Short communication
Resistance of microbial populations in DDT-contaminated
and uncontaminated soils
D. Kantachote
a,∗, R. Naidu
b, I. Singleton
a, N. McClure
c, B.D. Harch
daDepartment of Soil and Water, University of Adelaide, Waite Campus, Adelaide, SA 5064, Australia bCSIRO Land and Water, Private Bag No. 2, Glen Osmond, Adelaide, SA 5064, Australia
cSchool of Biological Sciences, Flinders University, Adelaide, SA 5001, Australia dCSIRO Mathematical and Information Sciences, Private Bag No. 2, Adelaide, SA 5064, Australia
Received 25 June 1999; received in revised form 3 March 2000; accepted 8 March 2000
Abstract
One DDT-contaminated soil and two uncontaminated soils were used to enumerate DDT-resistant microbes (bacteria, actinomycetes and fungi) by using soil dilution agar plates in media either with 150mg DDT ml−1 or without DDT at
different temperatures (25, 37 and 55◦C). Microbial populations in this study were significantly (p
<0.001) affected by DDT
in the growth medium. However, the numbers of microbes in long-term contaminated and uncontaminated soils were similar, presumably indicating that DDT-resistant microbes had developed over a long time exposure. The tolerance of isolated soil microbes to DDT varied in the order fungi>actinomycetes>bacteria. Bacteria from contaminated soil were more resistant to DDT than bacteria from uncontaminated soils. Microbes isolated at different temperatures also demonstrated varying degrees of DDT resistance. For example, bacteria and actinomycetes isolated at all incubation temperatures were sensitive to DDT. Conversely fungi isolated at all temperatures were unaffected by DDT. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: DDT; Microorganisms; Soil contamination
1. Introduction
Although DDT [1,1,1-trichloro-2,2-bis(p-chloroph-enyl) ethane] has been banned in developed countries due to its persistence and known bioaccumulation, it is still used in some developing countries to control agri-cultural pests and mosquitoes (Boul, 1995). Because of current or past use, DDT and its metabolites, such as DDD [1,1-dichloro 2,2-bis(4-chlorophenyl) ethane]
∗Corresponding author. Tel.:+61-8-8303-6530;
fax:+61-8-8303-6511.
E-mail address: [email protected] (D. Kantachote).
and DDE [1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene], have become a major environmental prob-lem in many countries throughout the world.
Most research has focussed on the short-term ef-fects of DDT and its metabolites on soil microbes (Lal and Saxena, 1982) and information on effects in long-term contaminated soil is limited. This is important as numerous researchers have shown that ageing of soil over a long period leads to a reduc-tion in the bioavailability of contaminants. For this reason we decided to investigate the effects of DDT on soil microbes in soil which had become contam-inated prior to the banning of DDT in South
tralia from June 1986 (Hopkins, 1988). Analysis of the effect of DDT on microbial diversity was seen as a first step towards the identification of resistant microbes that may be used for the remediation of DDT-contaminated sites. Hence, our aims were to (a) investigate the effect of DDT on soil microbial populations and (b) isolate DDT-resistant microbes from soils for further selection of DDT-degrading microbes.
2. Materials and methods
2.1. Samples
DDT-contaminated samples were collected (depth: 0–15 cm) from contaminated soil, which had been ex-cavated from its original site in Blackwood Forest and stored on a plastic liner and covered with plastic sheets at the Southern Waste Depot (S.W.D.), near Willunga, SA prior to this study. Eleven samples of approxi-mately 100 g each were taken from different parts of the stockpile to represent the range of properties of the contaminated soil. Uncontaminated surface soil sam-ples (A and B) (0–15 cm) were also taken from two areas as control samples. Each sample was analysed for a range of chemical properties including DDT and its residues (DDD and DDE) in duplicate. In addition, each sample was also used to study the effect of DDT on soil microbes.
