants in soil and sediments, namely:

• quantifying the ecological effects of contaminants by more mechanistic and/or compound-specific risk assessment,
  
• developing bioremediation technologies for contaminated site cleanup, and
  
• conducting process-based studies to advance our understanding of molecular interactions in soils and sediments.
  

Contaminants may be present in more than one labile or recalcitrant form within a sample matrix, where they are subjected to multiple attractive and/or binding processes. The problem currently confronting researchers is the development of systematic procedures for extracting these various compounds from soil and sediment (Jones et al., 1996).

Extraction of environmental solids for the analysis of organic contaminants has traditionally been accomplished by solvent extraction methods. These include batch solvent shaking extraction, continuous soxhlet extraction, and improved versions such as the soxtec extractor and accelerated solvent extraction (Northcott and Jones, 2000c). Differences in the ability of specific aqueous and organic solvents to extract compounds from environmental solids can provide information regarding compound retention mechanisms (Cheng, 1990; Hatzinger and Alexander, 1995; Kubiak et al., 1990). It is generally accepted that solvents extract the available compound fraction of contaminant and not the bound or sequestered residues. Various studies have used n-butanol (BuOH) as a "mild" solvent for extracting the bioavailable, or labile, fraction of polycyclic aromatic hydrocarbons (PAHs) in soil, and dichloromethane (DCM) as a "vigorous" solvent for the quantitative extraction of total extractable PAHs (Hatzinger and Alexander, 1995; Kelsey and Alexander, 1997; White et al., 1997).

Conventional and enhanced solvent extraction techniques do not extract contaminants that are sequestered within mineral and/or organic matter phases of the sample matrix. The extraction of "total" contaminant requires more astringent conditions such as acidic or alkaline solutions and methanolic saponification extraction (MSE) or hydrolysis extractions that release recalcitrant compounds from the sample matrix, or concurrently with it.

Methanolic saponification extraction has been used for the improved extraction of PAHs from marine particulate matter (Heemken et al., 1997), soils (Hartmann, 1996), and sewage sludge (Codina et al., 1994). Dissolution of organic matter (OM) by methanolic hydrolysis releases significant quantities of organic contaminants that are not readily extracted by most solvents because they are strongly adsorbed and/or occluded within soil organic matter (SOM) and sedimentary organic matter (SdOM) (Northcott and Jones, 2000c). Hydrolysis extraction has also been used in combination with solvent extraction methods to provide sequential extraction procedures for determining the bioavailability, sequestration, and formation of PAH-bound residues in soil (Eschenbach et al., 1998; Guthrie and Pfaender, 1998). However, these extreme extraction conditions may still not provide quantitative recovery of sequestered compound(s) in soil and sediment.

Modern extraction technologies, such as ultrasonication, microwave extraction, supercritical fluid extraction (SFE), and accelerated solvent extraction (ASE), enhance the kinetics of compound extraction and can improve compound recovery, in comparison with soxhlet extraction techniques (Northcott and Jones, 2000c). Improved compound extraction recoveries are obtained by using SFE at high temperatures and with the correct choice of modifier(s) (Langenfeld et al., 1993, 1994, 1995; Dean et al., 1995), or ASE at high temperatures (Dean, 1996; Hubert et al., 2000). Supercritical fluid extraction has also been successfully used to determine the desorption behavior and bioavailability of organic contaminants in soil and sediments (Bjorklund et al., 2000; Hawthorne and Grabanski, 2000; Weber and Young, 1997). However, the expense of these technologies means they are not readily available to many laboratories that continue to use traditional based solvent extraction methods.

We undertook a detailed study to examine the sequestration of ¹⁴C-labeled PAHs in sewage sludge–amended, arable soil by abiotic diffusive mechanisms such as intraparticulate diffusion (IPD) and intraorganic matter diffusion (IOMD) in the absence of microbial ameliorated processes. Repeated sludge application in arable soils increases the burden of organic contaminants in the soil as well as the potential for contaminant transfer into field crops and food chains. Therefore, sequestration may be an important process for mediating the availability of contaminants in these soils. We performed a long-term laboratory experiment to test the following hypotheses:

• Polycyclic aromatic hydrocarbon sequestration in soil can be followed by sequential solvent extraction procedures.
  
• Abiotic, diffusive processes play an important role in PAH sequestration in soil with moderate organic carbon content (2–5%).
  
• Progressive compound sequestration affects the dissolution behavior of PAHs aged in soil.
  

The objective of the experiments reported in this paper was to characterize comprehensively those solvent and saponification extraction methods that were available to us and would be used to extract, and determine the distribution of, aged ¹⁴C-labeled PAH residues in soil. To test our hypotheses we required reliable and reproducible experimental data from rigorously validated extraction methods. The following aspects of the extraction methodologies were considered:

• the kinetics of compound extraction, to ensure exhaustive compound extraction by a specific solvent;
  
• the reproducibility of extraction procedures, to determine statistically measurable changes in compound distribution as a function of aging, and to make comparative assessments between different compounds; and
  
• the limitations of each extraction method, to interpret the obtained experimental data.
  

