Environ. Sci. Technol. 1998, 32, 3224-3227
Evaluation of Spiking Procedures for the Introduction of Poorly Water Soluble Contaminants into Soil BRIAN J. REID, GRANT L. NORTHCOTT, KEVIN C. JONES, AND KIRK T. SEMPLE* Department of Environmental Science, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, LA1 4YQ, UK
The purpose of this study was to assess the suitability of various spiking procedures for the introduction of persistent organic pollutants (POPs) into soil environments. 14C-radiolabeled analogues of two representative polycyclic aromatic hydrocarbons (PAHs), phenanthrene (Phe), and benzo[a]pyrene (B[a]P), were introduced into soil using different spiking techniques, and the homogeneity of compound distribution in subsamples was assessed. It was established that under analogous spiking procedures dry soil could be spiked with greater homogeneity than wet soil. The procedure which gave the most homogeneous distribution of compound involved a single spiking/rehydration operation conducted on dry soil. Relative standard deviations of 2.40% for 14C-9-Phe and 3.65% for 14C-7B[a]P were obtained for this procedure. An optimum procedure for the spiking of wet soil was established, giving relative standard deviations of 4.1% for 14C-9-Phe and 3.7% for 14C-7-B[a]P. This procedure employed a highly spiked wet soil inoculum to distribute the compound throughout the soil system. The influence of carrier solvent on microbial cell numbers determined as colony forming units was also evaluated and shown to have a dramatic negative impact at high volumes.
Introduction There is currently considerable interest in the fate and behavior of persistent organic pollutants (POPs) within the soil environment. Processes such as sorption (1-4), desorption (5, 6), subsurface transport (7), and biodegradation (1, 5, 6, 8) have been the focus of much attention (9). Microcosms, often utilizing 14C-radiolabeled compounds, have frequently been employed in these investigations (6, 8). Such studies often depend on the introduction (spiking) of a compound of interest into the soil system. This immediately raises the question, can the spiking of nonpolar and often poorly water soluble compounds be achieved reliably, such that the compound is uniformly incorporated throughout the soil? A supplementary question relevant in many experimental systems, is does the spiking procedure effect the indigenous microbial community? Owing to the complex nature of soil, with its array of potential binding sites of varying affinity for POP molecules, the presence of organic matter and the potential for entrapment of POP molecules within soil micro- and nanopores, there are inherent difficulties associated with the homoge* Corresponding author telephone: 44 1524 594534; fax: 44 1524 593985; e-mail:
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neous introduction of nonpolar POPs of low aqueous solubility into soils. Clearly for most laboratory-based investigations of the fate and behavior of POPs in soil, it is necessary that the compound is distributed homogeneously. Should homogeneity fail to be achieved, data will not only be statistically unreliable, but the system may behave as a number of subsystems with zones of higher and lower localized concentrations. Sorption/desorption kinetics have been proposed to be concentration dependent (10) and biodegradation reliant on concentration gradients (11). Thus, the existence of such subsystems would hold implications for compound availability and biodegradability. In addition to homogeneous compound distribution it is advantageous that the integrity of the soil’s physical structure is maintained. It has been extensively reported that drying and rewetting of soils produces changes in soil organic matter (SOM), in particular increasing the water soluble or labile fraction (12-17). Drying and rewetting of air-dried soils has also been observed to influence soil pH (15). Alterations in the pH of soil will produce changes in the orientation and condensed state of SOM. This can affect the association between SOM and soil mineral components, water solubility of SOM, availability of SOM for microbial utilization, and the partitioning of organic contaminants to SOM (15). Dehydration of soil organic matter by drying results in the shrinkage of the polymeric structure of the OM into a more condensed state, due to the formation of strong intramolecular bonds (18). This process may result in the trapping or occlusion of organic contaminants within the condensed OM, therefore affecting contaminant release into the aqueous phase. The aim of this study was to assess the suitability of various spiking procedures for achieving homogeneous compound distribution in soil (applied to both wet and dry soil) previously described in the scientific literature. Additionally the impact of the carrier solvent employed on the microbial numbers was assessed by evaluating culturable microbial cell numbers. Two PAHs, phenanthrene (Phe) and benzo[a]pyrene (B[a]P), were selected as representative nonpolar persistent organic pollutants for use in this study.