2.2. Effects of DDT on soil microbes
The effect of DDT on soil bacteria, actinomycetes, and fungi was studied in diluted soil suspensions that were plated on selective media with or without DDT. The plate count technique was chosen because this technique provides viable organisms that can at least be used as indicators of microbial response to DDT and also permits isolation of microbes for subsequent studies. Microbes were counted and isolated using en-richment media specific for different microbial groups. Peptone glucose yeast extract agar, malt extract agar and starch salts agar were used for bacteria, fungi and actinomycetes, respectively (Alef, 1995). DDT dissolved in N,N-dimethylformamide, was added to the medium (0.5% v/v) to a final concentration of 150mg ml−1; controls included the same organic
sol-vent but no DDT. The spread plate technique was used for fungi and actinomycetes and two plates were used per dilution. For bacteria, the pour plate technique was used to isolate facultative anaerobes as several re-ports indicate that DDT transformation by facultative anaerobic bacteria yields DDD, which can undergo se-quential reactions until completely biodegraded (Boul, 1995). Four plates were used per dilution. Due to the potential use of isolated strains as inoculants during composting of DDT-contaminated soil, three identical sets of plates were incubated at 25, 37 and 55◦C. In-cubation times for bacteria were 4–5 days while acti-nomycetes and fungi were incubated for 4–10 days. The numbers of microbes were calculated per gram dry weight of soil.
2.3. Soil chemical analysis
Chemical analyses were carried out according to the Australian Laboratory Handbook of Soil and Water Chemicals Methods (Rayment and Higginson, 1992). Electrical conductivity (EC), pH and the composi-tion of soil water extracts were determined follow-ing overnight equilibration of soils (1:5 soil:water). The EC and pH were determined using an EC (Orion, model 160) and a pH meter (Orion, model EA 940). DOC (Dissolved Organic Carbon) was analysed using a total organic carbon analyser (Dohrmann, DC-180) and calculated as the difference between total carbon and inorganic carbon. NH4+-N and NO3−-N were
ex-tracted using 2 M KCl [10:1; KCl:fresh soil (v/w)] and then measured by auto analyser. Soil nitrogen was de-termined using the micro Kjeldahl method, while or-ganic carbon was determined by dichromate oxidation in the presence of concentrated sulfuric acid. The soil moisture content was estimated following overnight oven drying at 105◦C.
2.4. DDT extraction and analyses
DDT, DDD and DDE in soil and soil solution (1:5 soil:water) were extracted using hexane following the method of McDougall et al. (1995). The con-centration of DDT and its residues was determined by gas chromatography (Perkin-Elmer Auto System) using an electron capture detector, and a DB-5 col-umn (30 m×0.53 mm i.d., 1.5mm film thickness).
were set at 35 and 30 ml min−1, respectively; the column temperature was 200◦C, the injector temper-ature 260◦C, the detector temperature 330◦C, and the injection volume was 1ml. Standard solutions of
DDT and its residues (DDD and DDE) were pre-pared at five different concentrations ranging from 0.10 to 1.00 and 0.0025 to 0.10mg ml−1(hexane) for
soil and soil solution, respectively. Chromatographic peaks response versus concentrations were plotted and calibration was performed using linear regression analysis.
2.5. Statistical methods
There were two samples for uncontaminated soils and 11 samples for contaminated soil. The effects of temperature (25, 37 and 55◦C), media type with DDT/without DDT and soil samples were deter-mined using separate three-way analyses of variance (ANOVA) for each microbial group. Due to the im-balance associated with having four replicates for bacteria and two replicates for both actinomycetes and fungi, restricted maximum likelihood (REML) methods were employed. Preliminary analysis with three levels (two uncontaminated soils and the S.W.D. contaminated soil) revealed significant dif-ferences in mean microbial population levels among DDT-contaminated soil samples (p<0.001).
