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An in Situ Respirometric Technique to Measure Pollution-Induced Microbial Community Tolerance in Soils Contaminated with 2,4,6-Trinitrotoluene
Ecotoxicology and Environmental Safety 47, 96}103 (2000) Environmental Research, Section B doi:10.1006/eesa.2000.1934, available online at http://www.idealibrary.com on
An in Situ Respirometric Technique to Measure Pollution-Induced Microbial Community Tolerance in Soils Contaminated with 2,4,6-Trinitrotoluene1 Ping Gong,* Pietro Gasparrini,- Denis Rho,* Jalal Hawari,* Sonia Thiboutot,? Guy Ampleman,? and Geo!rey I. Sunahara* *Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, MontreH al, QueH bec, H4P 2R2, Canada; -Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, MontreH al, QueH bec, H3G 1M8, Canada; and ?Defence Research Establishment Valcartier, Department of National Defence, 2459 Pie XI Boulevard, Val-BeH lair, QueH bec, G3J 1X5, Canada Received November 30, 1999
due to individual acclimation, genetic or physiological adaptation, and loss of sensitive species as a result of longterm exposure to a toxicant or a mixture of toxicants (Posthuma, 1997). An elevated PICT can be used as an evidence that a toxicant has previously had contact with the community and may have caused structural changes. A variety of approaches to examine the tolerance or resistance of soil microorganisms to heavy metals and recal-citrant organic compounds have been developed. A conven-tional approach is to isolate and culture the tolerant microorganisms from soil samples (Klausmeier et al., 1974; Jordan and Lechevalier, 1975; Duxbury and Bicknell, 1983). However, an intrinsic shortcoming of the plate count tech-nique is that more than 99% of soil bacteria cannot be isolated on agar plates (Torsvik, 1995). Therefore, only a small proportion of soil microorganisms are available for further sensitivity testing. More recently, a thymidine or leucine incorporation technique (Bas as th, 1992; DmH az-Ravin a et al., 1994; DmH azRavin a and Bas as th, 1996; Pennanen et al., 1996) and Biolog microtiter plates (Rutgers et al., 1998; Siciliano et al., 2000) have been used to assess the tolerance of bacterial communities in soil. However, these two tech-niques also result in an incomplete evaluation of the entire microbial community because of insu$cient extraction of bacteria from soil (Rutgers et al., 1998; Siciliano et al., 2000; Witter et al., 2000). In a previous study, Witter et al. (2000) developed an in situ respirometric technique based on the analysis of the substrate-induced respiration (SIR) response to test the microbial tolerance to heavy metals at the community level in soil ecosystems. These workers demonstrated that microbial metal tolerance increased as soil metal concentrations increased (Witter et al., 2000). In the present study, this newly developed technique was used on soils contaminated with 2,4,6-trinitrotoluene (TNT) for several decades, since TNT is a proven toxicant to soil microorganisms and is very recalcitrant in soil (Klausmeier et al., 1974; Fuller and
Long-term exposure to 2,4,6-trinitrotoluene (TNT) can induce changes in the structure and activities of soil microbial communities. Such changes may be associated with an elevated microbial tolerance. An in situ respirometry technique based on the analysis of the substrate-induced respiration response to freshly added TNT was used to examine soil microbial tolerance to TNT at the community level. The speci5c growth rate derived by 5tting an exponential equation to respiration data was taken as the measurement endpoint. Microbial tolerance was evaluated using a tolerance index de5ned as the ratio of the speci5c growth rate at a spiking dose of 2000 lg TNT/g soil to that of the control with no spiked TNT. Three soils with long-term exposure histories (TNT level in soil: 1.5, 32, and 620 lg TNT/g, respectively) exhibited signi5cantly higher microbial community tolerance to TNT than two uncontaminated control soils. A soil containing 29,000 lg TNT/g exhibited the highest tolerance. Findings from this study support the hypothesis that pollutioninduced community tolerance can be used as a means of identifying those compounds that have exerted selective pressure on the community. 2000 Academic Press Key Words: soil microbial community; 2,4,6-trinitrotoluene; pollution-induced community tolerance; substrate-induced respiration; speci5c growth rate. INTRODUCTION
Pollution-induced community tolerance (PICT) has recently been used as an ecotoxicological tool for assessing the toxic e!ects of a pollutant on an ecosystem at the community level (Blanck et al., 1988; Dahl and Blanck, 1996; Rutgers et al., 1998; Siciliano et al., 2000). The PICT theory is founded on the principle that communities become tolerant Assigned NRCC Publication No. 43308. To whom all correspondence should be addressed. Fax: (514) 496}6265. E-mail: [email protected]. 96 0147-6513/00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved.
MICROBIAL COMMUNITY TOLERANCE TO TNT IN SOIL
Manning, 1997, 1998; Gong et al., 1999; Siciliano et al., 2000). The purpose of this study was to investigate the natural evolution of TNT tolerance of soil microorganisms at the community level. The hypothesis is that microbial communities that have undergone prolonged exposure to TNT contamination develop a concentration-dependent tolerance to TNT, which would further corroborate the PICT theory. MATERIAL AND METHODS
Soils Soils were collected from non-TNT-contaminated and TNT-contaminated sites. A garden soil (GS-3) and a forest soil (RAC) were sampled in the fall of 1998, and they had no history of exposure to TNT. Four TNT-contaminated soils (B23, B24, B25, B26) were sampled in July 1998 from a site around an abandoned TNT-manufacturing facility (Gong et al., 1999). All six soils were sieved ((2 mm) and stored at !203C. U.S. EPA Method 8330 was used to extract TNT from soil and to determine TNT concentrations in acetonitrile extracts using HPLC}UV (U.S. Environmental Protection Agency, 1997; Gong et al., 1999). The detection limit was about 0.5 mg TNT/kg soil. Some physiochemical properties of these soils are summarized in Table 1. Soil B23 was located in a nonvegetated area, while soils B24, B25, and B26 were all located within a vegetated area but varied in TNT concentration. More details about the sampling of TNT-contaminated soils and the "eld conditions can be found elsewhere (Gong et al., 1999; Siciliano et al., 2000). Tolerance Measurements Prior to tolerance testing, soils were thawed overnight in a refrigerator (43C). Twenty-gram (dry weight) soil samples
TABLE 1 Some Physical and Chemical Properties of the Soils Used in the Present Study
Soil GS-3 RAC B26 B24 B25 B23
Origin
Texture (%) TNT C @ USDA (lg/g) pH? (%) Sand Silt Clay classi"cation
Garden 0.0A Forest 0.0A IndustrialB 1.5 IndustrialB 32 IndustrialB 620 IndustrialB 29,000
6.5 7.7 6.7 6.4 6.2 6.9
8.7 4.1 6.4 15.6 11.5 NDC
15 85 80 70 69 ND
55.5 11.5 17 21 25 ND
29.5 Silty clay loam 3.5 Loamy sand 3 Loamy sand 9 Sandy loam 6 Sandy loam ND ND
? Measured in a 1:5 (v/v) H O suspension. @ Organic matter content, estimated from weight loss after 24-h ignition at 4303C. A Less than detection limit (&0.5 mg TNT/kg soil). B TNT-contaminated site. C Not determined.
