soil microbial biomass estimated by fumigation ...

0038-0717/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

Soi/ Bio/. Biochem. Vol. 25, No. 6, pp. 679--683, 1993 Printed in Great Britain. All rights reserved

SOIL MICROBIAL BIOMASS ESTIMATED BY FUMIGATION-EXTRACTION AND SUBSTRATE-INDUCED RESPIRATION IN TWO PESTICIDE-TREATED SOILS T. HARDEN,1 R. G. JoERGENSEN,1* B. MEYER 1 and V. WOLTERS2 1

Institut für Bodenwissenschaften, von-Siebold-Strasse 4, 3400 Göttingen and 2Zoologisches Institut, Saarstrasse 21, 6500 Mainz, Gerrnany (Accepted 1 January 1993)

Summary-An incubation experiment was perforrned with two soils and five pesticide treatments (benomyl, isoproturon, simazine, dinoterb and chloroforrn). Microbial biomass C and N was estimated by fumigation-extraction (FE). Two modifications of the substrate-induced respiration (SIR) method were used: (1) a continuous flushing system and (2) a static system with soil slurry. In addition, C02 production was measured. Estimates of biomass C by the two SIR methods were generally higher than those obtained by the FE method. The two SIR measurements and the two biomass estimates were closely correlated, indicating a similar response to pesticide treatments. Pesticide application almost always reduced the size of the microbial biomass. Of the different ways of measuring biomass, the FE method revealed the largest number of significant differences from the nil treatment. The biomass C-to-N ratio was only slightly influenced by soil or pesticide treatments. Apart from the benomyl treatment, pesticide application always increased soil respiration during the 0-10 day incubation.

INTRODUCTION

There has been considerable concem about the detrimental effects of pesticides on the soil microftora since the beginning of pesticide use (Greaves and Maikornes, 1980). Colony number, enzyme activity, nitrification and soil respiration have all been used in the search for such effects (Domsch et al., 1983). The development of methods for estimating the size of the soil microbial biomass during the 1970s provided new ways to measure the effects of pesticides on the soil microftora. Most of these measurements were carried out with the SIR method (substrate-induced respiration; e.g. Wardle and Parkinson, 1990). The applicability of the SIR method in pesticide contaminated soils, has however been doubted (Malkomes, 1986). One reason is that the calibration factor used to convert SIR to microbial biomass was established on soils that had not been exposed to pesticide contaminations (Anderson and Domsch, 1978). Another reason is that pesticide application may alter the proportion of organisms which are able to mineralize glucose. Recently a new method has been developed for estimating microbial biomass C and N in soil: the FE method (fumigation-extraction method; Vance et al., 1987). This method has proved to be robust against handling errors and to be applicable to a wide range of both natural and strongly disturbed soils (Joergensen and Brookes, 1991). In addition, the FE method, in contrast to the SIR method, has the advantage that it can be used soon after substrate

addition (Ocio and Brookes, 1990) and in the presence of growing plant roots (Mueller et al., 1992). There are good reasons to think that the FE method could also be used for biomass estimations in pesticide-contaminated soils. We therefore designed a laboratory experiment to answer the following questions: (1) How do the different ways of monitoring microbial performance reftect the effects of pesticides on the soil microbial biomass? (2) Is the calibration of the SIR method against measurements of microbial biomass C and N by the FE method affected by exposure of the soil to pesticides? One fungicide (benomyl) and three herbicides (isoproturon, simazine and dinoterb) were selected so as to give a range of chemical structures. Chloroform was also included, because its effects on microorganisms in soil are weil documented (Jenkinson, 1988; Joergensen et al., 1990) compared with those of the other four pesticides.

MATERIALS AND METHODS

Soils

An arable (Rasdorf, pH 6.8, l.48% organic C, 0.155% total N) and a grassland soil (Angerstein, pH 7.4, 2.45% organic C, 0.23% total N), were sampled (0-10 cm) near Göttingen. The two soils were Luvisols derived from Loess. They were carefully dried to reach approx. 40% of their water holding capacity (WHC), sieved (2 mm), incubated

• Author for correspondence. 679

T.

