Short Communication
Pentachlorophenol transformation in soil: a toxicological
assessment
R. McGrath
a, I. Singleton
b,*
a
Department of Industrial Microbiology, Bel®eld, University College, Dublin 4, Ireland b
Department of Soil and Water, Waite Campus, University of Adelaide, Glen Osmond, SA 5064, Australia
Accepted 21 December 1999
Abstract
Bioremediation is recognised as an economically viable method to treat contaminated soil and the use of microbial inoculants, in particular, white-rot fungi, has been proposed to enhance the remediation process. During bioremediation, a variety of pollutant transformation products will be created which may have toxic synergistic interactions and may not at all be detected by chemical analysis. This work examined the potential for formation of toxic breakdown products during pentachlorophenol (PCP) transformation in soil after inoculation withPhanerochaete chrysosporium; a frequently used fungal inoculant. To monitor toxicity during bioremediation, changes in soil dehydrogenase activity and eects of soil methanol extracts on the growth of a common soil bacteriumBacillus megateriumin liquid culture were determined. After 6 weeks of remediation, soil PCP levels had dropped from an initial 250 mg kgÿ1to 2 mg kgÿ1, and inoculation with P. chrysoporium did not improve PCP remediation
over uninoculated PCP-contaminated soil. Soil dehydrogenase activity remained very low in all soils containing PCP and did not recover throughout the experiment (6 weeks) despite the decrease in PCP levels. Soil methanol extracts varied in their toxicity towards growth ofB. megateriumand were most toxic after 6 weeks incubation when extracts obtained from PCP-contaminated soil inoculated with P. chrysosporiumcompletely inhibited B. megaterium. A longer incubation time would probably result in removal of toxic products and as soil methanol extracts were used in growth inhibition studies, the bioavailability of these toxic compounds remains in question. However, this work indicates that toxic transformation products may be formed during bioremediation and that ecotoxicological assays are useful to complement chemical analysis during bioremediation of contaminated soil.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Bioremediation; Soil; White-rot fungi; Toxicity; Pentachlorophenol;Phanerochaete chrysosporoium
Pentachlorophenol is a general biocide and has been used for a variety of purposes such as agriculture and timber preservation. Worldwide use of the chemical has led to severe contamination problems particularly around former timber treatment plants (Ceroici and Beresford, 1993; Yu and Shephard, 1997). The known toxicity (Megharaj et al., 1998) and persistence of PCP necessitates the clean-up of contaminated sites, es-pecially if they are to be redeveloped for housing or if
there are other human exposure risks involved. A var-iety of treatment options are available and bioremedia-tion has been used as a commercial remediabioremedia-tion method (Valo and Salkinoja-Salonen, 1986). The suc-cess of any commercial remediation operation is gener-ally determined by chemical analysis only and is based upon the successful removal of the parent compound, in this case, PCP. However, during the microbial transformation of PCP, a variety of breakdown pro-ducts may be formed some of which may be as toxic, or even more toxic than the parent product. It has been estimated that there are over 30 PCP transform-ation products (Middaugh et al., 1993) and toxicity of
Soil Biology & Biochemistry 32 (2000) 1311±1314
0038-0717/00/$ - see front matter72000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 1 5 - 8
www.elsevier.com/locate/soilbio
* Corresponding author. Fax: +61-8-8303-6511.
biodegraded PCP samples towards ®sh embryos ( Meni-dia beryllina) has been observed (Middaugh et al., 1993). Due to the heterogeneous (physical, chemical and biological) nature of soil it is likely that a variety of microbial (aerobic and anaerobic) and chemical transformations will occur during bioremediation, leading to the possible formation of many breakdown products. Chemical analysis of such mixtures is time consuming and also gives no indication of the possible synergistic toxic eects that may occur. Therefore, it appears that methods which give an indication of re-sidual soil toxicity after bioremediation will sup-plement more traditional chemical analysis and give a good estimate of the success of the reclamation pro-cess. However, it is ®rst necessary to demonstrate that toxic products are formed during bioremediation of contaminated soil. To examine this possibility it was decided to spike soil with a known amount of PCP and examine the toxicity of the soil using dehydrogen-ase activity as a measure of microbial activity over time. In addition, toxicity of soil methanol extracts was determined by addition to a culture of Bacillus megaterium previously found to be sensitive towards PCP (R. McGrath, unpub. Ph.D. thesis, University College Dublin, 1995).Bacillus megateriumwas chosen as representative of common soil bacteria since certain Bacillus species have been shown to be sensitive to PCP transformation products (Ruckdeschel and Renner, 1986). In addition,B. megateriumgrew well in the soil methanol extracts used for the toxicity assay.