The findings of this study are divided into the following sections:

• Experiment A: Evaluation and optimization of a butanol shake extraction and separation method for determining the "labile" extractable PAH fraction;
  
• Experiment B: Evaluation and optimization of a DCM soxtec extraction method for determining total solvent extractable PAHs, and
  
• Experiment C: Evaluation and optimization of an MSE method for determining the fraction of PAHs retained in soil organic matter after solvent extraction.
  

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Chemicals
Nonradiolabeled phenanthrene (Phe), pyrene (Pyr), benzo[a]pyrene (B[a]P), and the ¹⁴C-radiolabeled analogs ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P were obtained from Sigma-Aldrich Co. Ltd., Dorset, UK. Nonradiolabeled Phe (96% purity), Pyr (99% purity), and B[a]P (>98% purity), were obtained as crystalline solids, while ¹⁴C-9-Phe (98% radiopurity), ¹⁴C-4,5,9,10-Pyr (95% radiopurity), and ¹⁴C-7-B[a]P (98% radiopurity) were supplied in 1 mL of toluene with individual activities of 3.7 x 10⁶ Bq. Acetone, BuOH, DCM, methanol (MeOH), and toluene were obtained from Rathburn Chemicals Ltd., Walkerburn, UK. Sample oxidizer reagents, Carbosorb E and Permafluor-E, the organic-based combustion aid solution Combustaid, sample combustion cones and pads, ¹⁴C-Spec-Chec solution, Ultima Gold XR scintillation cocktail (UGXR), and 23-mL plastic scintillation vials were obtained from Canberra Packard (Meriden, CT). Anhydrous sodium sulfate and analytical grade potassium hydroxide were obtained from Merck (Darmstadt, Germany).

Individual 10 000 mg L^-1 stock solutions of Phe, Pyr, and B[a]P were prepared by dissolving 0.100 g of the solid in 10 mL of acetone in a volumetric flask. Due to limited solubility of B[a]P in acetone, the 10 000 mg L^-1 B[a]P stock solution was prepared by dissolving 0.100 g of solid B[a]P in a minimum volume of toluene, which was made to a final volume of 10 mL with acetone. Spiking solutions of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P were prepared by adding aliquots of each radiolabeled stock standard to 10 mL of the corresponding nonradiolabeled 10 000 mg L^-1 PAH standard in acetone. The activities of the working standards prepared in this manner were 79.6, 96.1, and 89.9 Bq µL^-1 for ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P, respectively.

Experimental
Soils
Field-wet, subsurface soil was collected from a 5- to 20-cm sampling depth at a rural hillside grassland plot (Lancaster University, Hazelrigg Field Station, UK-O.S. Sheet 102, Coordinates 493578) and a rural arable field (Wyre Valley, Lancashire, UK-O.S. Sheet 102, Coordinates 397338). The field-wet soils were passed through a 10-mm sieve to remove gravel and crop stalks followed by rubbing through a 2-mm sieve to remove small pebbles, and medium to large root and crop detritus. Soil dry weight was determined by drying the sieved soil to a constant weight at 105°C. Total carbon (TC) and total inorganic carbon (TIC) were measured with a Carlo Erba (Milan, Italy) CHNSO EA 1108-R elemental analyzer. Total C was measured by combusting approximately 25 mg of the previously oven-dried soil. A portion of this soil was combusted at 450°C for 6 h to remove organic carbon while retaining inorganic carbon (Ben-Dor and Banin, 1989; Donkin, 1991). Residual TIC was measured by elemental analysis of approximately 50 mg of the combusted soil residues. Total organic carbon (TOC) was calculated as TOC = TC - TIC. The Soil Survey and Land Research Centre, Cranfield University, Bedford, UK conducted the remaining chemical and physical analyses. The results of soil chemical and physical measurements are presented in Table 1.

Soil Spiking Procedures
Field-wet grassland and arable crop soil that had been amended with concentrated sewage sludge solids were spiked with the appropriate ¹⁴C radiolabeled and nonradiolabeled analog PAH using our previously evaluated and optimized procedure (Northcott and Jones, 2000b). Briefly, 250 g of field-wet soil was added to a blender containing 10 mL of acetone and 250 µL of the appropriate PAH spiking standard. The contents were blended to mix and transferred to amber glass microcosms. Grassland soil was spiked to a concentration that was representative of a marginally contaminated site within the UK (10 mg kg^-1) for Phe and B[a]P, and 50 Bq g^-1 (2986 disintegrations per minute [dpm] g^-1) and 43 Bq g^-1 (2580 dpm g^-1) for ¹⁴C-9-Phe and ¹⁴C-7-B[a]P, respectively. The arable soil was amended with 6.7 g of centrifuge-concentrated sewage sludge and spiked to a concentration of 10 mg kg^-1 with nonradiolabeled Phe, Pyr, and B[a]P and 79.6 (4776 dpm g^-1), 96.1 (5766 dpm g^-1), and 89.9 Bq g^-1 (5394 dpm g^-1) with ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P, respectively. Unspiked blanks containing arable soil amended with sewage sludge were prepared with the same spiking procedure and an equivalent volume of acetone. The prepared arable soil microcosms that were amended with sewage sludge were sterilized by irradiation (Northcott and Jones, 2000b).

