Abstract The objective of the study was to compare eco-toxicological data obtained from laboratory experiments onthe side-effects of three phenylamide fungicides, pure met-alaxyl (racemic mixture of R- and S-enantiomers), formu-lated metalaxyl and mefenoxam (only active R-enanti-omer) on the chemical and biochemical parameters of twosoils of different type and origin. The purpose of the com-parison was to determine to what extent mefenoxam, de-veloped as alternative to metalaxyl, can affect the activityof soil micro-organisms and their processes, and to eluci-date the differences between the effects of pure and formu-lated metalaxyl. The dynamics of the quantitative changesin biochemical parameters induced by the addition of thesefungicides at their recommended field rate were deter-mined in a sandy clay soil from Cameroon and a sandyloam soil from Germany, during a 120-day incubation ex-periment. The type of soil significantly influenced the ef-fect of these fungicides on the soil parameters studied. In-corporation of these fungicides generally stimulated the ac-tivity of phosphatases and ß-glucosidase, mineralizationand the availability of N and most plant nutrients in soils.The activity of dehydrogenase and the availability of NO3

–

were generally adversely affected. Among the fungicidestested, the stimulation was more pronounced with mefe-noxam followed by formulated metalaxyl.

Keywords Soil enzymes · Mefenoxam · Metalaxyl ·Plant nutrients · Soil biochemical parameters

Introduction

The phenylamide fungicide metalaxyl [methyl N-(2,6-di-methylphenyl)-N-(methoxyacethyl)-DL-alaninate], whichis the active ingredient of the fungicide Fonganil Neumanufactured by Novartis, is added to soil to control

fungal pathogens of the order Peronosporales, whichcause late blight, downy mildew, damping off and stemand fruit rots of many plants. These additions, while en-hancing the plant growth and crop yield (Urech et al.1977), can affect the homeostasis of the soil system(Kormondy 1976). Any perturbation is likely to lead to ashift in the equilibrium of the system while directly af-fecting the structure and function of the soil microbialcommunity. This is evidenced by the observations ofUsataya et al. (1993), who showed that applications ofmetalaxyl on vineyard soils over 3 years markedly de-creased microbial numbers and decreased the activityand increased the number of micro-organisms involvedin the mineralization of organic matter. Dvornikova et al.(1988) reported that the systemic application of metala-xyl induced a brief stimulation and a subsequent sup-pression of soil fungi and actinomycetes.

Mefenoxam, [methyl N-(2,6-dimethylphenyl)-N-(me-thoxyacethyl)-D-alaninate], is the R-enantiomer of met-alaxyl, introduced onto the market in 1996 under variousformulations and trade names including, Ridomil Gold,Fonganil Gold, Apron XL, Subdue, MAXX. It providesthe same level of efficacy as metalaxyl, but at half the ap-plication rate (Nuninger et al. 1996). Thus, the introduc-tion of mefenoxam may contribute to a reduction of theenvironmental risk associated with the use of metalaxyl(Nuninger et al. 1996). Consequently, mefenoxam will re-place technical metalaxyl in parts of the world where theregistration of metalaxyl has been cancelled (Johnson1996). Information on the effect of mefenoxam on soilquality is scarce. As phenylamide fungicides are amongthe most used fungicides worldwide, it is important toconsider their possible impact on soil quality and health.

Concern over the effects of these fungicides on soilprocesses is based on the fact that many of the reactionsin nutrient cycling are mediated by microbes (Hattori1973); there is also the possibility that these chemicalscan enter into the food chain and, thus, affect higher or-ganisms including humans. For example, metalaxyl hasbeen found in water supplies (Readman et al. 1997; Petrovic et al. 1998), and in food (Om et al. 1998). Fun-

A. Monkiedje · M. Spiteller (✉ )Institute of Environmental Research, University of Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germanye-mail: spiteller@infu.uni-dortmund.deTel.: +49-231-7554080, Fax: +49-231-7554085

Biol Fertil Soils (2002) 35:393–398DOI 10.1007/s00374-002-0485-1

O R I G I N A L PA P E R

Adolphe Monkiedje · Michael Spiteller

Effects of the phenylamide fungicides, mefenoxam and metalaxyl,on the microbiological properties of a sandy loam and a sandy clay soil

