Applied Soil Ecology 41 (2009) 293304

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Applied Soil Ecology

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Microbial toxicity and impacts on soil enzyme activities of pesticides used in potato cultivation
R. Maarit Niemi a,*, Ilse Heiskanen a, Jukka H. Ahtiainen a, Anne Rahkonen b, Keijo Mantykoski c, Leena Welling c, Pirkko Laitinen d, Pentti Ruuttunen d
a

Finnish Environment Institute, P.O. Box 140, FI-00251 Helsinki, Finland Potato Research Institute, Ruosuontie 156, FI-16900 Lammi, Finland skyla Institute for Environmental Research, P.O. Box 35, FI-40014 University of Jyva , skyla Finland , University of Jyva d MTT Agrifood Research Finland, Plant Production Research, FI-31600 Jokioinen, Finland
b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 November 2007 Received in revised form 25 November 2008 Accepted 8 December 2008 Keywords: Microbial effects Soil enzyme activities Luminescent bacteria test Pesticides Metribuzin Linuron Fluazinam Potato cultivation

In the conventional cultivation of potatoes, weed control and the control of potato late blight caused by Phytophthora infestans are carried out by the application of herbicides and fungicides. We investigated the impacts of the herbicides metribuzin and linuron and the fungicide uazinam on soil microbiota in microcosms, in mesocosms and in the eld. The toxicity of each pesticide in solution was assessed using the luminescent bacteria test and in soil by a solid phase modication. In microcosm tests, the microbial activity and biomass were estimated by measuring several soil enzyme activities together with soil ATP content. In the mesocosm tests, the separate addition of each pesticide and the simultaneous use of all the pesticides were investigated. We monitored the impacts on ten different soil enzyme activities and measured soil toxicity with the luminescent bacteria test separately in the 5 cm top layer and in the layer from 5 to about 20 cm below the surface. During one season, the impact of the use of pesticides was monitored in the eld in plots receiving pesticides for the third consecutive year and in control plots cultivated without the use of pesticides for the 3 preceding years. The pesticide concentrations were monitored in each experiment. The luminescent bacteria toxicity test revealed a strong inhibition by uazinam. In microcosms the herbicides increased several enzyme activities but metribuzin inhibited luminescence in the bacterial test. Fluazinam was highly toxic in microcosms. In the mesocosm with combined use of pesticides, decreased activities of some enzymes were observed rst in the surface soil and at harvesting also deeper in the soil. In the mesocosm experiment with separate use of pesticides, less impacts were observed. In the eld experiment the pesticides decreased seven enzyme activities, when calculated per soil fresh weight but activities of four enzyme decreases if calculated per soil organic matter. Controlling weeds by herbicides decreased weed growth and the decreases in enzyme activities were interpreted to be due to the lack of the stimulating impact of weed roots. A strong inhibition in the soil toxicity test and continuing bioavailability of uazinam were detected throughout the experiments even after winter in the eld. 2008 Elsevier B.V. All rights reserved.

1. Introduction The same elds are often used for crop cultivation for several consecutive years, and because herbicides are used to control weeds and fungicides in order to prevent potato late blight caused by the fungus Phytophthora infestans, it is necessary to evaluate harmful environmental impacts involved in the repeated use of pesticides.

* Corresponding author. Tel.: +358 400148661; fax: +358 204902290. E-mail address: maaritniemi@gmail.com (R.M. Niemi). 0929-1393/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2008.12.002

Herbicides and fungicides have been designed to specically affect certain enzyme activities in order to restrict their impacts to target weeds and plant pathogens, but non-target organisms in soil may also be affected. This can lead to reduced microbial diversity and possible decrease in soil fertility (Johnsen et al., 2001). Johnsen et al. pointed out the bioavailability aspects of pesticides in soil, and stressed the importance of combining laboratory and eld experiments and also the application of both traditional and molecular methods in studies on impacts of pesticides on microbial species diversity. The aim of this study was to assess the microbial effects in soil of two herbicides and one fungicide in common use in the

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cultivation of diverse crops. Metribuzin was selected because of its easy transfer as a water soluble compound (Hernandez et al., 1998), linuron for its persistence in the soil (Guzzella et al., 2006) and uazinam because of its persistence and wide taxonomic range to inhibit plant pathogenic fungi (Kimyoji et al., 1995) and simultaneous lack of information on its impacts on nontarget microbes. Metribuzin (4-amino-6-tert-butyl-4,5-dihydro-3methylthio-1,2,4-triazin-5-one) and linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea) inhibit electron transport at the photosystem II receptor site and are used to control weeds in several crops, including potato (Tomlin, 2002). Metribuzin is biodegraded rapidly in soil and photodecomposition is also efcient in the soil surface and in water. Microbial degradation is the primary factor in linuron disappearance from soil. Fluazinam (3-chloro-N-(3-chloro-5-triuoromethyl-2-pyridyl)-a,a,a-trifuoro-2,6-dinitro-p-toluidine) is a prophylactic fungicide with little curative activity but a good residual effect and rain fastness (Tomlin, 2002). It has an uncoupling activity on mitochondrial oxidative phosphorylation and is used as a fungicide to protect several crops including potato against P. infestans. Photodecomposition occurs in the soil surface. The herbicides metribuzin and linuron are usually sprayed in one treatment in the beginning of the growing season, whereas the fungicide uazinam is applied several times during the season. We assessed the impacts of the herbicides metribuzin and linuron and the fungicide uazinam in luminescent bacteria toxicity test, in its solid phase modication, on soil ATP content and functional diversity in soil, estimated as enzyme activities important in the mineralisation of P, N and S and biodegradation of common macromolecules cellulose, hemicellulose, xylan and starch, in microcosm, in mesocosm and in eld experiments in potato cultivation. Our hypothesis was that the sensitivity of different microbial processes varies and depends on the pesticide and exposure time. Direct impacts of pesticides on microbiota and indirect impacts due to plant growth can be differentiated by using micro- and mesocosm experiments. 2. Materials and methods 2.1. Experimental design The soil in the microcosm (50 g in 120 ml glass bottles) and mesocosm (18 l soil, 1720 cm depth) experiments was ne sand from two organically cultivated potato elds in Lammi representing soil previously unexposed to pesticides (Table 1). The soil used in the 2004 experiments had been under green fallow in 2002 and cultivated with potato in 2003. The soil for the experiments in 2005 had been used for oats (Avena sativa) cultivation in 2003 and for

