Effects of atrazine on microbial activity in semiarid soil

Applied Soil Ecology 35 (2007) 120–127 www.elsevier.com/locate/apsoil

Effects of atrazine on microbial activity in semiarid soil Jose´ L. Moreno a,*, Asuncio´n Aliaga a, Simo´n Navarro b, Teresa Herna´ndez a, Carlos Garcı´a a a

Department of Soil and Water Conservation and Organic Resources Management, CEBAS-CSIC, P.O. Box 164, 30100-Espinardo, Murcia, Spain b Department of Agricultural Chemistry, Geology and Pedology, University of Murcia, Campus Universitario de Espinardo, 30100-Espinardo, Murcia, Spain Received 27 February 2006; received in revised form 2 May 2006; accepted 4 May 2006

Abstract The effect of an atrazine formulation on microbial biomass, microbial respiration, ATP content and dehydrogenase and urease activity in a semiarid soil and the influence of time on the response of soil microbial activity to the herbicide treatment were assessed. The atrazine formulation was added to soil as aqueous solutions of different concentrations of active ingredient to obtain a range of concentrations in the soil from 0.2 to 1000 mg kg 1. Microcosms of soil with the different herbicide concentrations and untreated control soil were incubated for 6 h, 16 and 45 days. In general, an increase in the measured microbiological and biochemical parameters with atrazine concentration in soil was observed. The increase in microbial activity with atrazine pollution was noticeable after lengthy incubation. # 2006 Elsevier B.V. All rights reserved. Keywords: Atrazine; Soil microbial activity; Atrazine biodegradation; Metabolic quotient

1. Introduction Atrazine [2-chloro-4-ethylamino-6-isopropylamino1,3,5-triazine] is a herbicide widely used to control broad-leaved weeds especially in corn (Zea mays L.) and sorghum (Sorghum bicolor L. Moensch) production. Due to its widespread use, its relatively long halflife in agricultural soil and moderate soil mobility it has been detected in many environmental compartments, especially in surface water (Wenk et al., 1997). In recent years, atrazine use has decreased in favor of other striazine herbicides, and in many European countries it has been recommended to use atrazine only at rates

* Corresponding author. Fax: +34 968 396213. E-mail address: [email protected] (J.L. Moreno). 0929-1393/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2006.05.002

below 1.5 kg of active ingredient/ha per annum in agricultural crops (Seiler et al., 1992). Soil microorganisms and the processes that they govern are essential for long-term fertility of soil, and there is therefore, concern about the effects of herbicides on soil microbial biomass and activity (Perucci et al., 2000). Exposure to some xenobiotic organic compounds may change the resources which soil microorganisms use to obtain energy and nutrients, especially when the soil organic matter is scarce and these compounds are bioavailable in the media (Alexander, 1994) Specific microorganisms are able to detoxicate atrazine by N-dealkylation or dehalogenation reactions and this may imply the development of microbial communities that can utilize the N in the triazine ring (Haney et al., 2002). The concentration of atrazine in soil is an important factor which affects its biodegradation and the microbial response (Dzantor and

J.L. Moreno et al. / Applied Soil Ecology 35 (2007) 120–127

Felsot, 1991; Gan et al., 1996). Moreover, the conditions of a semiarid soil (low water content, and scarce nutrient content) may modify the atrazine degradation rate by microorganisms in comparison with other soil conditions. Several authors (e.g. Assaf and Turco, 1994; Gebendinger and Radosevich, 1999) have reported that the concentration and forms of C and N are major determinants of atrazine mineralization. Soil moisture and temperature directly affect many biological processes including microbial degradation of chemicals and could also influence atrazine persistence (Weber et al., 1993). However, literature on the effect of atrazine on microbial activity in semiarid soils is scarce. In this study, the effects of a range (from 0.2 to 1000 mg kg 1 soil) of atrazine concentrations on different microbial activity parameters of a semiarid soil, and the influence of time on the response of soil microbial activity to the herbicide were assessed.