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were weighed into 250-ml bottles, to which a volume of NPK solution (determined according to the ratio C:N:P:K"15:1:0.3:3.5, with respect to glucose-C) was added. The bottles were gently mixed for 2}3 min and then preincubated in darkness at 253C for 1}2 weeks. The "nal moisture was 45% of water-holding capacity of each soil. The TNT}glucose mixtures were prepared from a TNT stock solution of 0.25 g TNT/ml acetonitrile and D-(#)glucose (BDH, Darmstadt, Germany). Technical-grade TNT was obtained from ICI Explosives (McMasterville, PQ, Canada). Appropriate amounts of the TNT stock solution were added to glass jars, and the acetonitrile was allowed to evaporate prior to the addition of the corresponding amount of glucose. The TNT}glucose mixtures were blended thoroughly and stored at 43C in the dark until use. After preincubation, the soil samples were spiked with TNT}glucose mixtures (powder) and gently mixed for 2}3 min. Three soils (GS-3, RAC, B23) were spiked with TNT at six di!erent concentrations: 0, 125, 250, 500, 1000, and 2000 lg TNT/g. Because of limited quantities, the other three soils (B24, B25, B26) were spiked with only 0 and 2000 lg TNT/g. Glucose was added at a concentration of 10 mg/g soil (4 mg C/g soil), which induced the maximum speci"c growth rates of the GS-3 and RAC soils as determined in preliminary tests. Each treatment was prepared in quadruplicate. A specially designed respirometer similar to that described by Arulgnanendran and Nirmalakhandan (1998) was used to measure the O consumed in response to the TNT}glucose addition. A CO trap (10 ml of 1 M KOH) was placed in each sample and reference bottle, which was maintained at 253C in a thermostatically controlled water bath. Soil respiration was recorded for a minimum of 70 h. At the end of each test, the CO traps were titrated with 1 M HCl to determine the total amount of CO evolved. Data Analysis The time and cumulative amount of O fed were auto matically recorded by the respirometer. The oxygen consumption rate (y) was calculated and plotted against incubation time (t) to obtain a respiration curve (Fig. 1), from which the speci"c growth rate (l) was derived as the value of the exponent in the equation y"y e lt , where y is the O consumption rate when t"0 (Schmidt, 1992; Colores et al., 1996; Witter et al., 2000). The hourly O consumption rate was used for curve "tting (Fig. 1). The point of onset of exponential growth was estimated by a visible increase in the oxygen consumption rate after a lag
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RESULTS
GS-3 Garden Soil All of the O consumption rate curves produced for the control and those samples spiked with 125, 250, and 500 lg TNT/g soil were dominated by a single peak as illustrated in Fig. 1. However, at spiking doses of 1000 and 2000 lg TNT/g soil, two separate exponential growth phases were observed (Fig. 2a), from which two speci"c growth rates were derived. The speci"c growth rates for all treatments during the "rst or sole exponential growth phase were not statistically di!erent (P"0.40, ANOVA) (Fig. 3). Considering the second exponential phase at 1000 and 2000 lg TNT/g treatments, instead of the "rst, the speci"c growth rates decreased steadily as the concentration of spiked TNT increased, and the mean l values di!ered signi"cantly (P(0.01, ANOVA) (Fig. 3). FIG. 1. Substrate-induced microbial respiration curve in the GS-3 soil treated with 4 mg glucose-C/g and 125 mg TNT/g (data of one sample). Oxygen consumption rates per event and per hour are shown with curve "tting of the hourly O consumption rate (Hours 13 to 29) to an exponen tial equation.
RAC Forest Soil All samples of RAC soil experienced a single exponential growth phase. The mean speci"c growth rates decreased
period (Nordgren et al., 1988; Colores et al., 1996; Witter et al., 2000). The end of the exponential growth period was determined using the F test devised by Colores et al. (1996). The EC , de"ned as the e!ective TNT spiking concentra tion causing 20% inhibition of microbial respiration (evaluated by the speci"c growth rate), was calculated from a dose}response curve using ToxCalc (Tidepool Scienti"c Softwear, McKinleyville, CA). For each soil, the average l of the control samples was set to 100% and that of all other treatments was expressed as a percentage of the control. A linear interpolation method was used to derive the EC value. Experimental results were subjected to analysis of variance (ANOVA) using Statistica (Release 5.0, StatSoft, Tulsa, OK). Post hoc comparisons of the least signi"cant di!erence (LSD) or Sche!eH test were performed to examine the signi"cant di!erence between means. The tolerance index (TI) of a soil at a certain spiking dose was calculated as the ratio of the speci"c growth rate of the soil amended with the speci"c dose of TNT (k ) to that of the control (k ), i.e., 2 k TI" 2 . k Accordingly, TI represents the tolerance index to 2000 lg TNT/g soil. Since there were four replicates for each treatment, a maximum of 16 TI values could be obtained for each soil, from which the mean and standard deviation of the TI were estimated.
FIG. 2. Two exponential growth phases in (a) the GS-3 soil treated with 4 mg glucose-C/g and 2000 mg TNT/g and (b) the B23 soil treated with 4 mg glucose-C/g only (control). Both curves represent one sample of four replicates for the respective treatment.
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MICROBIAL COMMUNITY TOLERANCE TO TNT IN SOIL
FIG. 3. Response of the substrate-induced microbial community respiration to freshly amended TNT in the GS-3 and RAC soils. The speci"c growth rate is used as the measurement endpoint. These two soils were not exposed to TNT before testing. Each data point is the average of four replicates. Vertical bars represent SD.
rapidly from 0.15/h to 0.07/h as the concentration of spiked TNT increased from 0 to 250 kg/g (Fig. 3), which was then followed by a plateau. That is, the k values did not decrease any further when the spiking concentration was higher than 250 lg TNT/g. Statistically, the l values for the six treatments were signi"cantly di!erent (P(0.01, ANOVA).
FIG. 4. Microbial community response to freshly spiked TNT in the B23 soil. This soil initially contained 29,000 lg TNT/g. The speci"c growth rate is derived from the respiration curve of soil microbes induced by 4 mg glucose-C/g soil. Data are given as means$SD (n"4).
However, if the Sche!eH test is applied for the post hoc comparison, no signi"cant di!erence exists between B26, B24, and B25 soils. The EC of soil B23 was much higher than those of RAC and GS-3 soils (second peak), and RAC soil was more sensitive to TNT than was GS-3 soil (Table 2). DISCUSSION
Soils from TNT-Contaminated Site
Physiological Characteristics of Soil Microbes
There were two separate phases of exponential growth in all B23 soil samples (Fig. 2b). The "rst exponential phase had approximately half the length of duration and one-third the peak respiration rate of the second one. As illustrated in Fig. 4, the mean speci"c growth rates calculated for both phases were not signi"cantly di!erent between the six treatments (P'0.05, ANOVA). The other three site soils were tested only at the highest spiking rate, i.e., 2000 lg TNT/g soil. As for the RAC soil, only one exponential phase was observed in these soils.
The six soils used in the present study di!ered in their origin, texture, organic matter content, and degree of TNT
Comparison of Tolerance Index and EC20 between Soils Comparing the six soils tested in this study (considering only the second or sole peak in the GS-3 and B23 soils), a signi"cant increase in the TNT tolerance indices was observed, which was proportional to the magnitude of previous TNT contamination in soil (Table 2). Among the six soils tested, soil B23 exhibited the highest TNT tolerance, followed by B24, B25, and B26. The two uncontaminated soils had the lowest tolerance (ANOVA, post hoc test; LSD).
TABLE 2 E4ective Concentration Inhibiting Microbial Respiration by 20% (EC20 ) and Tolerance Index at a Spiking Dose of 2000 lg TNT/g Soil (TI2000 ) Calculated for the Six Soils Testeda EC
(lg TNT/g soil)
Soil
First peak
Second peak@
GS-3 RAC B26 B24 B25 B23
'2000 70 NA NA NA 1400
530 NAA NA NA NA 1300
TI First peak 0.99$0.12 0.50$0.04 0.61$0.12 0.77$0.13 0.70$0.13 0.93$0.30
A B C BC
Second peak@ 0.42$0.06 A NA NA NA NA 0.88$0.16 D
? Values followed by di!erent letters are signi"cantly di!erent (P(0.05, LSD). The second peak in the GS-3 and B23 soils is used for comparison. @ For the GS-3 soil, the "rst and sole peaks of 0}500 lg TNT/g treatments are used. A Not available.