Table 1 Characteristics of the soil in the micro- and mesocosm and eld experiments (as % with the exception of pH). Soil characteristics Micro- and mesocosm 2004, 020 cm pHH2 O pHKCl Loss on ignition Organic C Humus Clay Silt Sand Gravel
a b c

Field 015 or 020 cm 6.36.7 5.75.9 7.1 and 6.4b 3.4c 8.0 2.8 31 66 0.1

2005, 020 cm 5.3 4.7 6.97.1a 4.4 7.5 8.5 56 35 0.1

6.0 4.7 5.6 3.7 6.4 4.1 46 50 0.4

Higher 18th July and lower during the harvest. Control and treated, respectively. 3.3 0.3 in treated and 3.4 0.1 in control soil.

potato cultivation in 2004. Both experimental soils in 2004 and 2005 were fertilized with half of the nutrient levels recommended in accordance with the soil analysis and mixed for 5 min using a concrete-mixer. The normal application dose of the herbicide Senkor (metribuzin 700 g kg1) was 0.25 kg ha1 and of the herbicide Afalon (linuron 450 g l1) 1.5 l ha1 sprayed once on the soil surface when potato plants started to produce shoots. The normal application dose of the fungicide Shirlan (uazinam 500 g l1) was 0.4 l ha1, which in our mesocosm experiments was deliberately sprayed on the soil surface and not on the potato plants in order to mimic the worst case from the environmental point of view. The application of the fungicide was carried out once in the microcosm, ve times in the mesocosm experiments and six times in the eld experiment. In sampling and sample treatment cross contamination was avoided by using separate equipment for sampling augers and homogenization devices (sieving through a household colander and mixing with a buffer with stab homogenizer (Braun MR 400 Minipimer, 300w) for the control and each pesticide-treated soil. Soil dry weight (+105 8C overnight), loss on ignition (550 8C 1 h), pHH2 O and pHKCl (50 ml distilled water or 1 mol l1 KCl was added to 10 g of fresh soil) were measured from the soil samples. The toxicity of each pesticide was estimated with the luminescent bacterial toxicity test. The microbial effects of pesticides were studied in three replicate microcosms of 50 g of soil with each pesticide added into separate bottles in concentrations corresponding to those recommended for normal use and at tenfold concentrations. The controls were not treated with pesticides. In the rst test, the microcosms were incubated in a greenhouse in bottles covered with Paralm1 for 7 d, which caused draught veried by weighing the bottles. The test was repeated in an incubator (50% water holding capacity, +20 8C) in closed bottles in the laboratory and samples were analysed after 7 and 28 d incubation. The enzyme activities and ATP were analysed and a solid phase modication of the luminescent bacteria toxicity test was carried out after 7 and 28 d of incubation. The mesocosm experiment using separate additions of pesticides was carried out in three replicates using plastic boxes each with 18 dm3 soil with a depth of about 20 cm and with two potato plants (Solanum tuberosum, var Saturna) per pot in the open air with a clear plastic roof as a shelter against rain and with applications of each pesticide into separate mesocosms. The soil samples were taken separately from the top 5 cm and from the layer below 5 cm (to the bottom, soil depth from 17 to 20 cm) as composite samples consisting of ve sub-samples, using a 16 mm auger. For the herbicide study, the samples were taken 7, 28 and 78 d after spraying of the herbicides. For the fungicide study, the samples were taken 60 d after the rst application and 19 d after the last of the ve treatments. In addition to enzyme activity measurements from all the samples, soil toxicity was assessed during the harvest. The other mesocosm experiment was carried out in three replicates in a greenhouse (temperature between 4 a.m. and 9.30 p.m. +20 8C and during the night +16 8C, shading if radiation above 700 W m2, humidity 65%, irrigation manually every 2 d to the surface) as described for the rst experiment, but simulating the pesticide treatment used in the elds. Both the herbicides were sprayed on 16th June and the fungicide on 7th July, 16th July, 26th July, 5th August and 16th August 2004. The samples for enzyme activity measurements were taken as described for the rst experiment 1 d after the herbicide, 1 d after the last fungicide and 1 week after the last fungicide treatment and at harvesting 1 month after the last fungicide addition. In the eld experiment on mouldy ne sand, three replicate plots from the pesticide-treated and from the untreated control block were monitored in 2006. Potato was cultivated in both blocks