Experimental Section Materials. The PAHs, Phe and B[a]P, as both nonradiolabeled and 14C-radiolabeled analogues (14C-9-Phe and 14C-7-B[a]P) were obtained from Sigma Aldrich Co. Ltd., UK. Acetone, ethanol, and toluene used to produce standards were obtained from Merck, UK, and Rathburn Chemicals Ltd., UK. The sample oxidizer cocktails, Carbosorb-E and Permafluor-E, and the organic-based combustion aiding solution, Combustaid were obtained from Canberra Packard, UK. The nutrient agar used for the viable plate counting of microbial cell numbers was obtained from Merck, UK. Soil Type and Pretreatment. Subsurface soil (5-15 cm) was collected from a rural hillside environment (Lancaster University, Hazelrigg, Field Station, UKsO.S. sheet 97, [493578]). Field wet soil was passed through a 10-mm-gauge sieve. A portion (7 kg) of the soil was retained in this state, with the remainder of the soil (5 kg) being air-dried (over a 14-day period) on the bench and subsequently passed through a 2-mm-gauge sieve to remove stones and vascular material. Soil treated in this way was used in spiking protocols 1-7 and 10 (see below). The same field wet soil (4 kg) was force rubbed through a 2-mm-gauge sieve and used in spiking protocols 8, 9, and 11 (see below). The organic content of the soil (mass loss on ignition) was established to be 8.98% ( 0.24%. S0013-936X(98)00094-7 CCC: $15.00
1998 American Chemical Society Published on Web 08/29/1998
14 C-Radiolabeled and Nonradiolabeled Standards. A Phe or B[a]P concentration of 10 mg kg-1wet soil, indicative of a marginally contaminated soil and a concentration used in other studies (8), was employed in this investigation. Wet soil masses of 500 g (protocols 1-7 and 10) or 250 g (protocols 8, 9, and 11) were selected for the microcosms. Nonradiolabeled standards were prepared using a mixture (1:4) of toluene and ethanol (to ensure PAH solubilization), such that 1 µL would deliver the required amount of compound per 1 gwet soil. The 14C-radiolabeled PAH standards were prepared using toluene as a solvent such that 1 µL would deliver 950 Bq in the case of Phe and 1 µL would provide 732 Bq in the case of B[a]P. Soil Spiking Protocols. Protocols 1-4 and 10 describe procedures for spiking dry soil. Protocols 5-9 and 11 describe procedures for spiking wet soil. The dry soil was rehydrated in all cases to a final water content of 30% (dry weight). Mixing was accomplished in all procedures using a Waring commercial blender (model no. 35BL64). Protocol 1 (adapted from refs 8 and 19). Sufficient ethanol (17.5 mL) to provide an air-dried soil:solvent ratio of 1:0.045 (8) was placed inside the blender. To the ethanol the nonradiolabeled Phe (500 µL) and the 14C-9-Phe solution (27 µL) were added. Air dried soil (385 g) was blended with this solution in increments of 50-100 g for 1 min between additions of soil. When the addition of the soil into the spiked ethanol was completed, the soil was mixed thoroughly for 2 min. Complete mixing was ensured by turning any compressed soil in the blade with a nickel spatula; blending was then resumed. Turning/mixing was repeated twice. The soil was then transferred to an unsealed jar and the ethanol allowed to evaporate over 24 h in a fume hood. Deionized water (115 mL) was placed into the blender container and the spiked soil was slowly added to the water in increments of 50-100 g. Blending was sustained for 1 min between soil additions. When all of the soil had been added, the system was mixed thoroughly with turning for 2 min, a total of three times. The soil was then transferred to a glass jar with a screw cap seal and stored in darkness until analysis. Protocol 2 (adapted from ref 20). Sufficient water (115 mL) to rehydrate the air-dried soil was placed in the blender. The nonradiolabeled Phe (500 µL) and the 14C-9-Phe (27 µL) solutions were added to the water. To this solution was blended air-dried soil (385 g), in increments of 50-100 g. Complete mixing was accomplished as described in protocol 1. The soil was then transferred to a jar and sealed prior to analysis. Protocol 3 (adapted from ref 21). The nonradiolabeled Phe (500 µL) and the 14C-9-Phe (27 µL) were placed in a small beaker (100 mL). To this solution was mixed air-dried