Activity of Spiked Soil, Extracted Soil Residues, and Soil Solvent Extracts
An aliquot of 1 to 2 g of weighed sample (soil or extracted soil residue) was packed into paper combustion cones, capped with a combustion pad, and combusted for three minutes with a Canberra Packard Model 307 sample oxidizer. Filter papers containing retained solids were crushed by hand into paper combustion cones and oxidized. Combustion was assisted by the addition of 100 and 200 µL Combustaid to the soil and filter samples, respectively. Evolved ¹⁴CO₂ was trapped in 10 mL of Carbosorb E and eluted and mixed with 10 mL of Permafluor E scintillation cocktail in 23-mL scintillation vials. Trapping efficiency of the combustion process was established to be 98%. Activity in each sample vial was counted with a Canberra Packard Tri-Carb 250CA liquid scintillation analyzer for a period of 10 min or sufficient time to provide 50x background count values.

The activity of radiolabeled compound extracted by BuOH, DCM, and methanolic potassium hydroxide (KOH) was measured by pipetting 5 to 10 mL of solvent extract into 23-mL scintillation vials containing 10 to 17 mL of UGXR. Vials prepared in this manner were capped, shaken, and stored overnight in the dark to stabilize before the activity was measured by liquid scintillation counting (LSC). This stabilization period was necessary to counter the effects of chemiluminescence and photoluminescence induced by the interaction of ultraviolet radiation and various solvents with UGXR. Blank samples for total sample oxidation and solvent extraction were prepared with samples of field-wet soil blanks. Sample activity of oxidized samples was corrected for trapping efficiency and blank subtraction.

Individual quench curves were prepared with solvent or methanolic saponification extracts from unspiked soil blanks to correct variable chemical and color quenching in sample extracts. Mass balances were estimated for each compound after soxtec and saponification extraction by sample oxidation of soil residues.

Experiment A1: Soil n-Butanol Extraction
A previously reported BuOH soil extraction scheme was adopted for initial method evaluation (Hatzinger and Alexander, 1995). Approximately 2.5 g of nonsterile two-month-aged ¹⁴C-9-Phe spiked grassland soil was weighed in triplicate into individual 50-mL Teflon centrifuge tubes containing 5 mL of BuOH. Samples of unspiked soil were prepared as analytical blanks. The soil was extracted by shaking the sealed tubes on a rotating flat-bed shaker (IKA-Labortechnik KS 250; Janke and Kunkel, Staufen, Germany) at 150 rpm for times of 5 min, 15 min, and 1, 6, 12, and 24 h. The soil and BuOH were separated by filtration through a 150-mm Whatman (Maidstone, UK) 1 filter paper folded within a glass funnel. The centrifuge tube and filter paper were rinsed twice with 2.5 mL of BuOH to wash residual extracted compound directly into a scintillation vial. The filter paper was left to drain for 30 min, and 10 mL of UGXR scintillation fluid was added to the vial. The extracted ¹⁴C-9-Phe was measured by LSC.

Experiment A2: Recovery of ¹⁴C-9-Phenanthrene by Filter Paper and Centrifuge Separation
The following experiment was performed to identify and assess the loss of ¹⁴C-9-Phe during the filter paper separation procedure:

1. A 1.75-g aliquot of Na₂SO₄ was weighed into individual triplicate Teflon centrifuge tubes and 5 mL of BuOH and 5 µL ¹⁴C-9-Phe spike solution were added (5670 dpm ¹⁴C-9-Phe). The tubes were sealed, shaken for 60 min, and separated with the filter paper procedure described in Experiment A1. The scintillation vial was replaced with a fresh vial and the Teflon centrifuge tube and filter paper contents were rinsed four times with 2.5-mL individual BuOH rinses. The filter and contents were rinsed with two individual 10-mL aliquots of BuOH, which were also collected in individual scintillation vials. The ¹⁴C-9-Phe recovery standards were prepared by adding 5 µL x 1134 dpm µL^{-1 14}C-9-Phe standard directly into scintillation vials containing 10 mL of BuOH.
   
2. As above for Step 1 but without Na₂SO₄.
   

The recovery of ¹⁴C-9-Phe from the BuOH filtration procedures was compared with that obtained by direct sampling of butanol from centrifuge tubes. The ¹⁴C-9-Phe solution was spiked in sets of five replicates as described below.

1. A 5-µL aliquot of 1134 dpm µL^{-1 14}C-9-Phe solution was added to replicate 50-mL Teflon centrifuge tubes containing 5 mL of BuOH. The tubes were sealed and shaken for one hour on a flat-bed shaker. After shaking, the BuOH was poured directly into scintillation vials along with two 2.5-mL individual BuOH rinses of the centrifuge tubes.
   
2. A 5-µL aliquot of 1134 dpm µL^{-1 14}C-9-Phe solution was added to 10 mL of BuOH in 50-mL Teflon centrifuge tubes containing 1.75 g of Na₂SO₄. The tubes were sealed and shaken for one hour on a flat-bed shaker, and 5 mL of BuOH was transferred to scintillation vials by hand-operated pipette.
   