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from 1999 to 2002 with application of pesticides. In 2003 the crop plant was barley (Hordeum vulgare) and potato had been grown since then. Similar pesticide treatments had been applied in the pesticide-treated block in 20042006, whereas the control block had not received pesticides during this period. In 2004 and 2005 S. tuberosum, var Saturna and in 2006 var Tanu was grown. Altogether one herbicide application as a tank mixture of both linuron and metribuzin and six fungicide applications were carried out. At least 12 sub-samples were collected separately from the top 5 cm with a 34 mm wide auger and below 5 cm depth to the bottom of the ploughing layer, about 15 cm, with a 25 mm wide auger. The samples were transported to the laboratory within a few hours, cooled in insulation boxes and stored at +4 8C overnight. Soil samples were homogenized by sieving through a 4 mm sieve the day after sampling in the laboratory and 4 g aliquots were deepfrozen for the enzyme activity measurements and 10 g subsamples for the luminescent bacteria test. The rst sample was taken 7 d after the treatment with both of the herbicides, the second 7 d after the rst fungicide treatment, and the third at harvesting 4th September 2006, 1 week after the last fungicide application. The concentrations of the pesticides during each experiment were monitored. 2.2. Pesticides Linuron formulation used was Afalon1 Suspension Concentrate from Makhteshim-Agan Holland B.V. containing 450 g l1 linuron. Metribuzin formulation used was Senkor1 WG from Bayer CropScience, Germany, containing 700 g kg1 metribuzin, 2% alkylaryl sulphonate and 8% methylene-linked condensation product of arylsulphonic formaldehyde reaction products. Shirlan1 formulation of uazinam (Syngenta Crop Protection A/S) containing 50% (w/v) uazinam and less than 10% of ammonium salt of polyarylphenylether sulphate, was used. 2.3. Determination of pesticides in soil

E.C.3.2.1.91), -D-glucosidase (E.C.3.2.1.20) and a-D-glucosidase (E.C.3.2.1.20). 2.5. Soil ATP content The effects of the pesticides on active microbial biomass were estimated as soil ATP content after extraction in tri-chloroacetic acidEDTA solution and ltration (Schleicher & Schuell 604 lter paper), using a BioOrbit 1253 luminometer (BioOrbit, Turku, Finland) as described by Vanhala and Ahtiainen (1994). 2.6. Microbial toxicity tests The toxicity of the pesticides was rst ranked by the standard luminescent bacteria (Vibrio scheri) test in 2% NaCl solution (ISO, 1998) and the soil toxicity was estimated by a solid phase modication of the luminescent bacteria test from 0.5 g soil aliquots in 2% NaCl solutions at 15 8C for 30 min and measurement of luminescence using the reagents of Aboatox Co. (Lemminkaisenkatu 36, Fi-20520 Turku, Finland) and the BioOrbit 1253 luminometer (BioOrbit, Turku, Finland) (Ahtiainen et al., 2003). 2.7. Statistical tests KruskalWallis nonparametric analysis of variance was applied for the 7 d microcosm experiment, and one-way analysis of variance together with Dunnets multiple comparison with the control for the microcosm experiment including 7 and 28 d. For the mesocosm and eld experiments, general analysis of variance was run separately for each date to test for the signicance of treatment, soil depth and their interaction. Log10 transformation was used if the ShapiroWilk normality test indicated this to be necessary. Statistix1 Software Version 8 (Thallassee, FL 323172185, USA) was used. Only statistically signicant (P 0.05) impacts of pesticides are mentioned. 3. Results

Pesticide concentrations were analysed using a modication of the method developed by Andersson and Palsheden (1991). A 20 g aliquot of homogenized soil was extracted (sonication and shaking machine) with a mixture of ethyl acetate and acetone (5 + 1). After decanting and drying the sample with sodium sulphate, the internal standard (prothoate) was added to the sample and the solvent was evaporated with a rotary evaporator and gentle stream of nitrogen. Finally the concentrations of pesticides were determined by GCMS using a mass selective detector. It was feasible to analyze for the metabolites of metribuzin. 2.4. The enzyme activity prole From the microcosm and mesocosm experiments, enzyme activities were measured directly after sampling, whereas the deep-frozen 4 g aliquots of homogenized samples were used in the eld experiment. The soil enzyme activities were measured with the enzyme activity kit (ZymProler1) in multi-wells comprising 10 uorogenic enzyme activity measurements relevant for decomposition processes in soil at approximate in situ pH 6.0 in the modied universal buffer (Tabatabai, 1994) as described by Vepsalainen et al. (2001, 2004). The enzyme activities measured were arylsulphatase (E.C.3.1.6.1), phosphomonoesterase, PME (E.C.3.1.3.2), phosphodiesterase, PDE (E.C.3.1.4.1), leucine-AP (aminopeptidase) (E.C.3.4.11.1), alanine-AP (E.C.3.4.11.12), chitinase (b-N-acetylglucosaminidase; E.C.3.2.1.30), -D-xylosidase (E.C.3.2.1.37), cellulose 1,4-b-cellobiosidase (cellobiosidase,