Conse-quently, the soil samples were expanded to include 13 levels (two uncontaminated and 11 S.W.D. sam-ples). Separate analyses were conducted for each microbial group because significant differences were found amongst mean population levels for each of the three types of microbe investigated (p<0.001). The
Chi-square test was used to investigate the degree of DDT resistance in different microbial groups as indi-cated by growth response based on average microbial numbers in uncontaminated soils and contaminated soil. In addition, a log-linear model was fitted to the cross-classification (Deviance table) of the factors microbial type (Bacteria, actinomycetes, fungi), DDT effect (sensitive, unaffected, stimulated) and temper-atures (25, 37 and 55◦C) based on average microbial numbers in media containing DDT or without DDT of contaminated soil samples. This analysis was con-ducted to determine any dependency among these three factors. All statistical analyses were conducted using Genstat for Windows Version 5 Release 4.1.TM
3. Results and discussion
3.1. Soil properties
Table 1 lists the pertinent properties of the soils used in this work. As expected, DDT and its metabo-lites were not detected in the uncontaminated soils (minimum detection limit=2.5mg DDT kg−1). Mean
values of DDT, DDD and DDE in contaminated soil were 38.00, 2.34 and 2.61 mg kg−1, respectively while
DDT, DDD and DDE in soil solution were 1.38, 0.13 and 0.02 mg kg−1, respectively. Therefore,
percent-ages of DDT, DDD and DDE soluble in soil solution were 3.63, 5.56 and 0.77, respectively. Comparison of levels of DDT and its metabolites in the soils and soil solutions showed that DDE was present in the lowest amounts. This may be due to lower rates of microbial transformation of DDT to DDE or to strong binding of DDE to clay and/or organic matter. This could be one reason why DDE is more persistent than DDT and DDD in soils.
3.2. Distribution of soil microbes in uncontaminated and DDT-contaminated soils
The numbers of soil microbes in the uncontami-nated and long-term contamiuncontami-nated soils were similar
Table 1
Chemical characteristics of uncontaminated soils (Blackwood For-est: A and B) and DDT-contaminated soil from S.W.D.a
Chemical characteristics A (n=2) B (n=2) S.W.D. (n=22)
aTSS, DSS, ESS, mean DDT, DDD and DDE in soil solution,
respectively. Units for all parameters are mg kg−1 dry soil except
Table 2
Summary of the results of the three- way ANOVA of microbial population means (CFU per g dry soil×105) for determining the differences
amongst contaminated soil samples (LSD1) and all soil samples (LSD2)a
Soil sample DDT (mg kg−1) Incubation (◦C) Media DDT (
mg ml−1) Bacteria (n=4) Actinomycetes (n=2) Fungi (n=2)
Area A 25 0 275.9 143.6 4
0 150 96.6 145.1 7
37 0 249.5 143.6 7
150 135.8 93.7 7
55 0 36.2 28.6 0
150 21.7 21.5 0
Area B 25 0 94.4 37.2 18
0 150 62.7 52.2 16.1
37 0 83.9 26.1 5.2
150 44.1 10 7.8
55 0 12.7 2.6 0
150 4.7 3.1 0
S.W.D. 25 0 438.9 247 111
16 150 448.9 263.1 111
37 0 191.7 205.9 32.2
150 251 205.9 36.9
55 0 8 0 0
150 4.1 0 0
S.W.D. 25 0 1521.9 604.6 91.4
35 150 1303.6 653.1 104
37 0 571.9 228.3 156
150 417.2 228.3 130
55 0 14.3 2.6 2.6
150 3.8 5.2 2.6
S.W.D. 25 0 96.4 23.1 3.4
43 150 74.3 22.5 3.4
37 0 78.3 34.1 2.3
150 62.2 34.1 3.4
55 0 5.7 0 0
150 4.5 0 0
S.W.D. 25 0 1348.8 642.7 60.5
70 150 1204.4 640.8 74.3
37 0 673.6 203.2 25.1
150 452.6 160.6 37.2
55 0 4.9 14.8 1.2
150 5.3 14.8 1.2
LSD1 (5%) 0.1249 0.1039 0.1565
LSD2 (5%) 0.1286 0.0973 0.1624
aA selection of typical samples from contaminated soil are presented; LSD: least significant differences of means.
(Table 2), and were comparable with those reported in previous studies (e.g. Subba-Rao, 1995). The numbers of bacteria incubated at 25 and 37◦C were typically between 107–108 and 105–106cfu g−1 soil for 55◦C incubation (Table 2). Numbers of actinomycetes in all soil samples were consistent with previous find-ings (Dragun, 1998), and only a few actinomycetes
observa-tions that thermophilic fungi are rare in soils (Tate, 1995).