Sieved soil (2 mm, 40 g) was amended to 250 mg PCP kgÿ1
with PCP dissolved in 25 mM NaOH. Samples were then inoculated withPhanerochaete chry-sosporium CMI 174727 (a model fungal inoculant) grown on peat (2.5 g peat per 40 g soil) or left unino-culated. Uncontaminated soil samples (with or without peat addition) were also included as controls. Soil was collected from the campus at University College Dublin and was a clay loam (pH 6.4). Samples were incubated at 258C for 6 weeks and maintained at a constant moisture content (30% w/w) by regular ad-dition of sterile distilled water. Sterile soils (autoclaved on three consecutive days for 1 h at 1218C,) were also included to determine the amount of PCP transform-ation in the absence of microbial activity. Four repli-cates of each treatment were used. Analysis of test samples was carried out after 1 week of incubation and all subsequent analyses carried out on a fort-nightly basis.
PCP concentrations in soils were determined after soxhlet extraction and subsequent HPLC analysis (McGrath and Singleton, 1997). Anhydrous sodium sulphate (5 or 10 g) was added to a cellulose extraction thimble, overlaid with soil (5 or 10 g) at 25% (w/w) moisture content and extracted for 16 h with hexane. Extracts were concentrated to approximately 5 ml in a
rotary ®lm evaporator and transferred to volumetric ¯asks (10 ml). Evaporating ¯asks were rinsed with hex-ane and washings added to volumetric ¯asks. After equilibration at 208C volumes were made up to 10 ml. Samples were ®ltered through PTFE ®lters (Gelman, USA) and stored in glass Universal bottles at ÿ188C prior to analysis. HPLC analysis was carried out on an Envirosep-PP column (1253.2 mm, Phenomenex, England) maintained at 458C by a temperature control unit (Model 3, Waters Associates, USA). Flow rate was maintained at 1 ml minÿ1by a gradient controller (Model 680, Waters Associates, USA) and solvent delivery system (M45, Waters Associates, USA). Mobile phase was methanol:water 70:30. Samples (20 ml) were applied to the column by a Marathon auto-sampler (Spark Holland BV, The Netherlands). PCP was detected at 254 nm and readings integrated by a data module (Model 740, Waters Associates, USA). PCP concentrations were calculated by reference to appropriate standard PCP solutions.
Dehydrogenase activity in soil was assessed by the method of Bauer et al. (1991). In this, soil (5 g) was mixed with 5 ml of 2,3,5-triphenyl tetrazolium chloride reagent and incubated at 258C for 24 h. After this, 25 ml acetone was added and samples incubated for a further 2 h with periodic mixing. Samples were then ®ltered through Whatman No. 2 ®lter paper and diluted with acetone to bring readings within range of the standards. The absorbances of standards and samples were read at 546 nm on a LKB Biochrom Ultraspec 2 (LKB, England). Relevant controls allow-ing for background colour development in soils were included (Bauer et al., 1991).
The toxicity of soil towards B. megaterium was tested by the following method. Soil samples (10 g) from all test ¯asks were soxhlet extracted with metha-nol for 4 h. Extracts were concentrated till a ®nal volume of 10 ml was obtained. Samples (1 ml) of extracts were sterilised by ®ltration (0.2 mm, non-or-ganic binding ®lter, Gelman, USA) and added to 10% (w/v) Tryptone Soy Broth (TSB) (23 ml). To this was added B. megaterium inoculum (1 ml, Optical Density (OD) at 650 nm of 0.3) from a culture grown in 10% TSB for 18 h at 378C and 160 rpmÿ1. An OD of 0.3 at 650 nm was obtained by addition of sterile distilled water to the culture as required. Final volume of TSB, soil extract and inoculum was 25 ml. Cultures were subsequently incubated at 378C, 160 rpm in the dark for 8 h. OD at 650 nm was measured periodically on a Coleman spectrophotometer (Perkin Elmer, USA).
When required comparison between means was car-ried out using ANOVA followed by Tukey Multiple Comparisons test or Student t-test. All statistical analysis was carried out using Instat statistical package (Graph Pad, Intuitive Software for Science, USA).