121

Table 1 Soil characteristics pH (1:10 soil:water) ECa (1:10 soil:water) (dS/m) Total calcium carbonate (g kg 1) b Active calcium carbonate (g kg 1) Total organic carbon (g kg 1) Organic matter (g kg 1) Nitrogen (mg kg 1) Phosphorous (mg kg 1) Potassium (mg kg 1) Soluble sodiumc (mmol kg 1) Chloride (mmol kg 1) Sulphate (mmol gypsum kg 1) Coarse sand (2–0.25 mm) (%) Fine sand (0.25–0.05 mm) (%) Silt (0.05–0.002 mm) (%) Clay (<0.002 mm) (%) Texture a b c

7.5 0.29 554.6 142.5 5.2 8.0 300 313 910 4.82 2.5 3.1 8.65 16.82 44.69 29.62 Clay loam

Electrical conductivity. Values are expressed on a soil dry weight basis (105 8C). Sodium extractable with water (1:10 soil:water).

2. Materials and methods 2.1. Soil treatment In June 2002, the 0–10 cm depth layer of an agricultural soil from SE Spain, which had been abandoned several years ago after intensive utilization, was sampled. The climate regime of this region is semiarid, and it is characterized by sporadic rainfalls (annual precipitation: 200–300 mm) that in most cases are torrential, and moderate temperatures (19 8C of annual average). The plant cover of this soil was scarce and thus, the organic matter content was very low. The main characteristics of this soil are shown in Table 1. Two-hundred gram of sieved soil (<2 mm) were placed in plastic containers and distilled water was added in order to reach a soil moisture of 35–40% of the water holding capacity. This moist soil was pre-incubated for 7 days in order to activate the native microbial population. Then the soil was spiked using an atrazine commercial formulation (Beltrazina, 47% a.i., Probelte, S.A., Murcia, Spain). Several dilutions in water of this formulation were prepared to add to soil and thus a range of concentrations in soil from 0.2 to 1000 mg a.i./kg soil was obtained. Ten treatments of soil polluted with different atrazine concentrations (0.2, 0.4, 0.6, 1, 10, 50 100, 250, 500 and 1000 mg/kg soil) and one untreated control were established with three replicates per treatment, and were incubated at 28 8C. In order to obtain information on the effect of time of exposure to atrazine, the treatments were replicated three times, and the three series of containers were incubated for 6 h, 16 days and 45 days, respectively. The water content of soil was kept constant by adding

distilled water throughout the incubation. After incubation, each soil sample was homogenized and stored in plastic bags at 5 8C until analysis. 2.2. Soil microbial parameters Microbial biomass C was determined by the fumigation–extraction method (Vance et al., 1987). Ten gram samples were fumigated with chloroform and similar reference samples were not fumigated. C was extracted with 40 ml of K2SO4 (0.5 M) solution from fumigated and non-fumigated samples. The C content was measured in the centrifuged and filtered samples using a soluble-organic C analyzer (Shimadzu TOC5050A). Microbial biomass C (MBC) was calculated by the expression: MBC = C extracted  2.66 (Vance et al., 1987), where C extracted is the difference between C extracted from fumigated and non-fumigated samples. Soil respiration was analyzed by placing 50 g of soil with moisture content corrected to 30–40% of its waterholding capacity (water potential: 0.055 Mpa) in hermetically sealed flasks and incubating for 31 days at 28 8C. The CO2 produced was periodically measured (every day for the first 4 days and then weekly) using an infrared gas analyzer (Toray PG-100, Toray Engineering Co. Ltd., Japan). The data were summed to give a cumulative amount of CO2 evolved after 31 days of incubation, and soil respiration was expressed as mg CO2–C kg 1 soil per day. ATP was extracted from soil using the Webster et al. (1984) procedure and measured as recommended by