3. Spike recovery standards were prepared by spiking 5 µL x 1134 dpm µL^{-1 14}C-9-Phe solution directly into scintillation vials containing 10 mL of BuOH.
   

Ten milliliters of UGXR scintillation fluid was added to all vials and the activity of recovered ¹⁴C-9-Phe was determined by LSC.

Experiment A3: n-Butanol Extraction Kinetics
Triplicate 3.5-g samples of ¹⁴C-9-Phe- or ¹⁴C-7-B[a]P-spiked grassland soil were weighed directly into 50-mL Teflon centrifuge tubes. Thirty-five milliliters of BuOH was added to the centrifuge tubes, which were sealed and shaken for predetermined times. The tubes were centrifuged (MSE-Centaur 2; Sanyo Electric Co., Moriguchi City, Japan) at 3000 x g for 30 min to separate the soil and BuOH. Ten milliliters of BuOH was transferred to 23-mL plastic scintillation vials containing 10 mL of UGXR and compound activity was determined by LSC.

Experiment A4: Effect of Soil to n-Butanol Ratio
Aliquots of 1, 2.5, 5, 7.5, and 10 g ¹⁴C-7-B[a]P-spiked soil were extracted in triplicate with 35 mL of BuOH with the centrifuge tube extraction and separation technique (see Results section). The percentage of BuOH-extracted compound was calculated by dividing the total BuOH-extracted ¹⁴C-7-B[a]P activity by the total ¹⁴C-7-B[a]P activity in the mass of extracted soil.

Experiment B1: Optimization of Dichloromethane Soxtec Extraction of Polycyclic Aromatic Hydrocarbons in Soil
Approximately 2.5 g of ¹⁴C-9-Phe- or ¹⁴C-7-B[a]P-spiked grassland soil or sterile arable soil amended with sewage sludge was weighed in triplicate, then individually mixed and ground with 10 g of anhydrous sodium sulfate in a ceramic mortar and pestle to a dry, fine powder. The ground soil mixtures were transferred into 25- x 60-mm Whatman cellulose extraction thimbles and topped with a piece of DCM-extracted cotton wool. The thimbles were mounted in a Soxtec System HT 1043 extraction unit (Foss Tecator AB, Höganäs, Sweden) and lowered into beakers containing 40 mL of boiling DCM for 30 min. After 30 min the thimbles were raised to the rinsing position where they were continually extracted and rinsed with freshly distilled DCM. Extraction periods were 30 min, 1, 2, 4, 6, and 9 h. At the completion of extraction, the DCM extracts were transferred to 50-mL measuring cylinders with three 5-mL DCM rinses and adjusted to a final volume of 45 mL with DCM. Ten milliliters of DCM extract was transferred to a plastic scintillation vial containing 10 mL of UGXR scintillation fluid and the extracted compound determined by LSC.

The degree of compound carryover between extractions was periodically determined by replacing the extracted sample thimbles and DCM sample extracts with fresh blank thimbles and beakers of DCM immediately after the completion of sample extraction. The carryover samples were extracted for a total of 6 h and the activity determined as described above.

Experiment B2: Effect of Sample Drying on Extractability
A 30-g aliquot of wet soil was sampled from microcosms that contained sterile, arable sewage sludge–amended soil that had been spiked with ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P and aged for 50 d. The sampled, field-wet soil was weighed into disposable aluminium weighing pans, loosely covered with aluminium foil, and air-dried for 24 h in a fume hood. The dry weight of each soil sample was measured to convert dry weight results to a wet-weight basis for later comparison. The air-dried soil was ground to a fine powder with a mortar and pestle, then stored in 20-mL glass screw cap vials to maintain moisture content before analysis. The total activity of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P in the freshly sampled field-wet and corresponding air-dried samples were measured by sample oxidation (six replicates) followed by LSC. Total activities on a wet basis were calculated from the wet and dry measurements as above.

Six replicates of each wet soil sample were prepared for soxtec extraction as described for Experiment B2 and extracted for a total of 6 h. An additional six 2-g replicates of each air-dried soil sample were weighed directly into Whatman cellulose extraction thimbles and similarly extracted by DCM soxtec. After extraction the thimbles were left overnight in the fume hood to remove residual solvent, then the thimble contents were transferred to glass scintillation vials and weighed. The activity of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P remaining in the residues was measured by total sample oxidation followed by LSC. The remaining residues from wet-soil soxhlet extractions were combined for each individual PAH and used in the MSE optimization experiment.

Experiment C1: Optimization of Methanolic Saponification Extraction of Polycyclic Aromatic Hydrocarbon Residues in Solvent-Extracted Soil
Triplicates of approximately 2 g of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P-spiked residue from soxtec extractions were weighed into 50-mL teflon test tubes. Methanolic KOH lye (9 mL; 14:1 MeOH to 2 M KOH; pH 12) was added to the tubes, which were securely sealed and weighed before refluxing in a water bath at 95°C. Triplicate tubes containing ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P saponified soil residues were removed after 30 min, 1, 3, 5, and 7 h, and cooled in a sink of cold water. The centrifuge tubes were weighed to determine solvent losses during refluxing, with any loss corrected by the addition of MeOH. The tubes were centrifuged with a MSE-Centaur 2 at 3000 x g for 30 min to separate the saponification lye and extracted solid residue. Five milliliters of saponification lye was removed from each tube to a 23-mL scintillation vial containing 17 mL of UGXR scintillation fluid, then the activity was measured by LSC.

