Short communication
Pentachlorophenol utilization by indigenous soil microorganisms
Amar M. Chaudri*, Kirsten Lawlor, Steve P. McGrath
Soil Science Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK
Received 27 May 1999; received in revised form 19 August 1999; accepted 7 September 1999
Pentachlorophenol (PCP) has been used extensively world wide as a preservative by the timber and textile industries because of its antimicrobial properties. This has resulted in large areas of land being contaminated (Crosby et al., 1981; Sato, 1983), which can aect soil microbial communities and, thereby in¯uence the turn-over of nutrients and soil fertility (Domsch et al., 1983; Welp and BruÈmmer, 1992). Most organic com-pounds are not perceived to be a threat to soil mi-crobial processes (Wild et al., 1994). However, many xenobiotic organic compounds found in the environ-ment, are known biocides and may negatively aect the soil micro¯ora and its activities (Welp and BruÈm-mer, 1992; Harden et al., 1993; Chaudri et al., 1996).
The aim of this paper is to determine whether the indigenous soil microbial populations can survive and utilize PCP as a carbon source. In this study, non-con-taminated soil was amended with known concen-trations of PCP. The soil microbial biomass-C content and the total number of aerobic heterotrophic bacteria were determined after chronic exposure. In addition, we investigated the possible use of PCP when pre-sented as the sole carbon source, by the microbial populations that survived in the PCP-amended soils.
The soil used was a sandy loam (pH 7.0, 14% clay, 1.48% organic carbon, 0.15% total N) of the Cotten-ham series (Typic Udipsamment) from a long-term ley-arable ®eld experiment at Woburn, southeast England. The plot sampled was under continuous grass for 8 yr during which no nitrogen fertiliser was added. Prior to this, farm yard manure at 38 t haÿ1 was added 35 yr ago. The heavy metal concentrations in the soil were
(mg kgÿ1): 30 Zn, 4 Cu, 17 Ni, <0.2 Cd, 28 Cr and 31 Pb. Moist soil was separated initially into 1 kg por-tions (dry weight basis), before amending with 25, 50, 75, 100, 175 and 200 mg kgÿ1 PCP. The PCP (Sigma
pure grade) was dissolved in methanol, as it is rela-tively insoluble in water. The treatments included two controls with either water or methanol (10 ml kgÿ1)
added, to ensure that the eects observed on the microorganisms were not due to the solvent. During and after treatment, the soils were thoroughly mixed using an electronic food mixer, then placed in plastic bags, and their moisture contents adjusted to 50% water holding capacity. The bags were loosely tied and stored at 208C for 6 months in the dark.
Soil microbial biomass-C was determined using the method of Vance et al. (1987). Brie¯y, the same soil was split into two equal portions of known weight (on an oven dry basis), both at 50% water holding ca-pacity. One portion was extracted with 0.5 M K2SO4
(1:4 w/v) before fumigation with chloroform and the other after fumigation for 24 h. The dissolved organic carbon in the K2SO4 extracts was measured using a
high temperature carbon analyser (TOC 200, UV Developments, UK). The soil microbial biomass-C was then determined from the C ¯ush (Vance et al., 1987). Total aerobic heterotrophic bacteria (TSBA) were counted, after 10-fold serial dilutions of the soil using sterile deionised distilled water, and tryptone soy broth agar (TSBA, Difco, UK) as the plating medium. TSBA colonies were counted at the appropriate di-lution after 48 h incubation at 288C.
PCP utilization by microorganisms in the stored treated soils were measured in vitro using microtitre plates. Either 40 ml methanol (plate/methanol control) or methanol containing dissolved PCP (®nal concen-tration in the assay of 12.5 mg PCP lÿ1) were added to
the wells in the microtitre rows; a row of wells was left
Soil Biology & Biochemistry 32 (2000) 429±432
0038-0717/00/$ - see front matter#2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 7 1 - 6
www.elsevier.com/locate/soilbio
* Corresponding author. Tel.: +44-1582-763-133 ext 2792; fax: +44-1582-760-981.
without additions as a plate/water control. The metha-nol was allowed to evaporate completely for 12 h under vacuum, before adding 40 ml tetrazolium dye (0.1% w/v) and 120ml sterile water to each well. Then 40ml of a 1:100 (w/v) soil to water suspension of each of the PCP-amended soils was added to one column on the plate, giving 1 replicate well of each control and 6 replicates for each soil treatment. The bacterial counts in the 1:100 soil to water suspensions were stan-dardised to 8000 cells mlÿ1. The tetrazolium dye is
light yellow, but it reacts with electrons produced by microbial respiration to give a violet colouration of the solution in the well. The colour change is proportional to the amount of respiration. If no electrons are pro-duced, due to a lack of metabolic activity, then there is no or very little colour change. Any other eects, apart from respiration, of the soil in the assay were accounted for by the control wells which contained no carbon source. The microtitre plates were read ap-proximately every 12 h for 220 h after inoculation using a Multiskan RC microtitre plate reader (Labsys-tems, UK).