122

J.L. Moreno et al. / Applied Soil Ecology 35 (2007) 120–127

Ciardi and Nannipieri (1990). Twenty milliliters of a phosphoric acid extractant were added to 1 g of soil, and the closed flasks were shaken in a cool bath. Then the mixture was filtered through Whatman paper and an aliquot was used to measure the ATP content by the luciferin-luciferase assay in a luminometer (Optocomp 1, MGM Instruments, Inc.) Soil dehydrogenase activity (DH activity) was determined using 1 g of soil and the reduction of piodonitrotetrazolium chloride (INT) to p-iodonitrotetrazolium formazan was measured by a modification of the method reported by Von Mersi and Schinner (1991). Soil DH activity was expressed as mg INTF g 1 soil h 1. The urease activity in the soil was determined by the buffered method of Kandeler and Gerber (1988). In this procedure, 0.5 ml of a solution of urea (0.48%) and 4 ml of borate buffer (pH 10) were added to 1 g of soil in hermetically sealed flasks, and then incubated for 2 hours at 37 8C. The ammonium content of the centrifuged extracts was determined by a modified indophenol-blue reaction. Controls were prepared without substrate to determine the ammonium produced in the absence of added urea. 2.3. Atrazine extraction and analysis Atrazine extraction from soil samples was carried out using the Navarro et al. (2000) procedure. Briefly, atrazine was extracted by adding acetonitrile and dichloromethane to soil, and sonicating the mixture. Then the organic phase was filtered and evaporated to dryness. The residue was redissolved in acetone, and the solution was injected in to a GC- NPD system (HewlettPackard Model 6890 gas chromatograph equipped with a nitrogen–phosphorus detector) to determine the atrazine concentration.

Table 2 Atrazine concentration (mg kg 1 soil) in the assayed treatments after 6 h, 16 and 45 days of incubation Treatments (initial concentration)

Incubation time 6h

16 days

45 days

Control 0.2 0.4 0.6 1 10 50 100 250 500 1000

0.03 0.2 0.31 0.37 0.6 6.19 50 59.5 235.22 484.35 976

0.01 0.09 0.03 0.02

a

D.L. (Detection limit): 0.005 mg L 1.

50–90% of the atrazine added being recovered from the treated soils. After 16 days incubation only 2–50% of atrazine was recovered, and after 45 days, residues of this herbicide were undetectable in most cases. The effect of atrazine concentration (treatment) on microbial biomass C (Cmic), soil microbial respiration (SMR), ATP content and dehydrogenase (DH) and urease activity was different depending of the levels of the other factor (incubation time), as it can be deduced from the significant treatment  time interaction shown by ANOVA (Figs. 1–5). Fig. 1 shows the values of Cmic in the soils treated with atrazine at different rates. Cmic increased with the incubation time, but was often greater after 16 than 45 days, particularly in the soils

2.4. Data analysis Significant statistical differences between the means of the data were detected using a two-way ANOVA with treatments and incubation time as factors. Honestly significant differences (HSD) for a multiple mean comparisons were obtained using Tukey test at P < 0.5 level. This statistical analysis was performed with the program Statgraphics Plus 5.0 for Windows. 3. Results The atrazine concentration in soil decreased with the incubation time (Table 2). After 6 h, the degradation of this herbicide was minimal for most treatments, with

Fig. 1. Effect of different atrazine concentrations in soil and the incubation time on microbial biomass C, and result of two way ANOVA. Bars denote the S.D. of the mean (n = 3).

J.L. Moreno et al. / Applied Soil Ecology 35 (2007) 120–127

123

Fig. 2. Effect of different atrazine concentrations in soil and the incubation time on soil microbial respiration, and result of two way ANOVA. Bars denote the S.D. of the mean (n = 3).

Fig. 4. Effect of different atrazine concentrations in soil and the incubation time on soil dehydrogenase activity, and result of two way ANOVA. Bars denote the S.D. of the mean (n = 3).