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TABLE 3 Physiological Characteristics of the Substrate-Induced Exponential Growth of Soil Microbes?
Soil
Lag time (h)
GS-3 RAC B26 B24 B25 B23@ B23A
11$1 11$1 12$3 12$3 11$2 24$5 50$6
A A A A A B C
Speci"c growth rate (h\) 0.18$0.02 0.15$0.00 0.10$0.00 0.11$0.00 0.08$0.01 0.16$0.02 0.05$0.01
E D BC C B D A
Peak time Peak respiration rate (h) (mg O /h) 26$2 A 37$3 BC 30$3 AB 30$3 AB 40$4 C 40$6 C 80$13 D
4.7$0.9 1.9$0.1 3.0$0.7 5.4$1.2 4.6$1.0 0.5$0.2 1.8$0.8
C B B C C A AB
? Each soil received 4 mg glucose-C/g soil and equivalent nutrient elements of nitrogen, phosphorus, and potassium at a ratio of 15:1:0.3:3.5 (C:N:P:K). Values are given as means$SD (n"4). Values in the same column are signi"cantly di!erent if followed by di!erent letters (P(0.05, LSD). @ First peak. A Second peak.
contamination. Consequently, microorganisms in these soils demonstrated diverse responses to the addition of glucose, a readily usable substrate. GS-3 soil was the most active among the six tested, as re#ected by its short lag time and peak time and its high peak respiration and speci"c growth rates (Table 3). The relatively high organic matter content of soils B24 and B25 (and possibly soil B23) indicates an accumulation of organic matter or a decrease in organic matter mineralization, which might have resulted from TNT contamination (Table 1). It has been generally accepted that increasing environmental stress often causes a reduction in soil microbial activities such as respiration. In this study, aside from the decreased speci"c growth rate and the increased organic matter content at elevated soil TNT levels (Tables 1 and 3), there is no obvious relationship between TNT concentrations and other physiological parameters such as lag time and peak time (Table 3). Such parameters as lag time, speci"c growth rate, SIR, metabolic quotient (qCO ), and percentage of microbial biomass-C in total soil organic-C have been commonly used as indicators of soil contamination (Doelman and Haanstra, 1979; Haanstra and Doelman, 1984; Nordgren et al., 1988; Anderson, 1994). However, more recent studies have shown that some of these parameters are not consistently lower in contaminated soils (Dahlin et al., 1997; Dahlin and Witter, 1998; Giller et al., 1998; Witter et al., 2000). For instance, the toxicity of heavy metals to soil microbes measured by SIR, lag time, or speci"c growth rate can be confounded by soil organic matter (Witter et al., 2000). When the di!erent soils are compared, soil microbial activities may be a!ected by many environmental factors other than pollution (Welp and Brummer, 1999). Therefore,
measurements of soil microbial activities are insu$cient to establish a causal relationship between pollution and microbial response. In such cases, the PICT measurement on the soil microbial communities may be a useful tool to distinguish a polluted soil from a nonpolluted one (Rutgers et al., 1998; Posthuma, 1997). Specixc Growth Rate as Measurement Endpoint As an essential step in PICT measurements, an ecologically relevant parameter should be selected as the measurement endpoint that re#ects toxic e!ects at the community level. The speci"c growth rate was chosen for this research because its response to TNT challenge was more consistent and sensitive than other parameters such as lag time, peak time, SIR, peak respiration rate, total O consumed, and total CO produced (data not provided). The speci"c growth rate (l) is also biologically meaningful since SIR is directly related to the size of microbial biomass and the activity of soil microbial communities (Anderson and Domsch, 1978; Schmidt, 1992; Colores et al., 1996). Moreover, the l value can be determined precisely by curve "tting (Fig. 1) (Colores et al., 1996), whereas some of the other parameters (e.g., lag time and peak time) are estimated more or less visually. A signi"cant reduction in the speci"c growth rate was observed after the addition of TNT in "ve of the six soils tested in this study. Statistical analysis indicated that the inhibition of the speci"c growth rate was dependent on the dose of TNT added to the soil, but independent of soil type (ANOVA). A causal relationship can thus be established between the TNT spiking dose and the speci"c growth rate of the microbial communities in soil (Figs. 3 and 4, Table 2). Such a relationship was demonstrated previously in metalcontaminated soils (Witter et al., 2000). This causal relationship thus builds up an essential foundation for further testing of the PICT. Relation of Microbial Tolerance to TNT to PICT Theory In the present study, higher tolerance to TNT was observed at the community level in all of the four site soils that had long-term exposure to TNT, but was not seen in the two soils with no TNT exposure history. Although TNT-resistant microbial species have been found to exist in both polluted and unpolluted soils (Klausmeier et al., 1974), soils with high levels of TNT usually harbor a larger proportion of TNT-resistant bacteria than do soils with low levels of TNT (Fuller and Manning, 1998). A more recent investigation using Biolog microtiter plates demonstrated that microbial communities in soils slightly contaminated with TNT were more sensitive to freshly added TNT than those in highly TNT-contaminated soils (Siciliano et al., 2000). All these results corroborate the PICT theory (Blanck et al.,
MICROBIAL COMMUNITY TOLERANCE TO TNT IN SOIL
1988; Posthuma, 1997); that is, microbial communities resulting from long-term exposure to TNT possess relatively high overall tolerance to this pollutant. The absence of a clear concentration-dependent PICT is probably due to the fact that the sampling spots of the contaminated soils were so close to each other (5}6 m apart) that soil microorganisms might have migrated between them under "eld conditions. Accordingly, soils B24, B25, and B26 revealed a similar TNT tolerance. Additionally, the spiking level of 2000 lg TNT/g soil might not have been high enough to di!erentiate the TNT tolerance in these three soils. Even though the in situ TNT stress in soil B26 was low due to the very low level of TNT at the sampling time, this soil still exhibited a signi"cantly higher tolerance than did the GS-3 and RAC soils. This e!ect might also be attributed to inheritable genetic adaptation of soil microbes (Gadd, 1989). Binary Exponential Growth in GS-3 and B23 Soils At the two highest levels of TNT spiking (1000 and 2000 lg TNT/g soil) for the GS-3 soil and in all samples of the B23 soil, a second period of exponential growth was observed (Fig. 2). This phenomenon has been previously reported by Winkel and Wilke (1997). The appearance of a second exponential phase in soil GS-3 might be due to the growth of inherently TNT-tolerant microbial species. Since the "rst exponential growth phase in these samples treated with 1000 and 2000 lg TNT/g soil was so similar to the sole exponential growth phase in the control (the l values are not signi"cantly di!erent at P(0.001), the exponentially growing microorganisms might be of the same sensitive communities. Therefore, only the second growth period was