soil (10 g) in ∼1 g amounts. The mixture was then left unsealed overnight to allow the solvent to evaporate. This soil “inoculum” was then blended into the remaining soil (375 g), by adding ∼2 g of it to 50-100 g of air-dried soil and blending for 1 min between additions. After the addition was completed, the system was then thoroughly mixed with turning for 2 min. The mixture was then rehydrated with deionized water (115 mL), by blending the soil in increments of 50-100 g into the water. Mixing was accomplished as described in protocol 1. The soil was then transferred to a jar and sealed prior to analysis. Protocol 4 (adapted from ref 21). Protocol 3 was repeated using a portion of the original soil which had been ashed in a furnace overnight at 450 °C (10 g ashed weight). Protocols 5, 6, and 7. These procedures were repeats of protocols 1, 3, and 4 respectively using wet soil in place of the dry soil. Masses were adjusted to produce a system of total mass 500 g on completion of the spiking procedure; no water was added during these procedures.
Protocol 8. The nonradiolabeled Phe (250 µL) and the (13.5 µL) produced in ethanol were placed in a small beaker (100 mL) and mixed vigorously with 15 g of presieved field wet soil using a glass spatula for approximately 3 min. The resultant “inoculum” was then mixed with the remaining soil (235 g). This was achieved by adding approximately a quarter of the inoculum to one-quarter of the field wet soil and blending for 20 s. This procedure was repeated until all the soil and inoculum had been added and mixed. The mixed blender contents were then stirred with a spatula to break any large aggregates and given a final 20-s blend. The mixed contents were then transferred to a glass jar and sealed until analysis. Protocol 9. Protocol 8 was repeated using Phe standards produced in acetone. Protocol 10. Protocol 2 was repeated using a 14C-7-B[a]P standard produced in toluene and a nonradiolabeled B[a]P standard produced in ethanol:toluene (1:4). Protocol 11. Protocol 8 was repeated using a 14C-7-B[a]P standard produced in ethanol and a nonradiolabeled B[a]P standard produced in toluene. Analysis. The spiked soils were left for 21 d, to allow the compound to become incorporated/associated with the soil (42 days in the case of protocols 8, 9, and 11). After this conditioning period, a vertical core of soil was removed from each system using a stainless steel tube. The cored soil was extruded into a beaker and mixed with a nickel spatula. Six (1 g) samples were removed for analysis and packed into paper combustion cones. The samples were combusted using a Packard 307 sample oxidizer. The combustion process was conducted over 3 min, aided by the addition of Combustaid (100 µL) injected onto the samples prior to oxidation. Trapping efficiency of the sample oxidizer was assessed prior to the combustion of soil samples and was established to be greater than 97%. The resultant 14CO2 trapped and eluted solutions were counted using a Canberra Packard Tri-Carb 2250CA liquid scintillation analyzer for 10 min (sufficient time to provide 50× background values). Experimental blanks were prepared using blended wet soil and blended/rehydrated dry soil to which no solvent or PAH was added. Microbial Cell Number Monitoring. Indigenous cell numbers were established for both the wet and dry/ rehydrated soil before blending, 1 d after blending and after the 21-d equilibration period (the same time as the samples were analyzed for radioactivity). Cell numbers, determined as colony forming units (cfu), were enumerated using a viable plate technique whereby a soil sample (10 g) was extracted with 1% aqueous sodium chloride solution (100 mL) containing Tween 80 (0.004%) on a flat bed shaking platform at 150 rpm for 16 h. An aliquot of the supernatant was removed and serially diluted. The dilutions were then plated out in triplicate onto nutrient agar (2%) and incubated at 24 °C for 21 d, and microbial cultures were counted. 14C-9-Phe