R. McGrath, I. Singleton / Soil Biology & Biochemistry 32 (2000) 1311±1314
Analysis of raw data showed normal distribution allowing the above statistical tests to be carried out.
PCP concentrations in all non-sterile soils declined to approximately 2 mg PCP kgÿ1
after 6 weeks incu-bation at 258C (Fig. 1). Inoculation with P. chrysos-porium did not alter the ®nal amount of PCP degradation obtained. No PCP transformation was observed in sterile soils and PCP extraction eciencies of at least 90% were obtained. This demonstrates that despite the lack of previous exposure of this soil to PCP, an active soil microbial population capable of PCP transformation was present. We did not attempt to examine for the presence of PCP degradation pro-ducts. Clearly given the right environmental con-ditions, PCP is not persistent. However, higher PCP concentrations and repeated PCP addition coupled
with sorption of the chemical to soils high in organic matter (Lee et al., 1990) will alter persistence levels obtained.
Dehydrogenase activity decreased considerably in all soils contaminated with PCP (Fig. 2) and did not recover even though PCP concentrations had dropped to 2 mg kgÿ1 in soil after 6 weeks incubation. Dehy-drogenase activity re¯ects soil microbial activity (Ger-ber et al., 1991) and this result shows that there is some residual soil toxicity after 6 weeks bioremedia-tion, even though PCP concentrations were apparently very low. This suggests either that toxic PCP trans-formation products were formed or that the soil mi-crobes had not fully recovered from initial toxic responses towards PCP. Such a large decrease in dehy-drogenase activity was unexpected.
Methanol soil extracts were prepared from incu-bated control and test soil samples every 2 weeks and subsequently added toB. megateriumcultures to deter-mine the presence of methanol extractable toxic com-pounds. It was assumed that a decrease in growth of B. megaterium would indicate that toxic compounds (potential PCP transformation products) were present. The most apparent toxic eect was observed with extracts prepared from soils which had been incubated for 6 weeks. Methanol extracts from PCP-contami-nated soils with added fungal inocula completely inhib-ited the growth of B. megaterium (Fig. 3). Extracts from PCP-contaminated soils without fungal inocula gave approximately 40% inhibition of B. megaterium. Growth was signi®cantly lower (P < 0.05) than metha-nol extracts prepared from control ¯asks containing soil only (Fig. 3). The dilutions involved in preparing soil toxicity tests would have produced PCP concen-trations of less than 0.1 mg lÿ1
in growth media.
Pre-Fig. 1. Comparison of pentachlorophenol transformation in sterile soil with uninoculated and inoculated (fungal inoculant) non-sterile soil. Means of four replicates +/- SD.
QÐÐÐQ, sterile soil plus PCP, ^ÐÐÐ^, soil plus PCP plus fungal inoculum,qÐÐÐq, soil plus PCP.
Fig. 3. Eect of various soil methanol extracts on growth of B. megaterium. Methanol extracts were prepared from soil samples incubated for 6 weeks after initial PCP addition. Means of four repli-cates +/- SD. WÐÐÐW, soil plus PCP RÐÐÐR, soil alone,
qÐÐÐq, soil plus PCP plus fungal inoculum, XÐÐÐX, soil plus fungal inoculum.
Fig. 2. The eect of PCP and fungal inoculation alone and in combination, on dehydrogenase activity in soil after 6 weeks incu-bation. Means of four replicates+/- SD.WÐÐÐW, soil plus PCP
RÐÐÐR, soil alone,qÐÐÐq, soil plus PCP plus fungal inocu-lum, XÐÐÐX, soil plus fungal inoculum. TPF: Triphenyl forma-zan.
vious work (results not shown) demonstrating that 1 mg PCP lÿ1
was toxic but not completely inhibitory to B. megaterium, suggests that other toxic compounds extracted from soil were present in the growth media. These compounds may have been exerting synergistic toxic eects on B. megaterium. Overall it appears that during PCP transformation in soil, some toxic com-pounds were produced. This result is supported by evi-dence of the production and persistence of 3,4,5-trichlorophenol and 2,3,4,5 tetrachlorophenol from PCP in soil environments (Kuwatsuka and Igarashi, 1975; Baker and May®eld, 1980). These metabolites are considerably more toxic to fungi and bacteria than PCP (Ruckdeschel and Renner, 1986; Ruckdeschel et al., 1987), and mean that, even if present in small amounts, no reduction in soil toxicity may be achieved by bioremediation. Extreme care must be taken to avoid their production during bioremediation. The bioavailability of toxic metabolites must remain an issue, as methanol extracts were used to assess residual soil toxicity and compounds extracted by methanol may have been tightly bound to soil particles. How-ever, there is evidence suggesting that sorbed organic compounds are accessed by microbes (Calvillo and Alexander, 1996) and as soil dehydrogenase activity had decreased, it would appear as if toxic compounds were available to microorganisms. It is very likely that given more time the eventual toxicity of the soil would decrease to acceptable amounts, however, we consider that the evidence presented here suggests that toxico-logical measures of assessing the residual toxicity of soil after bioremediation are required. This is particu-larly so in the case of large-scale bioremediations where large volumes of soil are treated and it is di-cult to maintain optimal environmental conditions.