treated with 0.2, 0.6, and 1 mg atrazine kg 1 soil, and thus the values of Cmic after 16 and 45 days of incubation were higher than the untreated control. Soil microbial respiration was more or less constant at lower atrazine concentrations (Fig. 2), but in soil samples which were spiked with 500 and 1000 mg atrazine kg 1, SMR values were higher after 45 days of incubation than those after 6 h of incubation. The metabolic quotient (qCO2), which represents the amount of CO2–C evolved per unit of Cmic per hour, was significantly higher in the soil samples spiked with the highest amounts of herbicide than in the control soil for each incubation time (Table 3). The highest values

of qCO2 were obtained in the soil treated with 500 and 1000 ppm of atrazine after 16 days. Fig. 3 shows an increase in soil ATP content with incubation time for each treatment, with the highest values being obtained in soil treated with 1000 ppm of atrazine after 45 days of incubation. The ATP content per microbial biomass C unit (ATP:Cmic) was significantly higher in soil spiked with 1000 ppm of atrazine than in the control for each incubation time (Table 4), but this ratio was significantly lower than the control in some of the treatments with lower atrazine concentrations (0.4– 50 ppm atrazine) after 16 and 45 days. Soil dehydrogenase activity increased in all treatments with the incubation time (Fig. 4), the highest

Fig. 3. Effect of different atrazine concentrations in soil and the incubation time on soil ATP content, and result of two way ANOVA. Bars denote the S.D. of the mean (n = 3).

Fig. 5. Effect of different atrazine concentrations in soil and the incubation time on soil urease activity, and result of two way ANOVA. Bars denote the S.D. of the mean (n = 3).

124

J.L. Moreno et al. / Applied Soil Ecology 35 (2007) 120–127

Table 3 Effect of atrazine concentration and incubation time on the metabolic quotient (mg C–CO2 g 1 Cmic h 1) Treatments

Control 0.2 0.4 0.6 1 10 50 100 250 500 1000

Incubation time 6h

16 days

45 days

0.20 0.22 0.18 0.22 0.22 0.14 0.20 0.12 0.20 0.22 0.28

0.14 0.18 0.07 0.09 0.15 0.11 0.17 0.18 0.26 0.61 1.37

0.11 0.04 0.08 0.06 0.05 0.05 0.05 0.08 0.14 0.38 0.50

Factors

F-ratio

P-value

HSD

Treatment (A) Incubation time (B) Interaction (AB)

180.2 130.3 58.89

<0.001 <0.001 <0.001

0.065 0.024 –

Two way ANOVA results: F- and P-values for main factors and interactions, and HSD values.

Table 5 Effect of atrazine concentration and incubation time on soil dehydrogenase activity per unit of microbial biomass C ratio (mg INTF g 1 Cmic h 1) Treatments

Control 0.2 0.4 0.6 1 10 50 100 250 500 1000

Incubation time 6h

16 days

45 days

1.24 2.47 1.88 2.93 2.40 1.71 1.37 1.99 1.70 2.16 5.66

2.07 1.89 1.90 0.78 0.96 1.42 1.46 1.13 1.81 1.91 6.46

3.90 4.16 3.24 2.43 2.22 1.77 2.57 3.76 3.15 4.06 5.22

Factors

F-ratio

P-value

HSD

Treatment (A) Incubation time (B) Interaction (AB)

31.21 42.62 4.12

<0.001 <0.001 <0.001

0.959 0.360 –

Two way ANOVA results: F and P-values for main factors and interactions, and HSD values.

values being obtained in soil which was treated with 1000 ppm of atrazine for each incubation time. As can be observed in Table 5, the DH:Cmic ratio, which represents specific dehydrogenase activity, sharply increased in the samples containing the highest atrazine concentration (1000 ppm) in relation to control values,

but this increase was lower after the longest incubation time (45 days). No changes were observed in soil urease activity with increasing atrazine concentrations after 6 hours and 16 days of incubation (Fig. 5). Only the treatments

Table 4 Effect of atrazine concentration and incubation time on the ATP content per unit of microbial biomass C ratio (mg ATP g 1 Cmic)

Table 6 Effect of atrazine concentration and incubation time on urease activity per unit of microbial biomass C ratio (mg N–NH4+ g 1 Cmic h 1)