used for tolerance comparison. The second exponential phase in soil B23 was probably also due to the growth of dominant TNT-resistant microbes. Compared with the second peak, the "rst peak in soil B23 was much shorter and weaker, which might represent the growth of relatively sensitive microbes. In the presence of 29,000 lg TNT/g, these sensitive species, however, would soon die o! and be replaced by tolerant species. The "rst exponential phase in soil B23 was thus very transient and not used for tolerance comparison. The addition of up to 2000 lg TNT/g may not have changed the soil TNT level very much, given the extremely high background TNT level, so the substrate-induced microbial growth in this soil was not a!ected by the addition of a relatively small amount of TNT. In contrast to the work of Winkel and Wilke (1997), TNT was added together with glucose instead of 3}17 days prior to substrate addition. This di!erence allowed no or little time for TNT to exert any selective pressure on soil microbial communities. In addition, it was reported that the second phase of exponential growth became more signi"cant as the time between TNT spiking and glucose addition was in-
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creased (Winkel and Wilke, 1997). All the evidence suggests that the adaptation of TNT-tolerant communities is time dependent. TNT Bioavailability and Biotransformation Both solubility and soil adsorption limit the bioavailability of TNT, which necessitates the addition of large amounts of TNT to induce an observable inhibition. Like heavy metals, the amended TNT can be adsorbed and bound to both clay and organic matter and, thus, be rendered unavailable to soil microorganisms (Hundal et al., 1997; Li et al., 1997; Gong et al., 1999). Clay soils may thus be less sensitive than sandy soils, as is indicated by the higher EC for the GS-3 soil compared with the RAC soil. To avoid the in#uence of such factors, it seems appropriate to test soils at pollutant concentrations that can induce the maximum inhibition. By comparing the TI , GS-3 and RAC soils revealed similar TNT tolerance levels. Biodegradation of TNT may complicate the interpretation of results. Since microbial transformation takes place rapidly and extensively in soil under both aerobic and anaerobic conditions (Klausmeier et al., 1974; Lewis et al., 1997), the actual in situ concentrations of spiked and original TNT in soil are not known during and after the "rst or sole exponential growth period. Microbial transformation of TNT to less toxic metabolites could detoxify TNT (Sunahara et al., 1998) and therefore may confound the tolerance results. An observation of enhanced tolerance in one soil might be attributed to a higher biotransformation capacity of microbial communities in this soil. Therefore, it appears to be necessary to add TNT at the very onset of exponential growth so that no time was given for soil microbes to transform or detoxify TNT (Witter et al., 2000), especially when two exponential phases might appear. CONCLUSION
Long-term exposure to recalcitrant contaminants is thought to a!ect soil microbial communities by reducing their abundance and species diversity and selecting for resistant populations (Gadd, 1989). It is preferable to separate the target organisms from their inhabiting soil to study their tolerance, but there is no e!ective technique so far that can e!ectively extract entire microbial communities from soil. An alternative approach using the in situ respirometric technique has been developed and tested. Although the respirometric technique is more time consuming than some other techniques, such as the thymidine incorporation technique and the Biolog technique (Witter et al., 2000), its greatest advantage is that soil samples can be tested without extraction of microbial species. An evaluation of the overall tolerance of whole microbial communities can thus be made.
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By use of the in situ respirometric technique, it was revealed that the microbes in soil samples collected along a TNT gradient at a contaminated site possessed elevated tolerance to TNT in comparison with two uncontaminated soils. The most contaminated soil demonstrated the highest tolerance. Based on the results obtained from this and previous studies (Witter et al., 2000), it may be concluded that this respirometric technique is a promising tool for measuring PICT in both metal- and recalcitrant organic chemical-contaminated soils. However, caution should be taken in the interpretation of data, in view of the possible biotransformation of organic chemicals and the appearance of binary phases of microbial exponential growth. These problems can be avoided by technical adaptations. ACKNOWLEDGMENTS The authors thank Professor B.-M. Wilke, Technische UniversitaK t Berlin, Berlin, Germany, for his critical review of the manuscript and AndreH Chouinard, Marie-JoseH e Lorrain, Nathalie Matte, Jonathan Hodgson, Louise Paquet, and Serge Delisle for their technical assistance.
REFERENCES Anderson, J. P. E., and Domsch, K. H. (1978). A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biol. Biochem. 10, 215}221. Anderson, T.-H. (1994). Physiological analysis of microbial communities in soil: Applications and limitations. In Beyond the Biomass (K. Ritz, J. Dighton, and K. E. Giller, Eds.), pp. 67}76. Br. Soc. of Soil Science, Wiley}Sayce, London. Arulgnanendran, V. R. J., and Nirmalakhandan, N. (1998). Microbial toxicity in soil medium. Ecotoxicol. Environ. Saf. 39, 48}56. Bas as th, E. (1992). Measurement of heavy metal tolerance of soil bacteria using thymidine incorporation into bacteria extracted after homogenization}centrifugation. Soil Biol. Biochem. 24, 1167}1172. Blanck, H., WaK ngberg, S.-As ., and Molander, S. (1988). Pollution-induced community tolerance: A new ecotoxicological tool. In Functional ¹esting of Aquatic Biota for Estimating Hazards of Chemicals (J. Cairns, Jr., and J. R. Pratt, Eds.), ASTM STP 988, pp. 219}230. ASTM, Philadelphia. Colores, G. M., Schmidt, S. K., and Fisk, M. C. (1996). Estimating the biomass of microbial functional groups using rates of growth-related soil respiration. Soil Biol. Biochem. 28, 1569}1577. Dahl, B., and Blanck, H. (1996). Pollution-induced community tolerance (PICT) in periphyton communities established under tri-n-butylin (TBT) stress in marine microcosms. Aquat. ¹oxicol. 34, 305}325. Dahlin, S., and Witter, E. (1998). Can the low microbial biomass-C-toorganic C ratios in an acid and a metal contaminated soil be explained by di!erences in growth characteristics, substrate utilization e$ciency or maintenance requirements? Soil Biol. Biochem. 30, 633}641. Dahlin, S., Witter, E., Mas rtensson, A. M., Turner, A., and Bas as th, E. (1997). Where's the limit? Changes in the microbiological properties of agricultural soils at low levels of metal contamination. Soil Biol. Biochem. 29, 1405}1415. DmH az-Ravin a, M., and Bas as th, E. (1996). In#uence of di!erent temperatures on metal tolerance measurements and growth response in bacterial communities from unpolluted and polluted soils. Biol. Fertil. Soils 21, 233}238.