The solids remaining after saponification were recovered by filtration through Whatman 1 filter paper folded in a glass funnel. The teflon centrifuge tubes and the filter were rinsed three times with 3-mL aliquots of MeOH. The filter and contents were rinsed with another two 5-mL aliquots of MeOH and dried overnight in a fumehood, then the combined filter contents were oxidized to measure the nonextractable ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P residues.

Experiment C2: Polycyclic Aromatic Hydrocarbon Recovery during Methanolic Saponification Extraction
A spiking solution of 5 µL of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P in MeOH was individually added to 12 replicate teflon centrifuge tubes that contained 2 g of Na₂SO₄ and 9 mL of methanolic KOH. Six of the replicate tubes spiked with each PAH were subjected to MSE as described above, and the remaining six tubes were retained as controls. After MSE, 5 mL of saponification lye was subsampled from the six extraction and six control tubes and combined with 17 mL of UGXR in 23-mL scintillation vials. The Na₂SO₄ in each tube was recovered on filter paper and oxidized to determine the amount of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P activity retained by the solids during filtration. Standards for estimating spike recovery were prepared by adding 5 µL of the ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P solution directly into saponification lye and UGXR. Solution blanks were similarly prepared except for the radiolabeled spike. Activities of the compounds in each vial were determined by LSC.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Experiment A: Soil n-Butanol Extraction (Experiment A1)
A number of problems were observed with the BuOH extraction procedure. First, BuOH wicked up the filter papers and carried co-extracted, colored soil components that produced a chromatographic effect along the outer edges of the filter paper. Second, only 5 to 7 mL of BuOH was recovered, leaving a substantial portion of solvent and extractant on the filter paper, resulting in poor extraction reproducibility.

Recovery of ¹⁴C-9-Phenanthrene (Experiment A2)
¹⁴C-9-Phenanthrene continued to be released from the combined Na₂SO₄ filter papers with increasing volume of BuOH rinses. About 85% of the added ¹⁴C-9-Phe was recovered after 40 mL of BuOH rinses had been collected, and this increased to 91% in the absence of Na₂SO₄ when BuOH passed through the filter paper alone. This result indicates that a significant portion of extracted ¹⁴C-9-Phe remained in BuOH that was retained by the filtered solids and filter paper itself. Complete recovery of ¹⁴C-9-Phe was accomplished by spiking directly into BuOH in scintillation vials. Thus, ¹⁴C-9-Phe was not lost during dispensing of the standard. In contrast to the filtration separation procedure, 100% recovery of ¹⁴C-9-Phe was achieved by both centrifuge tube procedures.

Butanol Extraction Kinetics (Experiment A3)
The measured activity in each vial was scaled by the corresponding volumetric dilution to obtain total BuOH-extracted activity. This measured activity was divided by the total activity of the compound in the soil, measured by total sample oxidation, to obtain the percentage BuOH-extracted activity at each extraction time. The amount of ¹⁴C-9-Phe and ¹⁴C-7-B[a]P extracted from the spiked and aged soil continued to increase until six hours extraction time, after which the extraction remained statistically constant (Fig. 1) . To ensure effective and reproducible extraction of PAHs from soil with BuOH, a standard extraction time of 12 h was adopted.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Butanol extraction kinetics for 14C-9-phenanthrene (14C-9-Phe) and 14C-7-benzo[a]pyrene (14C-7-B[a]P). Data points represent the mean, and error bars the 95% confidence interval, of triplicate measurements.

• depleting the available BuOH-extractable compound fraction in the soil, or
  
• reaching and maintaining compound equilibrium between the soil and BuOH due to limited compound solubility in BuOH.
  

Soil spiked with ¹⁴C-7-B[a]P was used in this experiment because B[a]P has a substantially lower solubility in alcohol-based solvents than Phe and Pyr, and this decreased solubility allowed us to differentiate better the two processes described above.

The percentage of compound extracted with BuOH for each mass of soil was calculated by dividing the total BuOH-extracted ¹⁴C-7-B[a]P activity by the total ¹⁴C-7-B[a]P activity in the mass of extracted soil. The plot of percentage BuOH-extracted ¹⁴C-7-B[a]P and mass of extracted soil shows that a decreasing proportion of ¹⁴C-7-B[a]P was extracted with increasing soil mass (Fig. 2) . This trend implies that the solubility of ¹⁴C-7-B[a]P in BuOH influenced the amount extracted. If the amount of labile compound, not the amount of solvent, was the limiting factor, we would expect the proportion of extracted ¹⁴C-7-B[a]P to remain constant as the mass of extracted soil, and therefore the mass of extracted compound, increased.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Effect of soil mass on the amount of 14C-7-benzo[a]pyrene (14C-7-B[a]P) extracted in butanol. Data points represent the mean, and error bars are the 95% confidence interval, of triplicate measurements.