3.1. Toxicity and microcosms tests The potential toxicity of each pesticide was estimated as effective concentrations (EC20 for concentration causing 20% inhibition and EC50 for 50% inhibition). For metribuzin EC20 was 56 and EC50 199 mg l1, for linuron EC20 was 6.6 and EC50 23 mg l1, but for uazinam EC20 was 0.005 mg l1. The toxicity of each pesticide in soil and the impacts on soil microbial biomass (as ATP) and soil enzyme activities were measured from soil samples with normal and tenfold concentrations of the pesticides together with control without pesticide (Table 2). In the rst test, with additional stress on microbiota in the form of soil dryness, several signicant impacts of pesticides were observed. After 1 week, both herbicides increased several enzyme activities, without any signicant decreases and with no impacts on ATP and the luminescent bacteria toxicity test. The fungicide increased some enzyme activities but decreased leucine AP activity and ATP content and was toxic in the luminescent bacterial test. When the same test was repeated without moisture loss, signicant effects were less common after 7 d than in the previous test, but uazinam was again toxic in the luminescent bacteria test, appearing to decrease ATP content but to increase arylsulphatase and leucine AP activities. After 28 d, ATP content was higher in the uazinam-treated soil than in the control and no other signicant impacts were found. The lower concentration of metribuzin decreased the ATP content and was toxic in the luminescent bacterial test after

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Table 2 Signicant impact of pesticides on ATP content, luminescent bacteria toxicity and soil enzyme activities in fresh soil in microcosms.a Experiment Dry soilb 7d Treatment ATP Toxicity Arylsulphatase PME PDE Alanine AP Leucine AP Chitinase -Xylosidase Cellobiosidase -Glucosidase

a-Glucosidase

Metribuzin Metribuzin*10 Linuron Linuron*10 Fluazinam Fluazinam*10

+ +

+ + + + + +

+ + + +

+ + + +

+ + + + + +

Moisture controlc 7d Metribuzin Metribuzin*10 Linuron Linuron*10 Fluazinam Fluazinam*10 28 d Metribuzin Metribuzin*10 Linuron Linuron*10 Fluazinam Fluazinam*10

+ + +

+ + + + + +

+ +

+ +

nd nd nd nd nd nd nd nd nd nd nd nd

+ +

a b c

Signicant increase (+) and decrease () (or inhibition and stimulation of bioluminescense); nd: not done. KruskalWallis nonparametric analysis of variance, P < 0.05, n = 12. One-way analysis of variance with Dunnets multiple comparison with control, P < 0.05, n = 12.

7 d, but stimulation was observed after 28 d. Both herbicides increased some enzyme activities either after 7 d or 28 d incubation. 3.2. Mesocosm experiments When each pesticide was added to separate mesocosms, no impacts of herbicides were observed 1 week after addition, but, later on, a few impacts of pesticides were recorded (Fig. 1). Linuron increased PME activity after 78 d, metribuzin increased PDE activity in surface soil at 28 d, uazinam increased alanine AP activity after 78 d, metribuzin and linuron decreased leucine AP activity at 28 d but metribuzin increased it in subsoil after 78 d. Seasonal changes were common and depth-related differences were observed. Even the signicant impacts of pesticides were weak, and no impacts were observed for cellobiosidase, glucosidase or -xylosidase activities. Samples taken at the harvest were analysed for soil ATP content and toxicity (Fig. 2).

Linuron increased ATP content in the top soil. Fluazinam was highly toxic in the luminescent bacteria test. Pesticide contents were analysed from the top soil 7 d after herbicide treatment and at harvesting (Table 3). Metribuzin decreased in the top soil during 78 d to 9% of the original concentration and was not detected deeper in the soil. Linuron concentration decreased only to 42% during the 78 d exposure in the top soil and traces corresponding to 3% of the original top soil concentration and to 7% of the simultaneous concentration in the top soil were detected in soil below 5 cm. In the soil depth from 5 to 17 cm, uazinam concentration 1960 d after spraying corresponded to 3% of the content in the top soil. The irrigation was controlled so that no water ran out from the mesocosms. When the three pesticides were added to the same mesocosms, seasonal changes were observed and depth gradients developed for the same enzyme activities as in the other experiment (Fig. 3). The rst effects of pesticide treatment were observed 69 d after the herbicide and 48 d after the rst fungicide treatment, when PME

Table 3 Occurrence of pesticides (means of triplicates) in the two mesocosm experiments. The metabolites (metribuzin-desamino, metribuzin-desamino-diketo, metribuzin-diketo) of metribuzin were not detected. Experiment Pesticide Days after treatment 7 7 78 78 1960 1 1 69 69 848 90 90 2969 Concentration (kg1 dw) 0.265 1.750 0.025 0.735 0.820 0.14 0.49 <0.005 0.025 0.170 1.440 0.06 0.55 1.48 05 cm S.D. Concentration (mg kg1 dw) 517 cm S.D.

Each pesticide to the separate mesocosms

Metribuzin Linuron Metribuzin Linuron Fluazinam Metribuzin Linuron Fluazinam Metribuzin Linuron Fluazinam Metribuzin Linuron Fluazinam

0.035 0.212 0.007 0.078 0.130 0.03 0.14 0.007 0.057 0.325 0.057 0.255 1.428

<0.002 0.050 0.027

0.042 0.006

Pesticides to the same mesocosms

0.015 0.020 0.035 0.010 0.020 <0.005a

0.007 0.000 0.021 0.000 0.014

Traces observed but below the level of reliable detection.