3.3. Effect of DDT on soil microbial populations on agar plates
The results show that irrespective of the nature of the microbes and their origin, addition of DDT to growth media significantly affected populations. These results are consistent with those reported by Lal and Saxena (1982). Changes in numbers of colony forming units on DDT-contaminated and un-contaminated media were used as a measure of the effect of DDT on soil microbial populations. There are three possible effects of pesticides on microbes: inhibition, no effect and stimulation (Dragun, 1998). Data from the present study reveal that DDT had the full range of these effects on microbes (Table 2). Based on the means provided in Table 2, the mi-crobial groups were rated as either being sensitive, unaffected or stimulated by DDT at 150mg ml−1.
Chi-square tests to determine whether there was an association between the level of microbial growth (sensitive, unaffected, stimulated) and the type of soil microbe revealed the following trend in DDT resistance: fungi>actinomycetes>bacteria (Table 3). Moreover, the data show that bacteria from uncon-taminated soils were more sensitive to DDT than bacteria from contaminated soil, suggesting that a DDT-resistant population had developed in the con-taminated soil. These results are consistent with those reported by Kokke (1970) who found that the
Table 4
Short-term effects in soils spiked with 100 mg DDT kg−1 for 8 days and over 4 weeks, respectively, and long-term (this study) effects of
DDT on soil microbial populations (log CFU per g dry soil)
Bacteria Actinomycetes Fungi References
−DDT +DDT −DDT +DDT −DDT +DDT
7.7 7.7 6.8 6.7 5.4 5.3 Ko and Lockwood, 1968
8.3 6.6 Ha Lb H L Kahlon et al., 1990
8.0±7.8 7.9±7.7 7.5±7.5 7.5±7.5 6.8±6.7 6.8±6.8 This studyc, 25◦C 7.7±7.5 7.7±7.5 7.3±6.4 7.2±6.1 6.7±5.8 6.7±5.8 37◦C
6.0±6.1 5.9±6.1 5.5±5.8 5.6±5.8 4.6±4.9 4.7±4.9 55◦C
aH: higher populations. bL: lower populations.
cCounting microbes in media containing DDT 150 mg l−1and microbial populations (mean±S.D.) from 11 samples of contaminated
soil.
Table 3
Percentages of different microbial groups either sensitive, unaf-fected or stimulated by DDT in media isolated from contaminated (C) or uncontaminated soil (U)
Microbes Sensitive Unaffected Stimulated
U C U C U C
Bacteria 100 64 0 27 0 9
Actinomycetes 50 30 17 52 33 18
Fungi 0 9 67 58 33 33
proportion of DDT-resistant bacteria was markedly elevated in DDT-containing environments. The level of microbial resistance to DDT in this study was similar to that found in previous short-term stud-ies (Ko and Lockwood, 1968; Kahlon et al., 1990) (Tables 3 and 4).
In addition, results analysed by cross-classification based on means in media with DDT or without DDT in contaminated soil samples (Table 2) indicated that there was a significant influence of temperature on microbial numbers with microbes either being sensi-tive, unaffected or stimulated by DDT (p<0.05). For
years. However, DDT seemed to have less effect on bacterial populations in long-term than in short-term contaminated soils (Table 4), indicating that bacteria had adapted to survive in the environment contami-nated with DDT.
4. Conclusions
Our results show that microbial populations in a long-term DDT-contaminated soil were comparable with those in uncontaminated soils, and that this con-taminated soil was a good source of DDT-resistant microbes, with possible potential for use as inoculants for bioremediation of DDT-contaminated soils. Fungi were the most resistant to DDT, and DDT-resistant microbes were isolated at 25, 37 and 55◦C. These or-ganisms have potential as inoculants in bioremediation of soil by composting. Current work is examining the ability of DDT-resistant isolates to transform DDT in liquid and soil.
Acknowledgements
The Royal Thai government is thanked for a Ph.D. Scholarship for D. Kantachote. We would like to thank Dr. Megharaj Mallavarapu and Dr. Ken Lee for their helpful comments and also Julie Smith and Colin Rivers for some of the soil chemical analyses.
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