DmH az-Ravin a, M., Bas as th, E., and Frostegas d, As . (1994). Multiple heavy metal tolerance of soil bacterial communities and its measurement by a thymidine incorporation technique. Appl. Environ. Microbiol. 60, 2238}2247. Doelman, P., and Haanstra, L. (1979). E!ects of lead on soil respiration and dehydrogenase activity. Soil Biol. Biochem. 11, 475}479. Duxbury, T., and Bicknell, B. (1983). Metal-tolerant bacterial populations from natural and metal-polluted soils. Soil Biol. Biochem. 15, 243}250. Fuller, M. E., and Manning, J. F., Jr. (1997). Aerobic gram-positive and gram-negative bacteria exhibit di!erential sensitivity to and transformation of 2,4,6-trinitrotoluene (TNT). Curr. Microbiol. 35, 77}83. Fuller, M. E., and Manning, J. F., Jr. (1998). Evidence for di!erential e!ects of 2,4,6-trinitrotoluene and other munitions compounds on speci"c subpopulations of soil microbial communities. Environ. ¹oxicol. Chem. 17, 2185}2195. Gadd, G. M. (1989). Metal tolerance. In Microbiology of Extreme Environments (C. Edwards, Ed.), pp. 178}210. Open University Press, Milton Keynes. Giller, K. E., Witter, E., and McGrath, S. P. (1998). Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: A review. Soil Biol. Biochem. 30, 1389}1414. Gong, P., Siciliano, S. D., Greer, C. W., Paquet, L., Hawari, J., and Sunahara, G. I. (1999). E!ects and bioavailability of 2,4,6-trinitrotoluene in spiked and "eld contaminated soils to indigenous microorganisms. Environ. ¹oxicol. Chem. 18, 2681}2688. Haanstra, L., and Doelman, P. (1984). Glutamic acid decomposition as a sensitive measure of heavy-metal pollution in soil. Soil Biol. Biochem. 16, 595}600. Hundal, L. S., Shea, P. J., Comfort, S. D., Powers, W. L., and Singh, J. (1997). Long-term TNT sorption and bound residue formation in soil. J. Environ. Qual. 26, 896}904. Jordan, M. J., and Lechevalier, M. P. (1975). E!ects of zinc-smelter emissions on forest soil micro#ora. Can. J. Microbiol. 21, 1855}1865. Klausmeier, R. E., Osmon, J. L., and Walls, D. R. (1974). The e!ect of trinitrotoluene on microorganisms. Dev. Ind. Microbiol. 15, 309}317. Lewis, T. A., Ederer, M. M., Crawford, R. L., and Crawford, D. L. (1997). Microbial transformation of 2,4,6-trinitrotoluene. J. Ind. Microbiol. Biotechnol. 18, 89}96. Li, A. Z., Marx, K. A., Walker, J., and Kaplan, D. L. (1997). Trinitrotoluene and metabolites binding to humic acid. Environ. Sci. ¹echnol. 31, 584}589. Nordgren, A., Bas as th, E., and SoK derstroK m, B. (1988). Evaluation of soil respiration characteristics to assess heavy metal e!ects on soil microorganisms using glutamic acid as a substrate. Soil Biol. Biochem. 20, 949}954. Pennanen, T., Frostegas rd, As ., Fritze, H., and Bas as th, E. (1996). Phospholipid fatty acid composition and heavy metal tolerance of soil microbial communities along two heavy metal-polluted gradients in coniferous forests. Appl. Environ. Microbiol. 62, 420}428. Posthuma, L. (1997). E!ects of toxicants on population and community parameters in "eld conditions, and their potential use in the validation of risk assessment methods. In Ecological Risk Assessment of Contaminants in Soil (N. M. van Straalen and H. L+kke, Eds.), pp. 85}117. Chapman & Hall, London. Rutgers, M., van't Verlaat, I. M., Wind, B., Posthuma, L., and Breure, A. M. (1998). Rapid method for assessing pollution-induced community tolerance in contaminated soil. Environ. ¹oxicol. Chem. 17, 2210}2213.
MICROBIAL COMMUNITY TOLERANCE TO TNT IN SOIL Schmidt, S. K. (1992). A substrate-induced growth-rate method for estimating the biomass of microbial functional groups in soil and aquatic systems. FEMS Microbiol. Ecol. 101, 197}206. Siciliano, S. D., Gong, P., Sunahara, G. I., and Greer, C. W. (2000). Assessment of 2,4,6-trinitrotoluene toxicity in "eld soils by pollution induced community tolerance (PICT), denaturing gradient gel electrophoresis (DGGE) and seed germination assay. Environ. ¹oxicol. Chem. 19, 2154}2160. Sunahara, G. I., Dodard, S., Sarrazin, M., Paquet, L., Ampleman, G., Thiboutot, S., Hawari, J., and Renoux, A. Y. (1998). Development of a soil extraction procedure for ecotoxicity characterization of energetic compounds. Ecotoxicol. Environ. Saf. 39, 185}194. Torsvik, V. (1995). Cell extraction method. In Molecular Microbial Ecology Manual 1.3.1 (A.D.L. Akkermans, J. D. van Elsas, and F. J. de Bruijn, Eds.), pp. 1}15. Kluwer Academic, Dordrecht.
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U.S. Environmental Protection Agency (U.S. EPA) (1997). Nitroaromatics and nitramines by high performance liquid chromatography (HPLC). In ¹est Methods for Evaluating Solid =aste; S=-846 ;pdate; Part4:1(b). O$ce of Solid Waste, Washington, DC. Welp, G., and Brummer, G. (1999). E!ects of organic pollutants on soil microbial activity: The in#uence of sorption, solubility, and speciation. Ecotoxicol. Environ. Saf. 43, 83}90. Winkel, B., and Wilke, B.-M. (1997). Wirkung von TNT (2,4,6-Trinitrotoluol) auf Bodenatmung und Nitri"kation. Mitt. Deuschen Bodenkundlichen. Ges. 85, 631}634. Witter, E., Gong P., Bas as th, E., and Marstorp, H. (2000). A study of the structure and metal tolerance of the soil microbial community 6 years after cessation of sewage sludge applications. Environ. ¹oxicol. Chem. 19, 1983}1991.
An in Situ Respirometric Technique to Measure Pollution-Induced Microbial Community Tolerance in Soils Contaminated with 2,4,6-Trinitrotoluene1 Ping Gong,* Pietro Gasparrini,- Denis Rho,* Jalal Hawari,* Sonia Thiboutot,? Guy Ampleman,? and Geo!rey I. Sunahara* *Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, MontreH al, QueH bec, H4P 2R2, Canada; -Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, MontreH al, QueH bec, H3G 1M8, Canada; and ?Defence Research Establishment Valcartier, Department of National Defence, 2459 Pie XI Boulevard, Val-BeH lair, QueH bec, G3J 1X5, Canada Received November 30, 1999
due to individual acclimation, genetic or physiological adaptation, and loss of sensitive species as a result of longterm exposure to a toxicant or a mixture of toxicants (Posthuma, 1997). An elevated PICT can be used as an evidence that a toxicant has previously had contact with the community and may have caused structural changes. A variety of approaches to examine the tolerance or resistance of soil microorganisms to heavy metals and recal-citrant organic compounds have been developed. A conven-tional approach is to isolate and culture the tolerant microorganisms from soil samples (Klausmeier et al., 1974; Jordan and Lechevalier, 1975; Duxbury and Bicknell, 1983). However, an intrinsic shortcoming of the plate count tech-nique is that more than 99% of soil bacteria cannot be isolated on agar plates (Torsvik, 1995). Therefore, only a small proportion of soil microorganisms are available for further sensitivity testing. More recently, a thymidine or leucine incorporation technique (Bas as th, 1992; DmH az-Ravin a et al., 1994; DmH azRavin a and Bas as th, 1996; Pennanen et al., 1996) and Biolog microtiter plates (Rutgers et al., 1998; Siciliano et al., 2000) have been used to assess the tolerance of bacterial communities in soil. However, these two tech-niques also result in an incomplete evaluation of the entire microbial community because of insu$cient extraction of bacteria from soil (Rutgers et al., 1998; Siciliano et al., 2000; Witter et al., 2000). In a previous study, Witter et al. (2000) developed an in situ respirometric technique based on the analysis of the substrate-induced respiration (SIR) response to test the microbial tolerance to heavy metals at the community level in soil ecosystems. These workers demonstrated that microbial metal tolerance increased as soil metal concentrations increased (Witter et al., 2000). In the present study, this newly developed technique was used on soils contaminated with 2,4,6-trinitrotoluene (TNT) for several decades, since TNT is a proven toxicant to soil microorganisms and is very recalcitrant in soil (Klausmeier et al., 1974; Fuller and
Long-term exposure to 2,4,6-trinitrotoluene (TNT) can induce changes in the structure and activities of soil microbial communities. Such changes may be associated with an elevated microbial tolerance. An in situ respirometry technique based on the analysis of the substrate-induced respiration response to freshly added TNT was used to examine soil microbial tolerance to TNT at the community level. The speci5c growth rate derived by 5tting an exponential equation to respiration data was taken as the measurement endpoint. Microbial tolerance was evaluated using a tolerance index de5ned as the ratio of the speci5c growth rate at a spiking dose of 2000 lg TNT/g soil to that of the control with no spiked TNT. Three soils with long-term exposure histories (TNT level in soil: 1.5, 32, and 620 lg TNT/g, respectively) exhibited signi5cantly higher microbial community tolerance to TNT than two uncontaminated control soils. A soil containing 29,000 lg TNT/g exhibited the highest tolerance. Findings from this study support the hypothesis that pollutioninduced community tolerance can be used as a means of identifying those compounds that have exerted selective pressure on the community. 2000 Academic Press Key Words: soil microbial community; 2,4,6-trinitrotoluene; pollution-induced community tolerance; substrate-induced respiration; speci5c growth rate. INTRODUCTION
Pollution-induced community tolerance (PICT) has recently been used as an ecotoxicological tool for assessing the toxic e!ects of a pollutant on an ecosystem at the community level (Blanck et al., 1988; Dahl and Blanck, 1996; Rutgers et al., 1998; Siciliano et al., 2000). The PICT theory is founded on the principle that communities become tolerant Assigned NRCC Publication No. 43308. To whom all correspondence should be addressed. Fax: (514) 496}6265. E-mail: [email protected]. 96 0147-6513/00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved.