Fig. 1shows the eects of dierent concentrations of PCP in the soil after 6 months incubation on the soil microbial biomass-C and on the numbers of total aerobic heterotrophic bacteria. The methanol control contained 23% more biomass-C compared to the water control after 6 months. The soil microbial bio-mass-C was reduced by 56, 91, 77, 84, 69 and 22% at soil PCP concentrations of 25, 50, 75, 100, 175 and 200 mg kgÿ1, respectively, compared to the methanol
control (one-way ANOVA: variance ratio 190; P < 0.001). The increase in microbial biomass-C with methanol con¯icts with the known biocidal eects of methanol. It is possible that the methanol initially killed a portion of the microorganisms leading to par-tial sterilisation and subsequent release of carbon and nutrients. These could then be utilized by the surviving organisms to make more biomass, hence the overall increase after 6 months. However, the increase in bio-mass-C cannot be explained by this release concept alone, and therefore, it is possible that some of the methanol remained in the soil and was utilized as a carbon source by the remaining population. Alterna-tively the surviving population was more able to utilize soil carbon sources which they could not do when in competition with the organisms killed by the methanol treatment. However, this eect of methanol was not seen in the PCP treatments, which all showed signi®-cant reductions in biomass-C (P< 0.001) compared to methanol alone. The soil microbial biomass-C in the 25 and 175 mg PCP kgÿ1 soils was not signi®cantly dierent to each other, but it was signi®cantly greater than the 50, 75 and 100 mg kgÿ1 soils, which did not dier signi®cantly. The biomass-C in the 200 mg kgÿ1 treatment was greater than all other PCP treatments. It seems that the toxic eects of PCP were so great as to annul any stimulating eects of methanol as a car-bon source. Surprisingly, the lower concentrations of PCP were extremely deleterious to the soil microbial biomass.
Other workers have also found that small concen-trations of PCP applied to soil signi®cantly decrease the soil microbial biomass (Zeitz et al., 1987; SchoÈn-born and Dumpert, 1990). For example, 12 appli-cations of 5 g PCP mÿ2(calculated to be equivalent to approx. 25 mg PCP kgÿ1 per application) every 2
months over 24 months in the ®eld reduced the soil microbial biomass to such an extent that it took two years for it to fully recover (SchoÈnborn and Dumpert, 1990). SchoÈnborn and Dumpert (1990) suggested that the metabolites of PCP were likely to be more toxic to soil microorganisms. The half-life in soil of PCP has been determined to be 50 d in degradation studies, but the bacteria that degrade this compound are particu-larly sensitive to it (Sato, 1983). This may explain the lack of recovery by the microbial biomass seen in our experiments at lower concentrations, whereas the higher concentrations may have selected for microbial populations resistant to PCP. These could then grow with much decreased competition, and dominate and increase in number by utilising the PCP as a carbon source. Although the surviving microbial biomass was showing signs of recovery in soil containing the largest concentration of PCP (200 mg kgÿ1) it was still 22%
lower then the methanol control and as such,
accord-Fig. 1. Soil microbial biomass-C content and total numbers of aerobic heterotrophic bacteria (estimated from culturing on TSBA) 6 months after PCP was added to the soil. (Error bars represent stan-dard error of the mean).
A.M. Chaudri et al. / Soil Biology & Biochemistry 32 (2000) 429±432
ing to Domsch et al. (1983), it can be regarded as eco-logically critical.
The total aerobic heterotrophic bacterial counts (TSBA) followed a similar pattern to that of the soil microbial biomass-C (Fig. 1), with a marked decrease at 50, 75 and 100 mg PCP kgÿ1 soil. However, at 25,
175 and 200 mg PCP kgÿ1 soil, the counts were the
same as the control soils.
In our study, the PCP utilization assay tested the ability of the soil microorganisms from the PCP spiked soils, after 6 months incubation, to utilize PCP as a carbon source. When soil suspensions from the control soils (water and methanol only) were added to the water and methanol control microtitre wells containing no PCP or other carbon sources, there was very little change in optical density with time showing that there was no carbon available for microbial respiration (Fig. 2a). However, with soil suspensions from control soils, (incubated without PCP), but given PCP in the assay, small increases in absorbance occurred, showing that the soil microorganisms were capable of using PCP (Fig. 2b). With smaller soil concentrations (25, 50 and 75 mg kgÿ1) of PCP, there were only small increases in
the optical density suggesting that the soil
microorgan-isms were not particularly eective at utilizing PCP (Fig. 2c). With soil suspensions from soils incubated with higher concentrations of PCP (i.e. 100 mg kgÿ1
and above), the optical density readings increased sub-stantially, suggesting greater PCP utilization (Fig. 2d). The largest optical density increase occurred with soil suspensions from soils containing 175 and 200 mg PCP kgÿ1, con®rming the use of PCP as a carbon source by microorganisms in these soils. Regression analysis (Genstat 5 Committee, 1987) carried out on PCP utilization patterns, showed there were no signi®-cant dierences within the following groups of treat-ments: water and methanol controls; soil water and soil methanol controls; 25, 50 and 75 mg PCP kgÿ1; and 100, 175 and 200 mg PCP kgÿ1. Hence, a single regression line could be drawn through each group (Fig. 2). However, further analysis of the areas under the utilization curves, using one-way ANOVA (Gen-stat 5 Committee, 1987) showed PCP utilization between the treatment groups to be signi®cant dierent (P< 0.001).