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Fig. 1. Soil enzyme activities in the mesocosm experiment signicantly affected by separate pesticide treatments. M: etribuzin, L: linuron; F: uazinam; C: control; soil depth in cm. MUF: 4-methylumbelliferyl; AMC: 7-amido-4-methylcoumarin; AP: aminopeptidase.

and -xylosidase activities were decreased in the surface and cellobiosidase also in the sub-surface soil. At 90 d, even PME, xylosidase and -glucosidase activities were decreased in both the soil layers. At 69 d alanine AP was increased in the surface and decreased in the subsoil due to pesticide treatment. Pesticides in the soil samples were analysed from the mesocosms on three dates (Table 3), 1 d after the application of herbicides from the top soil only and later on separately from the top 5 cm and between 5 and 17 cm. Metribuzin content decreased to 18% and linuron to 35% in the surface soil during the 69 d period. In soil below 5 cm, the metribuzin concentration was 10% of the initial and the linuron concentration 4% of the initial content of the surface soil. However, relatively high concentrations of pesticides and signicant variation between the replicate samples were observed in the top soil at harvesting. About half of the initial metribuzin concentration and no decrease at all in linuron content was detected in the top soil, whereas deeper in the soil the concentrations were similar to those observed after 69 d. Only 2% of the uazinam content of the top soil was detected deeper in the soil 848 d after spraying and after 2969 d the concentration was below the detection level. However, in the top soil the uazinam concentration had not decreased during the 21 d period after the last addition. In the two mesocosm experiments, no metabolites of metribuzin were detected.

3.3. The eld experiment No difference in pHKCl was observed between pesticide-treated and control soil or between the top and deeper soil layers. pHKCl varied from 5.8 to 6.0 and pHH2 O form 6.0 to 7.0. Top soil was less moist than the deeper soil layer with the exception of the harvest period after recent rainfall. Soil organic matter content (as loss on ignition) was lower in the pesticide-treated soil than in the control soil in samples analysed for enzyme activities (P = 0.0007***, d.f. = 24). Luminescent bacteria toxicity test revealed persistent toxicity in the treated soil, increase in toxicity during the summer in top soil and higher level of toxicity in the top soil than below 5 cm (Fig. 4). Enzyme activities calculated per soil fresh weight yielded signicant decreases due to pesticides in the analysis of variance (Fig. 5). Arylsulphatase, cellobiosidase, -glucosidase, -xylosidase and a-glucosidase activities were always lower in the pesticidetreated soil, PME activity was lower with the exception of surface soil at 30 d and leucine AP activity with the exception at 7 d, whereas PDE, chitinase and alanine AP activities were not affected by pesticides. When a separate analysis of variance was run for the enzyme activities expressed per unit of soil organic matter, fewer decreases in enzyme activities were observed (Fig. 6).

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Pesticide contents were measured from both treated and control plots in the eld (Table 4). After the winter in May, traces of metribuzin and rather high concentrations of both linuron and uazinam were detected in plots treated with pesticides during the previous years. No metribuzin or its metabolites were detected in any soil layer on 9th June 2006 but some traces (even if below the level of reliable detection) of linuron were observed in treated soil in all soil layers, as well as traces of uazinam. Herbicide treatment on 12th June increased their concentrations in top soil and also in deeper layers, after which their concentrations started to decline, with some oscillation in the case of linuron. Late in September 14% of metrizubin and 24% of linuron persisted in top soil. The rst fungicide treatment on 11th July clearly increased its content in soil, and after the sixth treatment the concentration was about eightfold compared with the May level in top soil. On 4th September (8 d after the last application), fungicide concentration in top soil was 117% and at the harvest 126% of the concentration directly after the last spraying. The difference between pesticide-treated and control soil in vegetation was visible already 1 week after herbicide treatment, and a dense weed vegetation covered the control soil at harvesting. 4. Discussion The impacts of pesticides on soil microbes and enzyme activities are modulated by soil physical and chemical properties affecting their bioavailability and transportation, by composition of microbiota (occurrence of pesticide sensitive or tolerant and degrading species) and by vegetation (rhizosphere effects such as stimulation of microbiota and increase in excreted enzymes). Pesticide formulations contain potentially toxic impurities and degradation products of pesticides may be highly toxic. In the present study, soils used represent those preferably used in potato cropping in Finland. Soils used in micro- and mesocosms had not been previously exposed to pesticides and preselection of microbiota could be avoided. However, in the eld experiment even the control soil had been exposed to pesticides 3 years earlier. The inuence of vegetation could be discerned by using both soil

Fig. 2. Soil ATP content and soil toxicity in the luminescent bacteria toxicity test in the mesocosm with separate treatments with different pesticides at two soil depths at harvesting. Descriptions as in Fig. 1.

Arylsulphatase and cellobiosidase activities were decreased in pesticide-treated soil at 36 and 84 d; and -glucosidase activity in surface soil (and at 7 d also in subsoil). On the other hand, alanine AP activity was always higher in the pesticide-treated than in the control soil, and the impact on leucine AP activity varied with time.