MICROBIAL COMMUNITY TOLERANCE TO TNT IN SOIL
Manning, 1997, 1998; Gong et al., 1999; Siciliano et al., 2000). The purpose of this study was to investigate the natural evolution of TNT tolerance of soil microorganisms at the community level. The hypothesis is that microbial communities that have undergone prolonged exposure to TNT contamination develop a concentration-dependent tolerance to TNT, which would further corroborate the PICT theory. MATERIAL AND METHODS
Soils Soils were collected from non-TNT-contaminated and TNT-contaminated sites. A garden soil (GS-3) and a forest soil (RAC) were sampled in the fall of 1998, and they had no history of exposure to TNT. Four TNT-contaminated soils (B23, B24, B25, B26) were sampled in July 1998 from a site around an abandoned TNT-manufacturing facility (Gong et al., 1999). All six soils were sieved ((2 mm) and stored at !203C. U.S. EPA Method 8330 was used to extract TNT from soil and to determine TNT concentrations in acetonitrile extracts using HPLC}UV (U.S. Environmental Protection Agency, 1997; Gong et al., 1999). The detection limit was about 0.5 mg TNT/kg soil. Some physiochemical properties of these soils are summarized in Table 1. Soil B23 was located in a nonvegetated area, while soils B24, B25, and B26 were all located within a vegetated area but varied in TNT concentration. More details about the sampling of TNT-contaminated soils and the "eld conditions can be found elsewhere (Gong et al., 1999; Siciliano et al., 2000). Tolerance Measurements Prior to tolerance testing, soils were thawed overnight in a refrigerator (43C). Twenty-gram (dry weight) soil samples
TABLE 1 Some Physical and Chemical Properties of the Soils Used in the Present Study
Soil GS-3 RAC B26 B24 B25 B23
Origin
Texture (%) TNT C @ USDA (lg/g) pH? (%) Sand Silt Clay classi"cation
Garden 0.0A Forest 0.0A IndustrialB 1.5 IndustrialB 32 IndustrialB 620 IndustrialB 29,000
6.5 7.7 6.7 6.4 6.2 6.9
8.7 4.1 6.4 15.6 11.5 NDC
15 85 80 70 69 ND
55.5 11.5 17 21 25 ND
29.5 Silty clay loam 3.5 Loamy sand 3 Loamy sand 9 Sandy loam 6 Sandy loam ND ND
? Measured in a 1:5 (v/v) H O suspension. @ Organic matter content, estimated from weight loss after 24-h ignition at 4303C. A Less than detection limit (&0.5 mg TNT/kg soil). B TNT-contaminated site. C Not determined.
97
were weighed into 250-ml bottles, to which a volume of NPK solution (determined according to the ratio C:N:P:K"15:1:0.3:3.5, with respect to glucose-C) was added. The bottles were gently mixed for 2}3 min and then preincubated in darkness at 253C for 1}2 weeks. The "nal moisture was 45% of water-holding capacity of each soil. The TNT}glucose mixtures were prepared from a TNT stock solution of 0.25 g TNT/ml acetonitrile and D-(#)glucose (BDH, Darmstadt, Germany). Technical-grade TNT was obtained from ICI Explosives (McMasterville, PQ, Canada). Appropriate amounts of the TNT stock solution were added to glass jars, and the acetonitrile was allowed to evaporate prior to the addition of the corresponding amount of glucose. The TNT}glucose mixtures were blended thoroughly and stored at 43C in the dark until use. After preincubation, the soil samples were spiked with TNT}glucose mixtures (powder) and gently mixed for 2}3 min. Three soils (GS-3, RAC, B23) were spiked with TNT at six di!erent concentrations: 0, 125, 250, 500, 1000, and 2000 lg TNT/g. Because of limited quantities, the other three soils (B24, B25, B26) were spiked with only 0 and 2000 lg TNT/g. Glucose was added at a concentration of 10 mg/g soil (4 mg C/g soil), which induced the maximum speci"c growth rates of the GS-3 and RAC soils as determined in preliminary tests. Each treatment was prepared in quadruplicate. A specially designed respirometer similar to that described by Arulgnanendran and Nirmalakhandan (1998) was used to measure the O consumed in response to the TNT}glucose addition. A CO trap (10 ml of 1 M KOH) was placed in each sample and reference bottle, which was maintained at 253C in a thermostatically controlled water bath. Soil respiration was recorded for a minimum of 70 h. At the end of each test, the CO traps were titrated with 1 M HCl to determine the total amount of CO evolved. Data Analysis The time and cumulative amount of O fed were auto matically recorded by the respirometer. The oxygen consumption rate (y) was calculated and plotted against incubation time (t) to obtain a respiration curve (Fig. 1), from which the speci"c growth rate (l) was derived as the value of the exponent in the equation y"y e lt , where y is the O consumption rate when t"0 (Schmidt, 1992; Colores et al., 1996; Witter et al., 2000). The hourly O consumption rate was used for curve "tting (Fig. 1). The point of onset of exponential growth was estimated by a visible increase in the oxygen consumption rate after a lag
98
GONG ET AL.
RESULTS
GS-3 Garden Soil All of the O consumption rate curves produced for the control and those samples spiked with 125, 250, and 500 lg TNT/g soil were dominated by a single peak as illustrated in Fig. 1. However, at spiking doses of 1000 and 2000 lg TNT/g soil, two separate exponential growth phases were observed (Fig. 2a), from which two speci"c growth rates were derived. The speci"c growth rates for all treatments during the "rst or sole exponential growth phase were not statistically di!erent (P"0.40, ANOVA) (Fig. 3). Considering the second exponential phase at 1000 and 2000 lg TNT/g treatments, instead of the "rst, the speci"c growth rates decreased steadily as the concentration of spiked TNT increased, and the mean l values di!ered signi"cantly (P(0.01, ANOVA) (Fig. 3). FIG. 1. Substrate-induced microbial respiration curve in the GS-3 soil treated with 4 mg glucose-C/g and 125 mg TNT/g (data of one sample). Oxygen consumption rates per event and per hour are shown with curve "tting of the hourly O consumption rate (Hours 13 to 29) to an exponen tial equation.