Received: 3 December 2001 / Accepted: 9 April 2002 / Published online: 13 June 2002© Springer-Verlag 2002

gicides are generally used as formulated compounds tooptimize their effects on target organisms. The behaviour(e.g. penetration into plants and soil organisms, degrada-tion, leaching in soil) of the formulated compounds maybe different from that of the active ingredient. Conse-quently, ecotoxicity tests using active ingredients do notnecessarily represent the behaviour of the formulatedcompounds (Malkomes 1997).

The aim of this paper is to study the effect of pure andformulated metalaxyl on ammonification, nitrificationand on the activities of dehydrogenase, acid phosphatase,alkaline phosphatase and ß-glucosidase in soils of differ-ent type and origin and to elucidate differences betweenthe effects of pure and formulated metalaxyl. The effectof mefenoxam, as emulsifiable concentrate (EC) formu-lation, was also investigated. Ammonification is general-ly insensitive to stress whereas nitrification is sensitive;this is because only a few microbial species are involvedin the latter. Dehydrogenase activity is considered to be auseful index for measuring the side-effects of pesticideson microbial activity in soil (Schaffer 1993). Several en-zyme activities were measured simultaneously in orderto obtain a more valid estimate of the metabolic responseof soil to fungicide stress following a suggestion byNannipieri et al. (1990).

Materials and methods

Soils

The soil collected from Monheim, Germany (German soil) is usedregularly by Bayer for adsorption and degradation studies. Theother soil was collected in Cameroon (Cameroonian soil) from theexperimental research farm of the Institute of Agronomic Re-search for Development in Nkolbisson, near Yaounde. The redferralitic soil from this site covers 60% of the national surface areaof the country. These two soils, which had not received any pesti-cide applications for at least 5 years, were taken from the surfacelayer (0–10 cm) and then sieved (<2 mm). Their main propertiesare presented in Table 1.

Test chemicals

Pure metalaxyl, analytical standard grade (purity, >99%) (P-metala-xyl), was obtained from Riedel-de-Haen, Germany. Formulated met-alaxyl (F-metalaxyl) and mefenoxam were EC formulations contain-ing 24% and 48% metalaxyl and R-metalaxyl, respectively. Theywere obtained from Novartis Agro (Frankfurt). All other chemicalsused in the study were analytical grade from Merck or Aldrich.

F-metalaxyl contains metalaxyl as the active ingredient, whichis a racemic mixture of R- and S-enantiomers, whereas mefe-noxam contains only the active R-enantiomer. Both compounds,having the same molecular weight (279.34), empirical formula(C15H21NO4) and structural formulae (Fig. 1), have been com-pared on a microgram per gram basis.

Application of test substances to soil

P-metalaxyl and F-metalaxyl (144 µg active ingredient/100 g soilon a dry weight basis each) and mefenoxam (72 µg active ingredi-ent/100 g soil on a dry weight basis), respectively, were mixedthoroughly and separately into the soils at the recommended com-mercial application rate for cocoa crop in Cameroon of 1,080 g ac-

394

Table 1 Selected physico-chemical properties of soils Texture analysis (USDA) Sand clay loam Sandy loam

Clay (<2 µm) (%) 31 5Silt (<2–50 µm) (%) 24 23Sand (50–2,000 µm) (%) 45 72pH (water, 1:2.5) 4.80 7.20pH (0.01 M CaCl2, 1:2.5) 4.16 6.75Corganic (%) 3.01 1.69Ntotal (%) 0.17 0.09P (mg P2O5/100 g dry weight) 0.11 57Cation exchange capacity (mEq/100 g dry weight) 9.76 8.00Maximum water holding capacity (%) 54.4 34.4Density (g/ml) 1.35 2.50Origin Yaounde Monheim