Results and Discussion Homogeneity of 14C-9-Phe Distribution (Spiking Protocols 1-9). The homogeneity of compound distribution as a result of the nine spiking protocols tested, as determined from the six (1 g) replicates, varied greatly depending on the protocol adopted (Figure 1A,B). Relative standard deviation (RSD) values used to assess the proficiency of a given protocol are also presented in Figure 1A. Comparison of protocol 1 with protocol 5, protocol 3 with protocol 6, and protocol 4 with protocol 7 clearly indicates that under analogous spiking procedures dry soil can be spiked more homogeneously than wet soil. In the case of wet soil, spiking using a high proportion of excess ethanol (protocol 5) provided high compound homogeneity, with RSD of 7.97% (Figure 1B). The analogous procedure for dry VOL. 32, NO. 20, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Variations in number of colony forming units (cfu) before blending, 1 and 21 d after blending for wet soil (black bars) and dry/rehydrated soil (white bars).
FIGURE 1. (A) Mean activities (Bq g-1wet soil) for dry soil (white bars) and wet soil (black bars) relative standard deviations (% RSD - error bars) associated with the spiking protocols 1-9 for 14C9-Phe (n ) 6). (B) Relative standard deviations for dry soil (white bars) and wet soil (black bars). soil (protocol 1) also gave a high degree of compound homogeneity, 2.86% (Figure 1B). However, the one step spiking with rehydration procedure (protocol 2) proved the most effective with a RSD of 2.40% (Figure 1B). The use of a dry or ashed soil carrier matrix as employed in protocols 3 and 4 (for dry soil) and protocols 6 and 7 (for wet soil) proved less effective than the above procedures (Figure 1B), giving RSDs of 5.40% and 12.51% for the dry soils respectively and RSD of 11.77% and 43.04% for the wet soils, respectively. Ashing the carrier matrix appears to impede the homogeneity of compound distribution. This may be due to the widening of soil pores during the ashing process, which reseal when the spiked compound is added in liquid form, thus trapping the compound and inhibiting its distribution throughout the entire soil system. Alternatively, the removal of water may provide a larger number of sorption sites which could bind strongly with PAH molecules. Protocols 8 and 9 employ a wet soil inoculum as a carrier matrix for the spiking of field wet soils. These procedures provided a high degree of compound homogeneity, 4.1% and 4.7%, respectively. These results indicate no significant benefit in the use of ethanol (protocol 8) over acetone (protocol 9) for the production of standard solutions, as both solvents provide excellent tracer distribution. It should be noted that these protocols were conducted with half the masses used in protocols 1-7 i.e., 250 gsoil compared to 500 gsoil and therefore direct comparison cannot be drawn. Comparison of Figures 1 A & B reveal that where a high degree of homogeneity was obtained (protocols 1-3, 8, and 9) a consistent soil activity was observed. Conversely where poor homogeneity was obtained (protocols 4-7) an imprecise soil activity was observed. 3226
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FIGURE 3. Number of colony forming units (cfu) for the spiking protocols 1-7 for Phe, for dry soil (white bars) and wet soil (black bars). The use of 14C-radiolabeled compounds in this study facilitated the rapid determination of tracer homogeneity. It should be noted, however, that this does not indicate whether the 14C activity is associated with the parent compound. It is possible that degradation of the parent PAH molecules has occurred during the equilibration time. Half-lives of PAHs in soil have been estimated previously and shown to vary greatly [22-26]. Variability has been proposed to reflect soil type and compound availability. It is probable that some phenanthrene degradation occurred during the equilibration time, although B[a]P degradation is unlikely. Background Culturable Microbial Cell Number Dynamics during Blending. The viable plate counts reflecting microbial numbers for the blended wet soil and blended/ rehydrated dry soil which had not been amended with any compounds indicate that the blending process greatly influences cfus (Figure 2). Microbial cell numbers (cfus) increased after the blending procedure, as was particularly evident after the 21-d equilibration time. Dry/rehydrated