Acknowledgements
Ruth McGrath acknowledges provision of a Ph.D. scholarship from Bord Na Mona. John Flynn and Terry Union are thanked for technical assistance, and the Faculty of Agriculture, University College Dublin for soil analysis.
References
Baker, M.D., May®eld, C.I., 1980. Microbial and non-biological de-composition of chlorophenols and phenol in soil. Water, Air and Soil Pollution 13, 411±424.
Bauer, E., Pennerstorfer, C., Holubar, P., Plas, C., Braun, R., 1991. Microbial activity measurement in soil - a comparison of methods. Journal of Microbiological Methods 14, 109±117. Calvillo, Y.M., Alexander, M., 1996. Mechanism of microbial
utilis-ation of biphenyl sorbed to polyacrylic beads. Applied Microbiology and Biotechnology 45, 383±390.
Ceroici, W.J, Beresford C.R., 1993. Remediation of an abandoned wood preserving facility in Canada. UNEP Industry and Environment July±September, 21±26.
Gerber, H.R., Anderson, J.P.E., Bugeimogenson, B., Castle, D., Domsch, K.H., Malkomes, H.P., Somerville, L., Arnold, D.J., Vanderwerf, H., Verbeken, R., Vank, J.W., 1991. Revision of rec-ommended laboratory tests for assessing side eects of pesticides on the soil micro¯ora. Toxicology and Environmental Chemistry 30, 249±261.
Kuwatsuka, S., Igarashi, M., 1975. Degradation of PCP in soils II: the relationship between the degradation of PCP and the proper-ties of soils, and the identi®cation of the breakdown products of PCP. Soil Science and Plant Nutrition 21, 405±414.
Lee, L.S., Rao, P.S.C., Nkedi-Kizza, P., Del®no, J.J., 1990. In¯uence of solvent and sorbent characteristics on distribution of penta-chlorophenol in octanol-water and soil-water systems. Environmental Science and Technology 24, 654±661.
McGrath, R., Singleton, I., 1997. Analysis of pentachlorophenol in soils for use in bioremediation studies. In: Sheehan, D. (Ed.), Bioremediation Protocols. Methods in Biotechnology, Vol. 2. Humana Press, Totowa, pp. 169±176.
Megharaj, M., Singleton, I., McClure, N.C., 1998. Eect of penta-chlorophenol pollution towards microalgae and microbial activi-ties in soil from a former timber processing facility. Bulletin of Environmental Contamination and Toxicology 61, 108±115. Middaugh, D.P., Resnick, S.M., Lantz, S.E., Heard, C.S., Mueller,
J.G., 1993. Toxicological assessment of biodegraded pentachloro-phenol: Microtox and ®sh embryos. Archives of Environmental Contamination and Toxicology 24, 165±172.
Ruckdeschel, G., Renner, G., 1986. Eects of pentachlorophenol and some of its known and possible metabolites on fungi. Applied and Environmental Microbiology 51, 1370±1372. Ruckdeschel, G., Renner, G., Schwarz, K., 1987. Eects of
penta-chlorophenol and some of its known and possible metabolites on dierent species of bacteria. Applied and Environmental Microbiology 53, 2689±2692.
Valo, R., Salkinoja-Salonen, M.S., 1986. Bioreclamation of chloro-phenol-contaminated soil by composting. Applied Microbiology and Biotechnology 25, 68±75.
Yu, P.L., Shephard, J., 1997. Pentachlorophenol in New Zealand -biological treatment option. Australasian Biotechnology 7, 340± 344.
R. McGrath, I. Singleton / Soil Biology & Biochemistry 32 (2000) 1311±1314