Treatments

Treatments

Control 0.2 0.4 0.6 1 10 50 100 250 500 1000

Incubation time 6h

16 days

45 days

0.51 0.43 0.39 0.42 0.51 0.43 0.50 0.28 0.48 0.52 0.90

0.59 0.44 0.52 0.31 0.31 0.43 0.37 0.51 0.46 0.51 1.11

0.87 0.72 0.53 0.52 0.45 0.45 0.67 0.70 0.64 0.78 1.17

Control 0.2 0.4 0.6 1 10 50 100 250 500 1000

Incubation time 6h

16 days

45 days

33.26 34.37 32.25 30.02 36.40 25.72 23.94 22.85 27.62 33.51 36.16

17.56 10.64 14.11 7.86 6.34 8.94 7.87 9.00 12.89 10.31 19.74

11.85 14.44 9.67 8.17 8.06 7.68 9.20 11.48 17.38 9.80 21.07

Factors

F-ratio

P-value

HSD

Factors

F-ratio

P-value

HSD

Treatment (A) Incubation time (B) Interaction (AB)

24.67 32.04 2.08

<0.001 <0.001 0.0140

0.173 0.065

Treatment (A) Incubation time (B) Interaction (AB)

20.85 725.64 5.98

<0.001 <0.001 <0.001

2.203 1.150

Two way ANOVA results: F- and P-values for main factors and interactions, and HSD values.

Two way ANOVA results: F- and P-values for main factors and interactions, and HSD values.

J.L. Moreno et al. / Applied Soil Ecology 35 (2007) 120–127

containing 250 and 1000 ppm of atrazine showed higher urease activity than the rest of the treatments after 45 days of incubation. In all treatments, the trend for urease activity was a sharp decrease with incubation time. After 16 days, the values for the urease:Cmic ratio (Table 6) were significantly lower than control values, in all except the 1000 ppm treatment. Only after 45 days of incubation was this ratio higher than the control in soil with 250 and 1000 ppm of atrazine. A decrease in the urease:Cmic ratio (specific urease activity) was also observed in all soil treatments after 16 and 45 days of incubation in relation to soil samples after 6 h of incubation. 4. Discussion Atrazine persistence in soil is characterized by a moderate, short half-life of 35 to 50 days (Topp, 2001), although faster atrazine mineralization has been observed in soils which had been exposed to this herbicide as a normal agricultural practice (Pussemier et al., 1997; Houot et al., 2000). In our study, after 16 days of incubation there was, at a maximum, 50% of the added atrazine remaining. The biodegradation of atrazine was carried out by the native semiarid soil population under optimum conditions of soil water content and temperature, and thus faster atrazine mineralization took place in our study, but these optimum conditions differ from those which might occur in the field. Atrazine degradation in soil results from the activity of bacteria which are able to use the atrazine molecule as a C or N source (Mandelbaum et al., 1993). Differences detected in atrazine mineralization between the treatments were probably caused by the initial concentration of atrazine influencing rate of the atrazine biodegradation. Other authors have reported the influence of soil atrazine concentration on its biodegradation rate (Dzantor and Felsot, 1991; Gan et al., 1996). Cmic represents a sensitive indicator of changes in soil conditions, but biomass measurements have their limitations in soil pollution studies, because they do not permit evaluation of variation in the community structure of the microbial population (Brookes, 1995). In several cases a decrease in this parameter was observed with heavy metal pollution (Landi et al., 2000) and as a result of other xenobiotics such as herbicides (Perucci et al., 2000). In our case, an increase in Cmic was observed in some soil treatments with atrazine. In these treatments, the atrazine concentration must represent an amount of available C and N to permit the growth of a fraction of the microbial population. In the treatments with higher atrazine concentrations