In conclusion, the negative impacts of PCP at rela-tively low concentrations (50±100 mg kgÿ1 PCP in soil) on the soil microbial biomass-C and on TSBA numbers were severe. At higher concentrations, it would seem that a part of the microbial population re-sistant to the eects of PCP proliferated and were able to utilize the carbon from the PCP, as well as the car-bon liberated by the death of the sensitive populations. The soil used in this study has no previous history of PCP use as a pesticide, and therefore the surviving mi-crobial populations in the PCP treated soils must have been intrinsically resistant to its eects. This is import-ant as it shows that a soil when presented with an or-ganic contaminant may have microbial populations that will, to some extent, ameliorate the eects of the organic compound. Hence, over time, soils contami-nated with organic compounds may be detoxi®ed through soil microorganisms degrading and/or meta-bolising these compounds. However, this process of natural attenuation and recovery may take a long time, and will certainly depend on the soil type. This attenuating capacity of the soil should not be taken as a reason for allowing unrestricted entry of pollutants into soils.
Acknowledgements
IACR-Rothamsted receives grant-aided support from the United Kingdom Biotechnology and Biologi-cal Sciences Research Council.
Fig. 2. PCP utilization as a carbon source, in microtitre plates, by microorganisms from soil suspensions obtained from soils after 6 months incubation with dierent concentrations PCP. Treatments: (a) No PCP in assay; (w) plate water control, (*) plate methanol control. (b±d) contain 12.5 mg PCP lÿ1 as a carbon source in each
microtitre well; (b) (q) soil water control, (Q) soil methanol control: PCP treated soils (mg kgÿ1): (c) (
r) 25, (R) 50 and (T) 75, and (d) (t) 100, (r) 175 and (W) 200. Each line in the plots represents re-gression analysis of groups of treatments which were not signi®cantly dierent to each other. Between plots, the groups of treatments were signi®cantly dierent (P< 0.001) to each other as determined by one-way ANOVA of the areas under the utilization curves.
References
Chaudri, A.M., McGrath, S.P., Knight, B.P., Johnson, D.L., Jones, K.C., 1996. Toxicity of organic compounds to the indigenous population ofRhizobium leguminosarumbiovartrifoliiin soil. Soil Biology and Biochemistry 28, 1483±1487.
Crosby, D.B., Beynon, K.I., Greve, P.A., Korte, F., Still, G.G., Vonk, J.W., 1981. Environmental chemistry of pentachlorophe-nol. International Union of Pure and Applied Chemistry Report on Pesticides (14). Pure and Applied Chemistry, 53, 1051±1080. Domsch, K.H., Jagnow, G., Anderson, T.H., 1983. An ecological
concept for the assessment of side-eects of agrochemical on soil microorganisms. Residue Review 86, 65±105.
Genstat 5 Committee, 1987. Genstat 5 Reference Manual. Clarendon, Oxford, UK.
Harden, T., Joergensen, R.G., Meyer, B., Wolters, V., 1993. Soil mi-crobial biomass estimated by fumigation±extraction and substrate induced respiration in two pesticide treated soils. Soil Biology and Biochemistry 25, 679±683.
Sato, K., 1983. Eect of a pesticide, pentachlorophenol (PCP) on soil micro ¯ora, I. Eect of PCP on microbiological processes in soil percolated with glycine. Plant and Soil 75, 417±426.
SchoÈnborn, W., Dumpert, K., 1990. Eects of pentachlorophenol and 2,4,5-trichrolophenoxyacetic acid on the micro ¯ora of the soil in a beech wood. Biology and Fertility of Soils 9, 292±300. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction
method for measuring soil microbial biomass C. Soil Biology and Biochemistry. 23, 1097±1098.
Welp S.R., BruÈmmer G.W., 1992. Toxicity of organic pollutants to soil microorganisms. In: Hall., J.E. Sauerbeck, D.R., L'Hermite, P. (Eds.), Eects of Organic Contaminants in Sewage Sludge on Soil Fertility, Plants and Animals. Oce of the Ocial Publication of the European Communities, CEC publication EUR 14236 EN, Luxembourg, pp. 161±168.
Wild, S.R., Harrad, S.J., Jones, K.C., 1994. The in¯uence of sewage sludge applications to agricultural land on human exposure to polychlorinated dibenzo-p-dioxins (PCDDs) and -furans (PCDFs). Environmental Pollution 83, 357±369.
Zeitz, E., Dumpert, K., RoÈmbke, J., 1987. Eects of pentachlorophe-nol and 2,4,5-trichlorophepentachlorophe-nol on a soil ecosystem. I. Application and residue analysis. The Science of the Total Environment 61, 153±165.
A.M. Chaudri et al. / Soil Biology & Biochemistry 32 (2000) 429±432