The amount of ¹⁴C-9-Phe and ¹⁴C-7-B[a]P extracted from the spiked grassland soil remained statistically constant (p = 0.05) after two or one hours of soxtec extraction (Table 2). In comparison, the amount of ¹⁴C-9-Phe and ¹⁴C-7-B[a]P extracted from the sterile arable soil amended with sewage sludge increased over the first two hours and remained statistically constant (p = 0.05) after four or two hours of soxtec extraction (Table 2). Total sample oxidation of the spiked and sterilized arable soil showed that ¹⁴C-9-Phe and ¹⁴C-7-B[a]P activity was quantitatively maintained. The increased recovery of ¹⁴C-9-Phe in the sterilized arable soil results from the inhibition of microbiological activity by treatment with irradiation. A standard extraction time of six hours was adopted for subsequent extraction of ¹⁴C-9-Phe and ¹⁴C-7-B[a]P from the spiked and sterilized arable soil amended with sewage sludge.

Sample Drying and Extractability (Experiment B2)
This experiment was performed to determine the effect of drying on the extractability of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P spiked and aged in sterile arable soil that was amended with sewage sludge. The average dry weight of the three samples was 0.834 ± 0.012 kg kg^-1 sampled soil. To compare results this value was used to convert the mass of oxidized dried soil to a wet weight equivalent. Total oxidation results show that the activity of ¹⁴C-9-Phe and ¹⁴C-7-B[a]P in wet and air-dried soil were statistically indiscernible (p = 0.05) from each other (Table 3). There is a small but not significant statistical (p = 0.06) difference between the mean total activity of ¹⁴C-4,5,9,10-Pyr obtained for wet and air-dried soil. Therefore, drying soil under the specified conditions did not result in significant losses of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, or ¹⁴C-7-B[a]P by volatilization.

Optimization of Methanolic Saponification Extraction Procedure (Experiments C1 and C2)
The percentage of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P that remained in soil after MSE was calculated by dividing the compound activity in the MSE-extracted residues by that of the corresponding soxtec-extracted soil residues. The amount of nonextractable compound in the soil residues decreased through five hours of MSE extraction, then remained constant for all three compounds (Table 4). After 3 h of extraction there was no statistically significant increase in the amount of ¹⁴C-9-Phe or ¹⁴C-4,5,9,10-Pyr extracted by saponification. No further increase in the amount of saponification-extracted ¹⁴C-7-B[a]P occurred after 5 h.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

The results of varying the soil to BuOH ratio in Experiment A4 demonstrate the need to maintain a constant soil to BuOH ratio when extracting PAHs. This is critical if differences in the amount of compound extracted with BuOH are to be observed as a function of properties or aging of the extracted compound.

General Comments on the n-Butanol Extraction Methodology
Various soil–BuOH extraction methods have been reported in the research literature. These include mixing by inversion (Kelsey et al., 1997), shaking by hand for 2 min (Hatzinger and Alexander, 1995; White and Alexander, 1996), mechanical shaking for 24 h (Kelsey and Alexander, 1997), and vortex mixing for 30 s (White et al., 1998). Various soil to BuOH ratios have also been used, including 1:1.875 (White and Alexander, 1996), 1:2.5 (Hatzinger and Alexander, 1995; Kelsey and Alexander, 1997; Kelsey et al., 1997; White et al., 1997, 1998), and 1:5 (White et al., 1998). The separation of extracted soil and butanol has mainly been accomplished with filter paper methods (Hatzinger and Alexander, 1995; Kelsey et al., 1997; White and Alexander, 1996; White et al., 1997, 1998), and, in one case, by centrifugation (Kelsey and Alexander, 1997). The fraction of compound extracted by hand or mechanical shaking has been used to estimate the bioavailable fraction of compound in soil. However, the authors provide no explanation of the methodological variations they adopted or how these variations affect the amount of compound extracted by BuOH or the bioavailability of the compound. Our results clearly show that (i) the amount of compound extracted from soil by BuOH increases during six hours of shake extraction, then reaches an equilibrium concentration (Fig. 1); (ii) filtration does not provide quantitative compound recovery; and (iii) the amount of BuOH-extractable compound is inversely proportional to the soil to BuOH ratio. It is therefore difficult to compare directly the results obtained with the aforementioned methods. We believe our optimized BuOH soil extraction method provides a significant improvement over previous reported methods. Adopting a recognized and accepted soil to solvent ratio of 1:10 (as used in compound sorption–desorption experiments) provides direct comparison with results obtained in soil–water partitioning and sorption–desorption experiments. Furthermore, increasing the extraction time until equilibrium is reached between soil and BuOH provides a more reproducible and meaningful measure of the bioavailable or "labile" fraction of organic contaminant(s) in soil. Most importantly, a standardized and optimized BuOH extraction procedure will allow meaningful comparisons of different data sets.