125

added to soil, Cmic did not increase because the microbial population was not able to incorporate C from these high amounts of the atrazine for their proliferation and thus, a fraction of added atrazine was mineralized to obtain enough energy for their maintenance. The evidence for this fact was that higher CO2 evolution was observed in the treatments with high atrazine concentrations. Other authors reported that the microbial biomass was unaffected by atrazine although this herbicide caused significant enhancement of CO2 release (Ghani et al., 1996). The increase in soil microbial respiration (SMR) with the added concentration of atrazine may be due to the fact that this herbicide is a substrate for specific microorganisms such as Pseudomonas sp., which are able to mineralize the atrazine molecule completely (Mandelbaum et al., 1993; Ralebitso et al., 2002). In this study, the trend for SMR with incubation time in the treatments with high atrazine concentrations could be explained by only a small fraction of the soil microbial population being able to degrade atrazine completely to produce CO2 and H2O, after a short time of incubation (6 hours). In the course of incubation, the partial degradation of atrazine produced an increase in organic substrates which could be used by a greater range of soil microorganisms. However, after 45 days of incubation, exhaustion of these organic substrates was evident from SMR data obtained. The metabolic quotient (qCO2) is a useful tool to evaluate the microbial stress in a soil (Anderson and Domsch, 1993). An increase in qCO2 can represent a harmful effect of a herbicide on microorganisms, which are forced to use a great part of their energetic resources to survive, resulting in less organic C incorporation into the microbial biomass (Chander and Brookes, 1991). In our case, the microbial stress caused by atrazine increased after 16 days of incubation in the most polluted soils in comparison with the treatments after 6 h, but this microbial stress decreased after 45 days. The soil ATP content can represent an independent measure of microbial biomass when soil samples are pre-incubated under controlled conditions before analysis (Nannipieri et al., 1990). On the other hand, ATP measurements appear to reflect changes in the soil microbial activity in response to the effects of heavy metals and organic amendments (Tam, 1998). In this study, the soil samples were not pre-incubated for several days at 25 8C before ATP measurements, as done Jenkinson et al. (1979) to determine the correlation between ATP and biomass C. Soil ATP and Cmic values exhibited different trends in response to atrazine suggesting that the variation in soil ATP

126

J.L. Moreno et al. / Applied Soil Ecology 35 (2007) 120–127

content represents changes in the physiological state of the microbial community in response to the herbicide. Therefore, the ATP:Cmic ratio for the different soil treatments represents the ratio between microbial activity and biomass C. For all treatments, the ATP:Cmic ratio was very low in comparison with the values obtained by other authors, e.g. 5.5 mg ATP g 1 Cmic reported by Contin et al. (1991). The difference could be explained by the fact that the semiarid soil used in our study has a low organic C content and has suffered long periods of drought which favour the establishment of a native microbial community with a low activity compared with the soil used by the aforementioned authors. Nevertheless, the values of this ratio obtained in our study increased with the incubation time and in the most polluted treatment because there was an increase in metabolic activity of microorganisms, but these values were still lower than those reported by Contin et al. (1991), after 45 days of incubation. DH represents a group of intracellular enzymes present in active microorganisms in the soil (Nannipieri et al., 1990), which oxidize organic compounds and reflect the total oxidative activity of the soil microflora, and thus this enzymatic activity is considered an index of the metabolic activity of soil. From the data obtained, an increase in metabolic activity with atrazine concentration and with incubation time can be deduced. Some authors reported a positive correlation between DH and Cmic (Rossel et al., 1997); but other authors (e.g. Landi et al., 2000) have observed a decrease in the DH:Cmic ratio in response to soil Cd pollution. In our study, the correlation between those two parameters was not observed when the DH:Cmic ratio of different treatments with atrazine was compared, but an increase in this ratio was observed in the most contaminated soil. As was explained in the case of qCO2, the increase in DH:Cmic could indicate stress on the soil microbial population caused by the atrazine concentration. Soil urease activity includes, in great part, the extracelular activity which is fixed and protected on solid components of soil (clay and organic matter colloids), but this enzymatic activity is generated by the soil microorganisms. Thus, it has been reported that 79– 89% of the urolitic activity in a silty clay loam soil was due to extracellular urease (Paulson and Kuntz, 1969). High atrazine concentration added to soil did not interfere with the hydrolytic activity of urease after 6 hours and 16 days, but after 45 days of incubation an increase was observed in soil treated with 250 and 1000 ppm of atrazine This could be explained by atrazine degradation producing metabolites which were specific substrates for urease (Houot et al., 1998), and in