soil showed the greatest enhancement of 129 times increase, compared with a 50 times increase for wet soil. This enhancement is not entirely surprising, since increased aeration, deaggregation of the soil structure and enhanced nutrient release are favorable factors for microbial growth likely to occur from the blending process (27). Additionally, the deaggregation of soil structure may facilitate more efficient extraction of microorganisms from the soil. As a result the number of culturable microbial cells postblending would rise. Culturable Microbial Cell Numbers in the Soil Amended with 14C-9-Phe. The microbial cell numbers, as established by viable plate count, indicate that the nature of the protocol adopted to spike the soil had a bearing on the observed cell numbers (Figure 3). Protocols 1 (dry soil) and 5 (wet soil) gave greatly reduced microbial numbers; only 3% and 8% of the control values were observed, respectively. Interestingly, both of these protocols employ the use of a large volume of ethanol as a spiking solvent. The use of ethanol as a carrier solvent has been reported in the literature (8, 19). This result, however, poses the question, can a system which has been microbiologically altered, to such a large extent, be applied to the study of the natural environment? This point is perhaps especially important where the “aging” of PAHs is of concern, since microbiological processes may play an important role in this process (28). Comparison of protocol RSD with culturable microbial cell numbers. It can be seen that although protocols 1 (dry soil) and 5 (wet soil) provide low RSD associated with the spiking procedure (Figure 1), they also have the greatest impact on the culturable microbial cell numbers (Figure 3). This may, as already mentioned, be the result of a toxic effect exerted by ethanol. Protocol 2 appears to provide the most appropriate spiking procedure for air-dried soil with the highest homogeneity of compound distribution (RSD ) 2.40%) and the lowest impact on the microbial populations. Protocols 8 and 9 provide high homogeneity of compound distribution for wet soil (RSD ) 4.1% and 4.7%, respectively) and employ a minimal volume of solvent (equivalent to that used in protocol 2). As a result they are unlikely to impact dramatically on microbial populations. Verification of Suitability of Protocol 2 and 8 for B[a]P. On the basis of phenanthrene’s relatively high aqueous solubility (0.994 mg L-1) (29), benzo[a]pyrene (B[a]P) was selected as a PAH of considerably lower aqueous solubility (0.0038 mg L-1) (30) for greater applicability to particularly low solubility nonpolar compounds. Protocols 2 and 8 were repeated in an identical manner using B[a]P in place of Phe (protocols 10 and 11, respectively). On analysis of 14Cradioactivity in six (1 g) replicate samples the RSD were evaluated as 3.65% (protocol 10), cf. 2.40% (protocol 2), and 3.70% (protocol 11), cf. 4.1% (protocol 8). These high degrees of compound homogeneity further substantiate the use of protocol 2 (for air-dried soil) and protocol 8 (for field wet soil) for the introduction of low solubility nonpolar POPs into soil. Conclusions. The procedure used to incorporate POPs into soil has a bearing on the uniformity of the compound’s distribution. High volumes of tracer carrier solvent can have a significant impact on the number of cfus. The results of this study have indicated a suitable spiking protocol for the reliable introduction of PAHs into both dry and wet soil, employing minimal volumes of solvents. A single step spiking/rehydration operation conducted on dry soil provided the most appropriate means of incorporating PAHs into dry soil. A procedure involving a spiked wet soil inoculum provided the most appropriate means for the introduction of PAHs into wet soil.
Acknowledgments This work was financially supported by an award from the Natural Environmental Research Council (NERC), UK, and the Ministry of Food and Agriculture (MAFF), Food Contamination Division, U.K.
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Received for review January 29, 1998. Revised manuscript received May 22, 1998. Accepted July 16, 1998. ES9800941 VOL. 32, NO. 20, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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