Table 4 Metribuzin and metabolites, linuron and uazinam in soil, mg kg1 dw in the eld in 2006. Triplicate plots analysed, standard deviations in parentheses. State Depth (cm) Metribuzin Treated After the winter, 9th May Before treatment, 9th Junea Herbisides 0 dc Herbisides 7 dd Herbisides 25 dc Herbisides 29 da, fungicide 0 d Herbisides 36 dc, fungicide 7 d 05 015 015 05 05 015 05 015 05 05 015 0.003 (0.001) 0.002 (0.000) <0.002 0.123 (0.015) 0.047 (0.006) 0.023 (0.006) 0.047 (0.006) 0.017 (0.006) 0.030 (0.010) 0.027 (0.006) 0.007 (0.000) <0.002 <0.002 Control Linuron Treated 0.047 (0.028) 0.035 (0.011) <0.005 0.903 (0.035) 0.483 (0.035) 0.197 (0.057) 1.500 (0.000) 0.390 (0.011) 0.813 (0.011) 0.110 (0.057) 0.020 (0.000) <0.000b <0.000b Control Fluazinam Treated 0.097 (0.045) 0.083 (0.012) 0.013 (0.006) 0.009 (0.001) 0.017 (0.006) 0.037 (0.029) 0.013 (0.006) 0.213 (0.015) 0.257 (0.091) 0.065 (0.035) <0.005 Control

29th July27th August 5*uazinam Herbisides 76 da, fungicide 047 d Herbisides 78 da, fungicide 247 d Herbisides 84 da, fungicide 853 d Herbisides 103 da, fungicide 2772 d
a b c d

05 015 05 015 05 015

0.033 0.007 0.017 0.006

(0.006) (0.001) (0.006) (0.002)

<0.002

0.327 0.113 0.090 0.018

(0.011) (0.127) (0.134) (0.011)

<0.005

0.793 0.220 0.927 0.120

(0.163) (0.243) (0.338) (0.061)

<0.005

0.017 (0.006) 0.007 (0.002)

0.220 (0.127) 0.095 (0.011)

1.000 (0.622) 0.253 (0.143)

Metabolites of metribuzin not detected. Traces of linuron but below the reliable detection level and 0.010 in the 520 cm layer. Metabolites of metribuzin not analysed. Metribuzin-desamino and -diketo <0.01 in both soil layers; metribuzin-desamino-diketo 0.020 in upper 5 cm and 0.010 in the 520 cm layer.

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Fig. 3. Soil enzyme activities in the mesocosm experiment signicantly affected by joint pesticide treatments. Both herbicides applied 16th June and the fungicide ve times: 7th, 16th and 26th July and 5th and 16th August. The last sampling was on 14th September. T: pesticide-treated; C: control; soil depth in cm; MUF: 4-methylumbelliferyl; AMC: 7-amido-4-methylcoumarin; AP: aminopeptidase.

without plants (microcosms) and plant containing mesocosms. Of the pesticide formulations used, Sencor and Shirlan contained in addition to metribuzin and uazinam harmful constituents with possible impacts to the microbial responses observed. Estimation of the toxicity of pesticides, as commercial formulations, in solution in the luminescent bacteria test revealed metribuzin to be less toxic than linuron but uazinam to inhibit luminescence of V. scheri even in very low concentrations. In the soil microcosm experiments, distinct inhibition of luminescence by uazinam was observed repeatedly during 1 weeks exposure but after a month the inhibition was no longer signicant. Even the ATP content in the microcosm experiment appeared to decrease for 1 week but later on the ATP content appeared to increase. It is, however, possible that the measurement result was affected by direct inhibition of luciferase enzyme and the ATP content was not changed. In the mesocosm experiment at harvesting, uazinam was again clearly toxic in the luminescent bacteria test. In the eld experiment, uazinam had persisted from the spraying during previous year(s). Strong inhibition of luminescence was observed even in the subsoil and applications during the summer further increased toxicity in the surface soil; it was most pronounced at harvesting. This toxicity was attributed to uazinam rather than to the herbicides (Tables 2 and 4). Contrary to the toxic response of the fungicide, linuron in soil caused no inhibition in the

luminescent bacteria test and the only impact on ATP was an increase after 1 month in the tenfold concentration. Metribuzin had no impact on these variables in the rst microcosm experiment, but in the second experiment initial inhibition changed to stimulation within 1 month. In the microcosm and in the mesocosm experiments with separate pesticide treatments, impacts on soil enzyme activity patterns were observed. In the microcosms, both of the herbicides stimulated some enzyme activities, whereas some enzymes were not affected and none were inhibited. Stimulation depended on the enzyme, the herbicide, its concentration and duration of the exposure. In the mesocosm with separate herbicide treatments, no impacts were observed after 1 week of herbicide treatment but leucine AP activity decreased after 1 month in both linuron- and metribuzin-treated soil. However, at harvesting the impact on leucine AP had attenuated. Less stimulation by herbicides was recorded in the mesocosms than in the microcosms, possibly due to the compensation as absence of weed rhizosphere in the treated mesocosms when compared with the control mesocosms. The results of the mesocosm experiment with joint use of pesticides differed from the results with separate additions of pesticides. More decreases in enzyme activities were observed in the mesocosm and, especially, in the eld experiments with pesticides added to the same soil. This was the case even if, contrary to the soil

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Fig. 4. Loss on ignition and toxicity of the soil in the luminescent bacteria test (% of the bioluminescence in the respective control soil) in the eld experiment. Descriptions as in Fig. 3.