We adopted a standardized BuOH extraction procedure as described below, based on the findings presented earlier:

1. Soil (3.5 g) is extracted with 35 mL of BuOH for 12 h in 50-mL centrifuge tubes on a flat-bed shaker.
   
2. Extracted soil and butanol are separated by centrifugation at 3000 x g for 30 min.
   
3. Ten milliliters of BuOH is transferred by hand-operated pipette to a plastic scintillation vial containing 10 mL of UGXR scintillation fluid.
   

Since BuOH is miscible with water, this extraction method can be applied directly to wet soil and sediment samples, thereby eliminating artifacts introduced by sample drying. The described method was optimized and validated for the extraction of PAHs from two specific soil types and we recommend further method evaluation when using different soil types and/or other organic contaminant(s).

It is important to note that while BuOH solvent extraction has been used to provide estimates for the bioavailable fraction of contaminants in soils and sediments, the supporting experimental evidence appears confusing, and sometimes contradictory. Other techniques such as solid-phase microextraction (Mayer et al., 2000; Sijm et al., 2000), supercritical fluid extraction (SFE) (Bjorklund et al., 2000; Nilsson et al., 2002), and subcritical water extraction (Konda et al., 2002; Yang et al., 1995) may provide more meaningful chemical assays to predict the bioavailability of contaminants in soils and sediments.

Experiment B: Optimization of Dichloromethane Soxtec Extraction Procedure
Dichloromethane is an effective solvent for the extraction of PAHs from soil and sediment samples (Holoubek et al., 1990; Lopez-Avila et al., 1993; Wilcock and Northcott, 1995). Polycyclic aromatic hydrocarbons and other organic contaminants are commonly extracted from soil and sediment by soxhlet extraction. Efficient and rapid extraction of organic compounds occurs by immersing the sample directly into boiling solvent for a predetermined time (Lopez-Avila et al., 1993).

Spiked, nonsterile grassland soil and sterile, arable soil amended with sewage sludge were used in the experiment to estimate soxtec extraction kinetics and to determine whether different soil types, and the absence of biological activity, affected the rate and extent of PAH extraction. Extraction recoveries of 92 ± 2% and 75.5 ± 2.2% (mean ± 95% confidence interval, n = 3) were obtained for ¹⁴C-9-Phe or ¹⁴C-7-B[a]P respectively, from spiked and aged grassland soil. The total activity of ¹⁴C-9-Phe in this soil, as measured by total sample oxidation, was 20% of that measured immediately after spiking. This indicates that significant degradation of ¹⁴C-9-Phe occurred in the nonsterile grassland soil during the seven-month aging period following spiking. In comparison, 100% recovery of ¹⁴C-7-B[a]P activity was obtained by sample oxidation, indicating the recalcitrant nature and stability of higher molecular weight, polycondensed PAHs in microbially active soil. Similar recovery of ¹⁴C-7-B[a]P from both soils, as measured by soxtec extraction and total sample oxidation, indicates that microbial degradation was not responsible for the 75% recovery of ¹⁴C-7-B[a]P by soxtec extraction. This recovery suggests that sequestration of ¹⁴C-7-B[a]P within the organic carbon and/or mineral phases of soil limited its extractability in the biologically active grassland and sterile, arable soil amended with sewage sludge.

Soil and sediment samples are often dried and mixed to improve sample homogeneity and ease of handling, increase the mass of extracted substrate, and allow the use of water-immiscible extraction solvents. However, drying introduces significant changes in the partitioning behavior, bioavailability, sequestration, and extractability of organic contaminants (Altfelder et al., 1999; Kottler et al., 2001; Price and Birge, 1999; Shelton et al., 1995; White et al., 1997, 1998). A number of processes contribute to these changes and are discussed in detail elsewhere (Northcott and Jones, 2000a). The magnitude and type of changes in organic contaminant behavior introduced by soil drying are difficult to predict. Changes introduced by soil drying depend on the physical and chemical properties of the contaminants and soil (White et al., 1998).

It was surprising to find that 95% confidence intervals for total compound activity in air-dried soil were larger than those for wet soil. We expected to obtain a more homogeneous distribution of compound in the crushed, mixed, and air-dried soil samples. It appears that air-drying may have produced localized concentrations of radiolabeled PAHs, and contributed to a wider range of activity within the analyzed replicates.

The results of the soil drying experiment raise some important issues. While extractability of the compounds decreased with sample drying, there was no corresponding decrease in the total activity of ¹⁴C-9-Phe, ¹⁴C-4,5,9,10-Pyr, and ¹⁴C-7-B[a]P in air-dried soil. This effect has been previously observed for air-dried soil samples containing radiolabeled atrazine (Kottler et al., 2001; Shelton et al., 1995) and PAH residues (Kottler et al., 2001; White et al., 1997, 1998). Reduced compound extractability upon soil or sediment drying can result from:

• water removal from adsorption sites increasing the sorption capacity and energy of solute–sorbent interactions;
  
• dehydration and shrinkage–condensation and/or conformational changes of the humic macromolecular polymer promoting compound sequestration by intraorganic matter diffusion; and/or
  
• the collapse of aggregate structures and clay mineral nanopores and lattices to physically entrap compound molecules and increase the proportion of diffusion-retarded compound by intraparticle diffusion mechanisms.
  