Nkolbisson II Laacher Hof AxxaCameroon Germany

Designation Cameroonian soil German soil

Fig. 1 Structures of the two metalaxyl enantiomers

tive ingredient/ha for P-metalaxyl, F-metalaxyl and 540 g activeingredient/ha for mefenoxam (Novartis, personal communication).These application rates were based on the maximum single userate, assuming a soil depth of 5 cm and a soil density of 1.5 g/cm3.To avoid the potential effects of solvents upon the microbiologicalactivity of the soil, the calculated volumes of the application solu-tion (553 µl and 550.5 µl of the solution of P-metalaxyl in metha-nol for the German soil and for the Cameroonian soil, respective-ly; 949 µl and 814 µl of the solution of F-metalaxyl in water forthe German soil and for the Cameroonian soil, respectively;237.5 µl and 203.5 µl of the solution of mefenoxam in water forthe German soil and for the Cameroonian soil, respectively) weredispensed onto portions of ~30 g air-dry soil in porcelain dishes.The treated subsamples of soils were thoroughly mixed with aspatula until the solvent had completely evaporated (~20 min).The subsamples were subsequently added to the total soil mass ofthe corresponding field-moist soils (1,114 and 1,146 g for Germansoil and for Cameroonian soil, respectively, for P-metalaxyl; 1,912and 1,694 g for German soil and for Cameroonian soil, respective-ly, for F-metalaxyl and mefenoxam). Subsequently, the gross soilmass for each test chemical rate was mixed in a tumbling mixerfor 1 h. This was followed by adjustment of the moisture contentof the soil to 60% of the maximum water holding capacity, prior tosampling, to allow optimal conditions for activity of aerobic soilmicro-organisms. Batches of 100 g of each soil (equivalent dryweight) were incubated in the dark in 750-ml glass jars, undercontrolled temperature (20±2°C) for 120 days. The jars were kept

covered with perforated parafilm. The moisture content in eachflask was checked gravimetrically each week and at each samplingperiod. During the incubation, jars were removed periodically andsampled once for analyses of soil chemical and biochemical pa-rameters. Each experiment was carried out in duplicate.

Soil chemical analyses

Available N (NH4+ and NO3

–) were extracted in 2 M KCl as re-ported by Anonymous (2000), and quantified by colorimetricmethods (Kim 1996).

Biochemical analyses

Soil dehydrogenase activity was measured using 2,3,5-trip-henyltrazolium chloride (TTC) as the electron acceptor (Casida etal. 1964). Soil (20 g) was thoroughly mixed with CaCO3 (0.2 g) tofacilitate filtration of the soil suspension and subsequent extractionof the triphenyl formazan formed, and 6 g of this mixture was treat-ed in triplicate with 3% (w/v) TTC (1 ml) and incubated for 24 h at37±1°C. The triphenyl formazan formed was extracted quantita-tively from the reaction mixture with 100 ml methanol and assayedat 485 nm in a Shimadzu UV 1201 UV-VIS spectrophotometer.

Acid and alkaline phosphatase activities were determined ac-cording to a slight modification of the procedure of Tabatabai and

395

Fig. 2 Effect of fungicides onthe availability of NH4

+-N andNO3

–-N in the Cameroonian (a)and German (b) soils. *P<0.05between fungicide addition andno fungicide addition (pairedStudent t-test test)

Bremner (1969) and Eivazi and Tabatabai (1977) using p-nitro-phenylphosphate as substrate. Soil (1 g) was mixed with 4 mlmodified universal buffer (MUB) of pH 6.5 and pH 11 for acidand alkaline phosphatase assays, respectively, and 0.05 M p-nitro-phenyl phosphate (1 ml) and incubated for 1 h at 37±1°C. Then,0.5 M CaCl2 and 0.5 M NaOH (4 ml) were added and the mixturewas centrifuged at 3,000 r.p.m. for 10 min. The p-nitrophenol inthe supernatant was determined colorimetrically at 400 nm. Tolu-ene was not included in the enzyme assay because it might in-crease both acid and alkaline phosphatase activities and could beused as a C source by most soil micro-organisms (Kaplan andHartenstein 1979).