those treatments after 45 days, the atrazine metabolites must have been present at a concentration high enough to stimulate urease synthesis. The urease:Cmic ratio fluctuated with both atrazine concentration and incubation time. The reason for this may be that urease synthesis was activated by a higher content of its specific substrates in condition of low concentration of bioavailable ammonium but not by the size of the soil microbial population. From our results, urease activity cannot be considered as an index of soil microbial activity, because it only reflects variations in the specific substrates for this enzyme. Measurements of dehydrogenase activity, microbial respiration and ATP content are more sensitive to the microbial activity variation in response to atrazine addition. In conclusion, this study has shown that the variation in soil microbial activity reflects the capacity of microorganisms to respond to inputs of atrazine in a semiarid soil with a low organic matter content. The microbial activity increased as an adaptation to the stress caused by the high concentration of the xenobiotic added. This resilience capacity of microorganisms was developed under optimum conditions of temperature and soil moisture for its biodegradative activity. It is difficult to extrapolate this conclusion to the same soil under field conditions, because in a semiarid climate, these conditions are characterized by long dry periods. The results obtained in this study demonstrate a potential capacity for adaptation of the microorganisms in a common soil of a semiarid region when large amounts of atrazine were added. The capacity of the microbial population to respond varied in relation to the time after atrazine application. Microbiological parameters such as soil respiration, ATP content and dehydrogenase activity were sensitive bioindicators of the microbial activity response to atrazine inputs. References Alexander, M., 1994. Biodegradation and Bioremediation. Academic Press, San Diego, USA, 302 pp. Anderson, T.H., Domsch, K.H., 1993. The metabolic quotient for CO2 (qCO2) a specific activity parameter to access the effects of environmental conditions, such as pH, on the microbial biomass of forest soils. Soil Biol. Biochem. 25, 393–395. Assaf, N.A., Turco, R.F., 1994. Influence of carbon and nitrogen application on the mineralization of atrazine and its metabolites in soil. Pestic. Sci. 41, 41–47. Brookes, P.C., 1995. The use of microbial parameters in monitoring soil pollution by heavy metals. Biol. Fert. Soil 19, 269–275. Chander, K., Brookes, P.C., 1991. Microbial biomass dynamics during the decomposition of glucose and maize in metal-contaminated and non-contaminated soils. Soil Biol. Biochem. 23, 917–925.

J.L. Moreno et al. / Applied Soil Ecology 35 (2007) 120–127 Ciardi, C., Nannipieri, P., 1990. A comparison of methods for measuring ATP in soil. Soil Biol. Biochem. 22, 725–727. Contin, M., Todd, A., Brookes, P.C., 1991. The ATP concentration in the soil microbial biomass. Soil Biol. Biochem. 33, 701–704. Dzantor, E.K., Felsot, A.S., 1991. Microbial responses to large concentrations of herbicides in soil. Environ. Toxicol. Chem. 10, 649– 655. Gan, J., Becker, R.L., Koskinen, W.C., Bunte, D., 1996. Degradation of atrazine in two soils as a function of concentration. J. Environ. Qual. 25, 1064–1072. Gebendinger, N., Radosevich, M., 1999. Inhibition of atrazine degradation by cyanazine and exogenous nitrogen in bacterial isolate M91-3. Appl. Microbiol. Biotechnol. 51, 375–381. Ghani, A., Wardle, D.A., Rahman, A., Lauren, D.R., 1996. Interactions between C-14-labelled atrazine and the soil microbial biomass in relation to herbicide degradation. Biol. Fert. Soil 21, 17–22. Haney, R.L., Senseman, S.A., Krutz, L.J., Hons, F.M., 2002. Soil carbon and nitrogen mineralization as affected by atrazine and glyphosate. Biol. Fert. Soil 35, 35–40. Houot, S., Barriuso, E., Bergheaud, V., 1998. Modifications to atrazine degradation pathways in a loamy soil after addition of organic amendments. Soil Biol. Biochem. 30, 2147–2157. Houot, S., Topp, E., Yassir, A., Soulas, G., 2000. Dependence of accelerated degradation of atrazine on soil pH in French and Canadian soils. Soil Biol. Biochem. 32, 615–625. Jenkinson, D.S., Davidson, S.A., Powlson, D.S., 1979. Adenosine triphosphate and microbial biomass in soil. Soil Biol. Biochem. 11, 521–527. Kandeler, E., Gerber, H., 1988. Short-term assay of soil urease activity using colorimetric determination of ammonium. Biol. Fert. Soil 6, 68–72. Landi, L., Renella, G., Moreno, J.L., Falchini, L., Nannipieri, X., 2000. Influence of cadmium on the metabolic quotient, L-D-glumatic acid respiration ratio and enzyme activity:microbial biomass ratio under laboratory conditions. Biol. Fert. Soil 32, 8–16. Mandelbaum, R.T., Wackett, L.P., Allan, D.L., 1993. Mineralization of the s-triazine ring of atrazine by stable bacterial mixed cultures. Appl. Environ. Microbiol. 59, 1695–1701. Nannipieri, P., Ceccanti, B., Grego, S., 1990. Ecological significance of the biological activity in soil. In: Bollag, J.-M., Stotzky, G. (Eds.), Soil Biochemistry, 6. Marcel Dekker, New York, pp. 293–355. Navarro, S., Oliva, J., Barba, A., Garcı´a, C., 2000. Determination of simazine, terbuthylazine, and their dealkylated chlorotriazine