RAC Forest Soil All samples of RAC soil experienced a single exponential growth phase. The mean speci"c growth rates decreased
period (Nordgren et al., 1988; Colores et al., 1996; Witter et al., 2000). The end of the exponential growth period was determined using the F test devised by Colores et al. (1996). The EC , de"ned as the e!ective TNT spiking concentra tion causing 20% inhibition of microbial respiration (evaluated by the speci"c growth rate), was calculated from a dose}response curve using ToxCalc (Tidepool Scienti"c Softwear, McKinleyville, CA). For each soil, the average l of the control samples was set to 100% and that of all other treatments was expressed as a percentage of the control. A linear interpolation method was used to derive the EC value. Experimental results were subjected to analysis of variance (ANOVA) using Statistica (Release 5.0, StatSoft, Tulsa, OK). Post hoc comparisons of the least signi"cant di!erence (LSD) or Sche!eH test were performed to examine the signi"cant di!erence between means. The tolerance index (TI) of a soil at a certain spiking dose was calculated as the ratio of the speci"c growth rate of the soil amended with the speci"c dose of TNT (k ) to that of the control (k ), i.e., 2 k TI" 2 . k Accordingly, TI represents the tolerance index to 2000 lg TNT/g soil. Since there were four replicates for each treatment, a maximum of 16 TI values could be obtained for each soil, from which the mean and standard deviation of the TI were estimated.
FIG. 2. Two exponential growth phases in (a) the GS-3 soil treated with 4 mg glucose-C/g and 2000 mg TNT/g and (b) the B23 soil treated with 4 mg glucose-C/g only (control). Both curves represent one sample of four replicates for the respective treatment.
99
MICROBIAL COMMUNITY TOLERANCE TO TNT IN SOIL
FIG. 3. Response of the substrate-induced microbial community respiration to freshly amended TNT in the GS-3 and RAC soils. The speci"c growth rate is used as the measurement endpoint. These two soils were not exposed to TNT before testing. Each data point is the average of four replicates. Vertical bars represent SD.
rapidly from 0.15/h to 0.07/h as the concentration of spiked TNT increased from 0 to 250 kg/g (Fig. 3), which was then followed by a plateau. That is, the k values did not decrease any further when the spiking concentration was higher than 250 lg TNT/g. Statistically, the l values for the six treatments were signi"cantly di!erent (P(0.01, ANOVA).
FIG. 4. Microbial community response to freshly spiked TNT in the B23 soil. This soil initially contained 29,000 lg TNT/g. The speci"c growth rate is derived from the respiration curve of soil microbes induced by 4 mg glucose-C/g soil. Data are given as means$SD (n"4).
However, if the Sche!eH test is applied for the post hoc comparison, no signi"cant di!erence exists between B26, B24, and B25 soils. The EC of soil B23 was much higher than those of RAC and GS-3 soils (second peak), and RAC soil was more sensitive to TNT than was GS-3 soil (Table 2). DISCUSSION
Soils from TNT-Contaminated Site
Physiological Characteristics of Soil Microbes
There were two separate phases of exponential growth in all B23 soil samples (Fig. 2b). The "rst exponential phase had approximately half the length of duration and one-third the peak respiration rate of the second one. As illustrated in Fig. 4, the mean speci"c growth rates calculated for both phases were not signi"cantly di!erent between the six treatments (P'0.05, ANOVA). The other three site soils were tested only at the highest spiking rate, i.e., 2000 lg TNT/g soil. As for the RAC soil, only one exponential phase was observed in these soils.
The six soils used in the present study di!ered in their origin, texture, organic matter content, and degree of TNT
Comparison of Tolerance Index and EC20 between Soils Comparing the six soils tested in this study (considering only the second or sole peak in the GS-3 and B23 soils), a signi"cant increase in the TNT tolerance indices was observed, which was proportional to the magnitude of previous TNT contamination in soil (Table 2). Among the six soils tested, soil B23 exhibited the highest TNT tolerance, followed by B24, B25, and B26. The two uncontaminated soils had the lowest tolerance (ANOVA, post hoc test; LSD).
TABLE 2 E4ective Concentration Inhibiting Microbial Respiration by 20% (EC20 ) and Tolerance Index at a Spiking Dose of 2000 lg TNT/g Soil (TI2000 ) Calculated for the Six Soils Testeda EC
(lg TNT/g soil)
Soil
First peak
Second peak@
GS-3 RAC B26 B24 B25 B23
'2000 70 NA NA NA 1400
530 NAA NA NA NA 1300
TI First peak 0.99$0.12 0.50$0.04 0.61$0.12 0.77$0.13 0.70$0.13 0.93$0.30
A B C BC
Second peak@ 0.42$0.06 A NA NA NA NA 0.88$0.16 D
? Values followed by di!erent letters are signi"cantly di!erent (P(0.05, LSD). The second peak in the GS-3 and B23 soils is used for comparison. @ For the GS-3 soil, the "rst and sole peaks of 0}500 lg TNT/g treatments are used. A Not available.
100
GONG ET AL.
TABLE 3 Physiological Characteristics of the Substrate-Induced Exponential Growth of Soil Microbes?
Soil
Lag time (h)
GS-3 RAC B26 B24 B25 B23@ B23A
11$1 11$1 12$3 12$3 11$2 24$5 50$6
A A A A A B C
Speci"c growth rate (h\) 0.18$0.02 0.15$0.00 0.10$0.00 0.11$0.00 0.08$0.01 0.16$0.02 0.05$0.01
E D BC C B D A
Peak time Peak respiration rate (h) (mg O /h) 26$2 A 37$3 BC 30$3 AB 30$3 AB 40$4 C 40$6 C 80$13 D
4.7$0.9 1.9$0.1 3.0$0.7 5.4$1.2 4.6$1.0 0.5$0.2 1.8$0.8
C B B C C A AB
? Each soil received 4 mg glucose-C/g soil and equivalent nutrient elements of nitrogen, phosphorus, and potassium at a ratio of 15:1:0.3:3.5 (C:N:P:K). Values are given as means$SD (n"4). Values in the same column are signi"cantly di!erent if followed by di!erent letters (P(0.05, LSD). @ First peak. A Second peak.