in micro- and mesocosms, the control soil in the eld had also received pesticides a few years previously, although not during the 3 previous years. Therefore, some adaptation to the pesticides could have occurred. In the mesocosms, the rst pesticide-related impacts, decreased activities, were observed 10 weeks after herbicide and 1 week after fungicide treatments, typically in the surface soil. At harvesting, the same enzyme activities were also decreased deeper in the soil. In the eld, the activities (per fresh soil) of several enzymes were decreased in pesticide-treated soil. S, P and N mineralisations, cellulose, hemicellulose and xylan biodegradation and even starch degradation were all decreased. The slightly higher organic matter content of the control samples for enzyme activity measurement contributed to the higher enzyme activities in control soil. At the onset of the eld experiment, soil organic C in the 20 cm surface layer of the treated soil did not signicantly differ from the control soil. During the sampling for microbial tests, loss on ignition in the treated 5 cm surface soil was 90% and in the 515 cm layer 92% of the corresponding values in the control soil. The dense weed vegetation in control soil may have contributed to loss on ignition estimates even if the formation of organic soil is known to be a very slow process. When the enzyme activities were calculated per organic fraction of the soil, fewer and less consistent impacts of pesticide treatments were observed. Tu (1992) observed that herbicides including linuron and metribuzin caused temporary effects, both inhibition and stimulation, on soil enzyme activities and soil processes relevant for the fertility of soil. Our microcosm results conrm his observations but our mesocosm and eld experiments also revealed gradual decrease in enzyme activities. The use of phenylurea herbicides, including linuron, for 10 years decreased bacterial diversity (16S rDNA DGGE) and affected substrate utilisation patterns (El-Fantroussi et al., 1999). Linuron decreased nodulation by

Rhizobium strains (Haider et al., 1991; Fernandez-Pascual et al., 1988) and severely inhibited soil bacterial survival (Leach et al., 1991). Katayama et al. (2001) observed that some pesticides both decreased respiratory quinones (an estimate of microbial biomass) and affected their proles (species composition), whereas linuron had no impact. Junnila et al. (1993) observed nitrication inhibition even during the following spring after application of metribuzin. Lupwayi et al. (2004) observed no impact on either microbial biomass C or microbial diversity after metribuzin treatment, but herbicide-induced shifts in microbial composition were revealed. Fluazinam has been shown to have a broad fungicidal spectrum in antifungal tests in vitro and good protection against plant diseases caused by different fungal genera (Kimyoji et al., 1995). Couderchet (2003) evaluated environmental aspects of the use of fungicides in vineyards. He regarded uazinam as a potential soil and water contaminant because of its long detention, despite its low water solubility. There seems to be no data available on impacts of uazinam on benecial microbes in soil, but the tests with plant pathogenic fungi revealed fungicidal activity against phycomycetes, ascomycetes, basidiomycetes and deuteromycetes (Kimyoji et al., 1995) and it is hardly possible that benecial fungi are not affected. The mechanisms of stimulation of some enzyme activities in microcosms due to herbicides and uazinam in the present study are not known. Direct stimulation of enzyme activities is not probable because no impact was observed 1 d after pesticide treatment but clearly after 7 d. In the light of published literature on other pesticides the metabolic shifts probably indicate changes in microbial species composition. Roberts et al. (1998) and Cullington and Walker (1999) isolated bacterial strains capable of linuron degradation and Mochida et al. (1993) isolated and puried an enzyme from a coryneform soil bacterium degrading linuron. Linuron can be utilised as the sole C and N source by Variovorax sp. (Srensen et al., 2005). Roberts et al. (1993) observed that a consortium of soil bacteria is needed for the complete degradation of linuron and that a Pseudomonas strain is involved. El-Fantroussi (2000) observed that linuron degradation capacity by soil microbiota was related to long-term use of this herbicide and that a consortium of microbes was needed for the complete transformation. Bordjiba et al. (2001) identied fungal species capable of both linuron and metribuzin biodegradation and Shelton et al. (1996) isolated a Stretomyces strain with similar capability. Di et al. (1998) tested degradation rates of pesticides including linuron and metribuzin in surface and sub-surface soil. No consistent relationship was observed between eld and laboratory degradation rates, probably due to changes in soil microbial activities and organic matter content affecting sorption. Although initial degradation of metribuzin was rapid in sub-arctic soils (24% persisted after 35 d), subsequent decomposition was slower and 5% still remained after 468 d (Conn et al., 1996). Slower biodegradation in sub-surface soil has been explained by smaller microbial populations and activity (Allen and Walker, 1987; Moorman and Harper, 1989; Mueller et al., 1992). Degradation due to sunlight may also have contributed to the higher degradation rates observed in the surface soil. Appropriate plant residues have also been observed to enhance bioremediation in metribuzincontaminated soil (Wagner and Zablotowicz, 1997). Bending et al. (2007) observed that degradation of fungicides and herbicides was fastest in soils with high organic matter and microbial biomass content. Dehydrogenase activity was reduced in soil with low organic matter content but unaffected in soil with more organic matter. No impact on bacterial community structure was observed but a few protozoa and fungi were absent after fungicide treatment by azoxystropin, tebuconazole and chlorothalonil. All the soils of the present study contained more organic matter than the soils studied by Bending et al. and, therefore, sufcient microbial biomass and diversity might have been

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Fig. 5. Enzyme activities per soil fresh weight affected signicantly by pesticide treatments in the eld experiment. PDE, chitinase and alanine AP activities were not signicantly changed due to pesticide treatment. Descriptions as in Fig. 3.