Investigations where organic chemicals in soil and sediment are measured by solvent extraction methods are unable to quantify sequestered or bound compound fractions. Reductions in the amount of compound extracted on sample drying are generally attributed to compound volatilization. This has been supported by observations of increased apparent loss of more volatile compounds within a specific compound class (PAHs and polychrlorinated biphenyls [PCBs]) on drying soil or sediment (Auer and Malissa, 1990; Berset et al., 1999; Chiarenzelli et al., 1996). However, we believe that reduced compound extractability on drying soil and sediment samples results from the formation of nonextractable residues by sequestration of parent compound or compounds within the soil or sediment matrix, and loss by volatilization (Altfelder et al., 1999; Shelton et al., 1995; White et al., 1997, 1998). Compound volatilization may be the dominant "loss" process for volatile compounds and when astringent drying procedures are employed, that is, freeze-drying and forced-air oven drying (Berset et al., 1999; Guggenberger et al., 1996). However, our data show that compound sequestration is the dominant process that reduces the extractability of PAHs and most likely other semivolatile compound classes on controlled soil drying. Therefore, if it is necessary to remove water from soil or sediment samples before solvent extraction, we recommend mixing the sample with anhydrous Na₂SO₄.

We standardized the DCM soxtec extraction procedure as described below based on the findings presented above:

1. Field-wet soil (2.5 g) was mixed and ground with 10 g of anhydrous Na₂SO₄ with a mortar and pestle to obtain a dry free-flowing powder.
   
2. The soil–Na₂SO₄ mixture was soxtec-extracted with 40 mL of DCM by immersion and boiling for 30 min followed by 5.5 h solvent rinsing.
   
3. After returning to room temperature the DCM extract was transferred to a 50-mL measuring cylinder with three 5-mL DCM rinses and adjusted to 45 mL with DCM.
   
4. Ten milliliters of DCM was transferred by hand-operated pipette to a plastic scintillation vial containing 10 mL of UGXR scintillation fluid.
   

Experiment C: Optimization of the Methanolic Saponification Extraction Procedure
Benzo[a]pyrene required an extraction time of five hours compared with three hours for Phe and Pyr before a constant amount was released from the soil matrix by MSE (Table 4). This may reflect the greater affinity (K_oc) of B[a]P for soil organic matter phases compared with ¹⁴C-9-Phe and ¹⁴C-4,5,9,10-Pyr, and the time required for complete extraction of hydrolyzable soil organic matter and corresponding sequestered ¹⁴C-7-B[a]P. An extraction time of five hours was previously used for MSE of PAHs from solvent-extracted soil residues (Eschenbach et al., 1994). Our results confirm that five hours is an optimum time for MSE of PAH residues from previously extracted soil residues.

Compound recovery obtained by MSE showed, for all test compounds, that no measurable losses occurred by volatilization or retention–sorption to experimental materials. This finding contrasts with the retention of PAHs observed during the BuOH extraction procedure and demonstrates the efficiency of MeOH for rinsing extracted compounds from the filter paper and retained solids.

Summary of Optimized Extraction Methods
Some aspects of the optimized extraction scheme require further discussion (Fig. 3) . After BuOH extraction, the centrifuged soil plug retained traces of BuOH. This precluded sample oxidation of the BuOH-extracted soil residues because the high temperatures (900°C) produced an explosive BuOH mixture. Furthermore, due to the presence of BuOH traces and the corresponding extracted compound in the soil plug, we did not carry out a sequential extraction with DCM. Instead we used separate subsamples for soxtec extraction. The activity of compound in DCM soxtec and MSE extracts was determined by LSC, and the amount of compound remaining in extracted soil residues by sample oxidation and LSC. Excellent agreement was achieved between the activity measured in these extract solutions and the calculated difference in the activity of residues oxidized before and after extraction (<2% difference).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Flow chart of the combined soil extraction scheme. PAH, polycyclic aromatic hydrocarbon; BuOH, butanol; DCM, dichloromethane; UGXR, Ultima Gold XR scintillation fluid; MSE, methanolic saponification extraction.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
This paper presents a combined procedure for determining the extractability of labile and sequestered forms of PAHs in soil. The validated extraction protocol (Fig. 3) combines the optimized efficiency for each individual procedure with other important advantages. The soils are extracted with solvent without prior drying, which introduces significant changes to compound extractability. By using DCM soxtec extraction and a modified small-scale MSE procedure, we have significantly reduced the volume of solvent required to achieve exhaustive compound extraction and the exposure risks to laboratory personnel. The variability of experimental data obtained by each extraction method is small and further validates our optimized procedures. While new extraction technologies are available, many laboratories do not yet have access to them and traditional solvent-based extraction methods remain their method(s) of choice. Our optimized extraction procedure represents a refinement and enhancement of traditional solvent extraction methods.

	ACKNOWLEDGMENTS

 
The authors wish to acknowledge the Food Contaminants Division of the Ministry of Agriculture, Fisheries and Food, UK, for funding this research.

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