680

HARDEN

for 10 days at 25°C and then stored at 4°C until the experiment started. App/ication of pesticides and incubation procedure

An incubation experiment with six treatments was carried out: nil, benomyl (3 mg kg- i soil, Benlate, Du Pont; methyl-1-(butylcarbamyl)-2-benzimidazolcarbamate], dinoterb [70 µl kg- 1 soil, Flüssig Herbogil, Bayer; 2,4-dinitro-6-tert.-butylphenol], isoproturon [65 µl kg- 1 soil, Graminon 500, Ciba Geigy; N-(4isopropyl-phenyl-)-N', N'-dimethylurea], simazine [40 µl kg- 1 soil, Simazin 500 flüssig, Spiess-Urania; 4,6-diethylamino-2-chloro-s-triazine) and chloroform [CHC1 3 LiChroso/v, Merck No. 2444). The pesticide concentrations were 10 times the amount usually applied in the field. Field application rates were related to quantities used in the laboratory, assuming an even distribution of the pesticide in the 0-5 cm layer and a soil bulk density of 1.5 g cm- 3• The pesticides were sprayed, in aqueous suspension, onto a thin layer of the soil spread on a 40 x 50 cm polyethylene sheet and then carefully homogenized. The pesticides were kept in suspension by magnetic stirring. The amount of suspension added was calculated to bring the water content to 50% WHC. Chloroform fumigation was performed as described for fumigation extraction (see below) but using two bulks of soil (about 400 g). After the pesticide application, each treatment was divided into five replicates. The soil (80 g, on an oven dry basis), contained in a glass vial, was placed in 1 litre bottles, each containing 10 ml 1 M NaOH in a 50 ml beaker, plus 10 ml water at the bottom of the bottle. The soils were kept at 25°C for 20 days in the dark. The microbial biomass contents of the variously-treated soils were measured at the end of this 20 day incubation. Analytical procedures

Carbon dioxide evolved during incubation was calculated from the quantity of 0.1 M HCl required to bring the pH of two 0.5 ml aliquots of the NaOH solution to pH 8.3. Substrate-induced respiration was measured after amending moist soil (20 g, on an oven-dry basis) with a powder containing 60 mg glucose and 200 mg talcum (Anderson and Domsch, 1978). The C02 production rate was measured hourly, using the method ofHeinemeyer et al. (1989), in which each sample was continuously purged with ambient air (250 ml min- 1 ) and the evolved C0 2 was measured using an i.r.detector. Soil microbial biomass C by SIRAo was calculated (Kaiser et al., 1992) from the maximum initial respiratory response, where: Biomass C (µg C g- 1 soil) =

(µl C0 2 g- 1 soil h- 1 ) x 30.

Substrate-induced respiration was also measured as described by West and Sparling (1986). Moist soil

et al.

(1 g, oven-dry basis) was placed into a 25 ml volumetric ftask (total volume 29.7 ml). Glucose solution was added to give a fluid volume of 2 ml at a concen-tration of 20 mg glucose g- 1 soil. The bottles were closed by rubber seals and kept for 3 h at 25°C in a water-bath with gentie shaking. i mi of air was taken from the headspace 1 and 3 h after glucose addition for measurement of C0 2 concentration by gas chromatography. The respiration rate measured over 2 h by SIRws was converted (Sparling et al., 1990) to microbial biomass C using the formula: Biomass C (µg C g- 1 soil)

= (µl

C02 g- 1 soil h- 1 ) x 50.

Fumigation and extraction were performed as described by Brookes et al. (1985) and Vance et al. (1987). Moist soil [40 g, on an oven-dry basis (ca 24 h at l05°C)) was split into two parts, each (20 g, on an oven-dry basis) placed in a 250 ml bottle. The nonfumigated control was immediately extracted with 80 ml 0.5 M K 2S04 for 30 min on an oscillating shaker at 200 rev min- 1 and then filtered through a paper filter (Whatman 42). For the fumigated treatment, bottles containing the field-moist soil were placed in a desiccator containing wet tissue paper and a via! of soda lime. A beaker containing 25 ml ethanol-free CHC1 3 (stabilized with 20 µl 2-methyl-2-butene 1- 1 ) was added and the desiccator evacuated until the CHC1 3 had boiled vigorously for 2 min. The desiccator was then kept in the dark at 25°C for 24 h. After fumigation, CHCl 3 was removed by repeated evacuation and the soil then extracted. The final K 2S0 4 extracts were stored at -15°C prior to analysis. Organic C in the K 2S04 soil extracts was measured by an automated u.v.-K2SP8 oxidation procedure (Wu et a/., 1990), using a Dohrman DC 80 automatic organic C analyser. Soil microbial biomass C was calculated (Wu et al., 1990) from the relationship: Biomass C = 2.22 Ec, where Ec is [(organic C extracted from fumigated soil) minus (organic C extracted from non-fumigated soil)). NH 4-N, N0 3-N and organic N were measured one after another by steam distillation as described by Joergensen and Meyer (1990). Total N was calculated by adding NH4-N, N0 3-N and organic N. Soil microbial biomass N was measured (Jenkinson, 1988) from the relationship: Biomass N = 2.22 EN, where EN is [(total N extracted from fumigated soil) minus (total N extracted from non-fumigated soil)). RESULTS