present to allow some biodegradation of pesticides. In the present study, no decrease in uazinam content in surface soil occurred before harvesting, but in the subsoil a clear decrease was evident in the mesocosm. Fluazinam concentration appeared to increase in the surface soil even after the last spraying at harvest in the eld. The apparent lack of decrease of uazinam content in surface soil may have been caused by release of uazinam from plant material. In the rst mesocosm experiment, both metribuzin and linuron concentrations in the surface soil had decreased after 10 weeks but elevated concentrations were observed after 13 weeks at harvesting. The use of composite samples aimed at controlling the impact of spatial variation in pesticide occurrence. Standard deviations

were rather high in linuron and metribuzin measurements in the rst mesocosm experiment in September and also in linuron and uazinam measurements in August and September in the eld. This may indicate the establishment of sites with exceptionally high concentrations, possibly due to bioaccumulation, during the summer (Su and Zhu, 2007). We were able to monitor only for the metabolites of metribuzin, which were detected 7 d (but not anymore 15 d) after spraying in the eld after several years of application in the whole ploughing layer but not at all in the mesocosm studies with only one treatment. It seems probable that metribuzin metabolites had no important impacts to the microbial responses measured in our

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Fig. 6. Enzyme activities per loss on ignition affected signicantly by pesticide treatments (P 0.05) in the eld experiment. PME, PDE, chitinase, -xylosidase and aglucosidase activities were not signicantly changed due to pesticide treatment. Descriptions as in Fig. 3.

study. US EPA National Pesticide Information Center (http:// www.npic.orst.edu/tech.htm) lists 25 metabolites of the degradation of linuron. Guzzella et al. (2006) observed two major metabolites from diuron and linuron in soil columns, DCPMU [3-(3,4-dichlorophenyl)-1-methylurea] and DCA (3,4-dichloroaniline). Enhanced biodegradation of linuron was observed to develop with successive eld treatments, but considerable in-eld spatial heterogeneity in the degradation rate still existed (Rasmussen et al., 2005). Their results suggested that intrinsic soil properties affected the linuron-metabolising bacterial population and thereby determine the spatial variability in the linuron mineralisation activity. Spatial heterogeneity of pesticide degraders could have contributed to the increase in variation of pesticide concentrations during summer in the present study. The indirect impact of herbicides in our mesocosm and eld experiments as gradual decreases in some enzyme activities during the summer, when compared with control soil with weed rhizosphere, is plausible because herbicides in microcosms tended to increase many enzyme activities immediately after application. Naturally, plant cover including weeds strongly enhances soil microbiota and its activities. The slight and often transient impacts of linuron and metribuzin on soil enzyme activities in microcosms may not indicate a clear inuence on soil biodegradation capacity. However, these changes show a metabolic transition possibly involving changes in species distribution

towards pesticide-resistant microbiota and even selection of species capable of pesticide biodegradation. The autumnal increase of pesticides after the initial decrease was observed both in the mesocosm and in the eld experiment. A plausible explanation to increased concentrations and increased spatial heterogeneity in the autumn is root exudation and release from plant vacuoles (Saxena and Stotzky, 2000; Dinelli et al., 2007; Schroder et al., 2007). There is hardly any published literature on the fate and impacts of uazinam in the environment. Although uazinam was designed to affect plant pathogenic fungi, it had a consistently strong inhibitive action towards the gram negative bacterium V. scheri used in the luminescent bacteria toxicity test. The prevalence of traces of uazinam and inhibition throughout the year even below top soil with increased impact during the growing season in potato cultivation, some indication of inuence on microbial biomass (or luciferase activity) and even slightly on soil enzyme activities, clearly reveals the need for further investigation of its impacts on microbial species distribution. Both bacterial and fungal DNA ngerprinting and identication of affected species could reveal the impact on microbial diversity. It would be important to test the impacts of the pesticide on sensitive functions, such as N-transformation processes simultaneously (Kotzerke et al., 2008). The persistence of linuron and uazinam and movement of metribuzin were conrmed in the present study.

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All the pesticides studied in this work were simultaneously studied in two other eld sites and a manuscript is in preparation. 5. Conclusions The treatments, especially with the herbicides, appeared to increase mainly temporarily some enzyme activities in soil without rhizosphere. The denser plant cover in the control soil as compared with herbicide-treated soil may explain the higher activities of some enzymes in mesocosms and in the eld. Metribuzin, linuron and uazinam caused changes in enzyme activities, and these metabolic shifts probably indicate changes in the microbial composition also. Soil toxicity testing with luminescent bacteria indicated bioavailability of the fungicide and severe toxic effects throughout the experiments. Although soil biodegradation capacity relevant for the fertility of soil appeared not to be seriously affected by the herbicides, the observed impacts are in agreement with published literature on impacts on soil microbial biodiversity. A further study is needed on the effect of uazinam on soil microbial diversity, including fungal species composition. Acknowledgements The Ministry of Agriculture and Forestry supported this study nancially. MTT Agrifood Research Finland was responsible for the coordination and organised eld experiments jointly with the Potato Research Institute. Pesticide analyses were carried out at the University of Jyvaskyla and microbiological investigations at the Finnish Environment Institute. Daniel Richterlich is warmly thanked for his advice in the greenhouse experiment carried out at the University of Helsinki. Timo Myyrylainen and Tomi Myyr ylainen are thanked for providing the experimental eld site and for the operation of the eld experiment. Our thanks are due to Minna Madsen, Niina Lehtonen, Anuliina Putkinen, Susanna Vahakuopus, Minna Rannali, Virva Uotinen and Anna Packalen for the micro- and mesocosm experiments, sampling and carrying out microbiological analyses. We thank Seppo Niemela, Hannu Rita and Hannu Sirvio for the discussion on statistical aspects. Michael Bailey is acknowledged for the correction of the language. References
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