127

metabolites in soil using sonication microextraction and gas chromatography. J. AOAC Int. 83, 1239–1243. Paulson, K.N., Kuntz, L.T., 1969. Locus of urease activity. Proc. Am. Soil Sci. Soc. 33, 897–901. Perucci, P., Dumontet, S., Bufo, S.A., Mazzatur, A., Casucci, C., 2000. Effects of organic amendment and herbicide treatment on soil microbial biomass. Biol. Fertil. Soil 32, 17–23. Pussemier, L., Goux, S., Vanderheyden, V., Debongnie, P., Tresinie, I., Foucart, G., 1997. Rapid dissipation of atrazine in soils taken from various maize fields. Weed Res. 37, 171–179. Ralebitso, T.K., Senior, E., Verseveld, H.W., 2002. Microbial aspects of atrazine degradation in natural environments. Biodegradation 13, 11–19. Rossel, D., Tarradellas, J., Bitton, G., Morel, J.-L., 1997. Use of enzymes in soil ecotoxicology: a case for dehydrogenase and hydrolytic enzymes. In: Tarradellas, J., Bitton, G., Rossel, D. (Eds.), Soil Ecotoxicology. CRC-Lewis Publishers, Boca Raton, FL, pp. 179–206. Seiler, A., Brenneisen, P., Green, D.H., 1992. Benefits and risk of plant protection products-possibilities of protection drinking water: case atrazine. Water Supply 10, 31–42. Tam, N.F.Y., 1998. Effects of wastewater discharge on microbial populations and enzyme activities in mangrove soils. Environ. Pollut. 102, 233–242. Topp, E., 2001. A comparison of three atrazine-degrading bacteria for soil bioremediation. Biol. Fert. Soil 33, 529–534. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring microbial biomass C. Soil Biol. Biochem. 19, 703–707. Von Mersi, W., Schinner, F., 1991. An improved and accurate method for determining the dehydrogenase activity of soils with iodonitrotetrazolium chloride. Biol. Fert. Soil 11, 216–220. Weber, J.B., Best, J.A., Gonese, J.U., 1993. Bioavailability and bioactivity of sorbed organic chemicals. In: Linn, D.M. (Ed.), Sorption and degradation of pesticides and organic chemicals in soil. American Society of Agronomy, Soil Science Society of America, Madison, Wisconsin, USA, pp. 153–196. Webster, J., Hampton, G., Leach, F., 1984. ATP in soil: a new extractant and extraction procedure. Soil Biol. Biochem. 16, 335–342. Wenk, M., Bourgeois, M., Allen, J., Stucki, G., 1997. Effects of atrazine-mineralizing microorganisms on weed growth in atrazine-treated soils. J. Agric. Food Chem. 45, 4474–4480.