Soil microbial biomass

Biomass C estimates by the two SIR methods were generally higher than those obtained by the FE method, with the SIRws method giving the highest

Microbial biomass estimation

681

Table 1. Microbial biomass C estimated by SIRAo (Anderson and Domsch, 1978; Kaiser et al., 1992), SIRws (West and Sparling, 1986; Sparling et al., 1990) and FE (Vance et al., 1987; Wu et al., 1990), microbial biomass N by FE (Brookes et al., 1985; Jenkinson, 1988) Biomass C (SIRAo)

Biomass C (SIRwsl

Treatment

Biomass C (FE) µgg- 1 soil

Biomass N (FE)

Biomass C:N (FE)

Soii Rosdorf Nil Benomyl Simazine Isoproturon Dinoterb Chloroform LSD' (P < 0.05)

301 330•• 306 301 139** s1•• 12

470 470 462 437 241** 102•• 26

285 273 247** 246** 148** 58** 20

59 63 55 61 18** 14•• 13

4.7 4.4 4.8 4.1 8.4„ 4.0 1.6

Soil Angerstein Nil Benomyl Simazine lsoproturon Dinoterb Chloroform LSD (P < 0.05)

726 700 673* 696 256** 128** 45

1781 1766 1601** 1730 871** 276** 107

511 523 556** 504 296** 87** 16

136 116 109* 134 87** 21••

3.8 4.5** 5.1•• 3.8 3.4 4.0 0.5

20

•Least Significant Difference. Significant difference compared to the respective nil treatment: • P < 0.05; ••p < 0.01.

values (Table !). Irrespective of method, the grassland soil Angerstein contained more biomass C and N than the agricultural soil Rosdorf All SIR and FE biomass estimations were significantly correlated, with one exception (Table 2). This indicates that measurements by the four different methods showed very similar responses to pesticide treatment. However, biomass C estimations by means of the FE method revealed the largest number of significant deviations from the nil treatment (Table 1). With a few exceptions, pesticide application reduced the size of the microbial biomass with dinoterb and chloroform inducing the strongest biomass depression in both soils. Biomass C-to-N ratio was little affected by pesticide application (Table !). lt was significantly greater in the agricultural soil treated with dinoterb and the grassland soil treated with benomyl and simazine. A one-way ANOVA calculated separately for each soil revealed that the relative deviation from the nil treatment was not systematically affected by the method used to estimate biomass. A veraging the relative effects on SIRAD• SIRw8, FEc and FEN for the two soils, the benomyl treatment lead to a 2%

reduction, isoproturon 4%, simazine 5%, dinoterb 51 % and chloroform 80%. Soil respiration

With the exception of the benomyl treatment, pesticide application increased soil respiration during the 0-10 day incubation (Table 3). This effect was very marked in the dinoterb and chloroform treatments of both soils and in the isoproturon treatment of the grassland soil. In contrast, pesticide effects on soil respiration were less consistent during the 10-20 day incubation. C0 2 evolution was still significantly increased in the dinoterb treatment ofthe agricultural soil, but significantly reduced in the dinoterb and in the chloroform treatment of the grassland soil. The inverse effects of pesticide application on biomass estimations and on C02 evolution during the first 10 days of incubation lead to highly-significant negative correlations (Table 2). In contrast, there were no significant correlations between biomass estimations and C0 2 evolution over the 10-20 day period with the agricultural soil. With one exception (biomass N) both estimates were positively correlated in the grassland soil.

Table 2. Spearman rank correlation matrix

Soil Rasdorf (n = 30) Biomass N(FE) Biomass C(FE) Biomass C(SIRAol Biomass C(SIRwsl COr.C (0-10 d)

Soil Angerstein (n

Biomass C (FE)

Biomass C (SIRAo)

Biomass C (SIRws)

C02-C (0-lOd)

0.74***

0.62** 0.12•••

0.68** 0.82••• 0.87•••

-0.74••• -0.90••• -0.80••• -0.78***

0.19 -0.21 0.03 -0.04 0.25

0.39

0.68** 0.57*

0.18••• 0.52* 0.84•••

-0.13•••

0.25 o.55* 0.53* 0.52* -0.56*

= 30)

Biomass N(FE) Biomass C(FE) Biomass C(SIRAol Biomass C(SIRwsl cor.c (0-10 d) Level of significance

•p < 0.05; ••p < 0.01; •••p < 0.001.

-0.19••• -0.82••• -0.80•••

C02-C (10-20d)

T.

682

HARDEN

Table 3. Respiration rates

C02-C (µgg- 1 soil) 0-10 d 10-20 d Soil Rasdorf Nil Benomyl Shnazine Isoproturon Dinoterb Chloroform LSD• (P