contamination. Consequently, microorganisms in these soils demonstrated diverse responses to the addition of glucose, a readily usable substrate. GS-3 soil was the most active among the six tested, as re#ected by its short lag time and peak time and its high peak respiration and speci"c growth rates (Table 3). The relatively high organic matter content of soils B24 and B25 (and possibly soil B23) indicates an accumulation of organic matter or a decrease in organic matter mineralization, which might have resulted from TNT contamination (Table 1). It has been generally accepted that increasing environmental stress often causes a reduction in soil microbial activities such as respiration. In this study, aside from the decreased speci"c growth rate and the increased organic matter content at elevated soil TNT levels (Tables 1 and 3), there is no obvious relationship between TNT concentrations and other physiological parameters such as lag time and peak time (Table 3). Such parameters as lag time, speci"c growth rate, SIR, metabolic quotient (qCO ), and percentage of microbial biomass-C in total soil organic-C have been commonly used as indicators of soil contamination (Doelman and Haanstra, 1979; Haanstra and Doelman, 1984; Nordgren et al., 1988; Anderson, 1994). However, more recent studies have shown that some of these parameters are not consistently lower in contaminated soils (Dahlin et al., 1997; Dahlin and Witter, 1998; Giller et al., 1998; Witter et al., 2000). For instance, the toxicity of heavy metals to soil microbes measured by SIR, lag time, or speci"c growth rate can be confounded by soil organic matter (Witter et al., 2000). When the di!erent soils are compared, soil microbial activities may be a!ected by many environmental factors other than pollution (Welp and Brummer, 1999). Therefore,
measurements of soil microbial activities are insu$cient to establish a causal relationship between pollution and microbial response. In such cases, the PICT measurement on the soil microbial communities may be a useful tool to distinguish a polluted soil from a nonpolluted one (Rutgers et al., 1998; Posthuma, 1997). Specixc Growth Rate as Measurement Endpoint As an essential step in PICT measurements, an ecologically relevant parameter should be selected as the measurement endpoint that re#ects toxic e!ects at the community level. The speci"c growth rate was chosen for this research because its response to TNT challenge was more consistent and sensitive than other parameters such as lag time, peak time, SIR, peak respiration rate, total O consumed, and total CO produced (data not provided). The speci"c growth rate (l) is also biologically meaningful since SIR is directly related to the size of microbial biomass and the activity of soil microbial communities (Anderson and Domsch, 1978; Schmidt, 1992; Colores et al., 1996). Moreover, the l value can be determined precisely by curve "tting (Fig. 1) (Colores et al., 1996), whereas some of the other parameters (e.g., lag time and peak time) are estimated more or less visually. A signi"cant reduction in the speci"c growth rate was observed after the addition of TNT in "ve of the six soils tested in this study. Statistical analysis indicated that the inhibition of the speci"c growth rate was dependent on the dose of TNT added to the soil, but independent of soil type (ANOVA). A causal relationship can thus be established between the TNT spiking dose and the speci"c growth rate of the microbial communities in soil (Figs. 3 and 4, Table 2). Such a relationship was demonstrated previously in metalcontaminated soils (Witter et al., 2000). This causal relationship thus builds up an essential foundation for further testing of the PICT. Relation of Microbial Tolerance to TNT to PICT Theory In the present study, higher tolerance to TNT was observed at the community level in all of the four site soils that had long-term exposure to TNT, but was not seen in the two soils with no TNT exposure history. Although TNT-resistant microbial species have been found to exist in both polluted and unpolluted soils (Klausmeier et al., 1974), soils with high levels of TNT usually harbor a larger proportion of TNT-resistant bacteria than do soils with low levels of TNT (Fuller and Manning, 1998). A more recent investigation using Biolog microtiter plates demonstrated that microbial communities in soils slightly contaminated with TNT were more sensitive to freshly added TNT than those in highly TNT-contaminated soils (Siciliano et al., 2000). All these results corroborate the PICT theory (Blanck et al.,
MICROBIAL COMMUNITY TOLERANCE TO TNT IN SOIL
1988; Posthuma, 1997); that is, microbial communities resulting from long-term exposure to TNT possess relatively high overall tolerance to this pollutant. The absence of a clear concentration-dependent PICT is probably due to the fact that the sampling spots of the contaminated soils were so close to each other (5}6 m apart) that soil microorganisms might have migrated between them under "eld conditions. Accordingly, soils B24, B25, and B26 revealed a similar TNT tolerance. Additionally, the spiking level of 2000 lg TNT/g soil might not have been high enough to di!erentiate the TNT tolerance in these three soils. Even though the in situ TNT stress in soil B26 was low due to the very low level of TNT at the sampling time, this soil still exhibited a signi"cantly higher tolerance than did the GS-3 and RAC soils. This e!ect might also be attributed to inheritable genetic adaptation of soil microbes (Gadd, 1989). Binary Exponential Growth in GS-3 and B23 Soils At the two highest levels of TNT spiking (1000 and 2000 lg TNT/g soil) for the GS-3 soil and in all samples of the B23 soil, a second period of exponential growth was observed (Fig. 2). This phenomenon has been previously reported by Winkel and Wilke (1997). The appearance of a second exponential phase in soil GS-3 might be due to the growth of inherently TNT-tolerant microbial species. Since the "rst exponential growth phase in these samples treated with 1000 and 2000 lg TNT/g soil was so similar to the sole exponential growth phase in the control (the l values are not signi"cantly di!erent at P(0.001), the exponentially growing microorganisms might be of the same sensitive communities. Therefore, only the second growth period was used for tolerance comparison. The second exponential phase in soil B23 was probably also due to the growth of dominant TNT-resistant microbes. Compared with the second peak, the "rst peak in soil B23 was much shorter and weaker, which might represent the growth of relatively sensitive microbes. In the presence of 29,000 lg TNT/g, these sensitive species, however, would soon die o! and be replaced by tolerant species. The "rst exponential phase in soil B23 was thus very transient and not used for tolerance comparison. The addition of up to 2000 lg TNT/g may not have changed the soil TNT level very much, given the extremely high background TNT level, so the substrate-induced microbial growth in this soil was not a!ected by the addition of a relatively small amount of TNT. In contrast to the work of Winkel and Wilke (1997), TNT was added together with glucose instead of 3}17 days prior to substrate addition. This di!erence allowed no or little time for TNT to exert any selective pressure on soil microbial communities. In addition, it was reported that the second phase of exponential growth became more signi"cant as the time between TNT spiking and glucose addition was in-
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creased (Winkel and Wilke, 1997). All the evidence suggests that the adaptation of TNT-tolerant communities is time dependent. TNT Bioavailability and Biotransformation Both solubility and soil adsorption limit the bioavailability of TNT, which necessitates the addition of large amounts of TNT to induce an observable inhibition. Like heavy metals, the amended TNT can be adsorbed and bound to both clay and organic matter and, thus, be rendered unavailable to soil microorganisms (Hundal et al., 1997; Li et al., 1997; Gong et al., 1999). Clay soils may thus be less sensitive than sandy soils, as is indicated by the higher EC for the GS-3 soil compared with the RAC soil. To avoid the in#uence of such factors, it seems appropriate to test soils at pollutant concentrations that can induce the maximum inhibition. By comparing the TI , GS-3 and RAC soils revealed similar TNT tolerance levels. Biodegradation of TNT may complicate the interpretation of results. Since microbial transformation takes place rapidly and extensively in soil under both aerobic and anaerobic conditions (Klausmeier et al., 1974; Lewis et al., 1997), the actual in situ concentrations of spiked and original TNT in soil are not known during and after the "rst or sole exponential growth period. Microbial transformation of TNT to less toxic metabolites could detoxify TNT (Sunahara et al., 1998) and therefore may confound the tolerance results. An observation of enhanced tolerance in one soil might be attributed to a higher biotransformation capacity of microbial communities in this soil. Therefore, it appears to be necessary to add TNT at the very onset of exponential growth so that no time was given for soil microbes to transform or detoxify TNT (Witter et al., 2000), especially when two exponential phases might appear. CONCLUSION
Long-term exposure to recalcitrant contaminants is thought to a!ect soil microbial communities by reducing their abundance and species diversity and selecting for resistant populations (Gadd, 1989). It is preferable to separate the target organisms from their inhabiting soil to study their tolerance, but there is no e!ective technique so far that can e!ectively extract entire microbial communities from soil. An alternative approach using the in situ respirometric technique has been developed and tested. Although the respirometric technique is more time consuming than some other techniques, such as the thymidine incorporation technique and the Biolog technique (Witter et al., 2000), its greatest advantage is that soil samples can be tested without extraction of microbial species. An evaluation of the overall tolerance of whole microbial communities can thus be made.
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By use of the in situ respirometric technique, it was revealed that the microbes in soil samples collected along a TNT gradient at a contaminated site possessed elevated tolerance to TNT in comparison with two uncontaminated soils. The most contaminated soil demonstrated the highest tolerance. Based on the results obtained from this and previous studies (Witter et al., 2000), it may be concluded that this respirometric technique is a promising tool for measuring PICT in both metal- and recalcitrant organic chemical-contaminated soils. However, caution should be taken in the interpretation of data, in view of the possible biotransformation of organic chemicals and the appearance of binary phases of microbial exponential growth. These problems can be avoided by technical adaptations. ACKNOWLEDGMENTS The authors thank Professor B.-M. Wilke, Technische UniversitaK t Berlin, Berlin, Germany, for his critical review of the manuscript and AndreH Chouinard, Marie-JoseH e Lorrain, Nathalie Matte, Jonathan Hodgson, Louise Paquet, and Serge Delisle for their technical assistance.
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