ß-Glucosidase activity was determined using p-nitrophenyl-ß-D-glucopyranoside as the substrate (Eivazi and Tabatabai 1988).Four millilitres of MUB (pH 6.0) and 0.025 M p-nitrophenyl-ß-D-glucopyranoside (1 ml) were added to soil (1 g) and the reactionmixture was incubated at 37±1°C for 1 h. The rest of the methodwas the same as described above for acid and alkaline phosphataseactivities. Toluene was not used in this assay either because of theshort incubation period.

Results of enzyme activities are reported on an oven driedweight basis, determined by drying the soils for 24 h at 105°C.

Statistical analysis

The results for each sampling period and from over the total incu-bation period were reported as a percentage of the control andcompared using ANOVA, with treatment as the independent vari-able. The means were subsequently analysed by way of multiplecomparisons using the paired Student t-test procedure (Dunnett1985).

Results and discussion

Effect on available N

The changes of NH4+ and NO3

– content in soils duringthe incubation are shown in Fig. 2. In the shorter term(3–30 days), F-metalaxyl and P-metalaxyl caused a sig-nificant decrease in NH4

+ content of both soils. Mefe-noxam, while causing a significant decrease in the Ger-man soil NH4

+ content, stimulated that of the Cameroon-ian soil as early as 14 days after application. The inhibi-tory effects of these fungicides on short-term exposurewere reversible on long-term incubation (especially after120 days) as evidenced by the significant increase inNH4

+ content in both soils (Fig. 2a, b). This increase wasmore pronounced with mefenoxam followed by F-met-alaxyl. Probably mefenoxam is more bioavailable to bac-teria than F-metalaxyl and P-metalaxyl. This assessmentis supported by the findings of Pai et al. (2001) who re-ported that after 21 days of incubation 78% of mefe-noxam was degraded by rhizosphere microbial popula-tions. Metalaxyl has been reported to have DT50 values(the time taken for 50% active ingredient to be metabo-lized) in soil ranging from 3 to 8 weeks (Sharom andEdgington 1982).

Similar trends were recorded for the NO3– content in

Cameroonian soil (Fig. 2a). This indicated that thesecompounds stimulated the growth and the activities ofammonifying and nitrifying bacteria, which were mainlyresponsible for the mineralization of organic N to NH4

+

and oxidation of NH4+ to NO3

–, respectively. The inhibi-

tory effects of all these chemicals on nitrification weremore pronounced in German soil, even on the 120th dayof incubation (Fig. 2b), but generally mefenoxam wascomparatively less inhibitory and even showed a signifi-cant increase on the 75th day of incubation (Fig. 2b). P-metalaxyl and F-metalaxyl effects resulted in delays of30 days in the recovery of NH4

+ and NO3– in the Camer-

oonian soil (Fig. 2a). These effects can be considerednormal according to the theoretical framework for test-ing the side-effects of pesticides, proposed by Domsch etal. (1983). As delays of recovery of this available Nwere >60 days in the German soil (Fig. 2b), the effectsof P-metalaxyl and F-metalaxyl were considered criticalin this soil (Domsch et al. 1983).

Effect on soil enzyme activities

Pesticides have been shown to have direct and indirect ef-fects on soil enzyme activity (Nannipieri 1994). The ad-dition of fungicides, in general, stimulated the activitiesof phosphatases and ß-glucosidase in both soils as shownin Figs. 3, 4, 5. Dehydrogenase activity was generallynegatively affected, especially in the Cameroonian soiltreated with F-metalaxyl and P-metalaxyl (Figs. 4, 5).Mefenoxam significantly stimulated the activity of acid

396

Fig. 3 Effect of mefenoxam on enzyme activities in the Camer-oonian (a) and German (b) soils. *P<0.05 between fungicide addi-tion and no fungicide addition (paired Student t-test test)

and alkaline phosphatases and ß-glucosidase in both soils(Fig. 3). Dehydrogenase activity was the most affected ofall enzyme activities under mefenoxam stress. This inhib-itory effect was more pronounced in the German soil.This difference in the dehydrogenase activity in the twosoils may be ascribed to the difference in the decomposi-tion rates of fungicides or their transformation to less tox-ic by-products in both soils as suggested by Nannipieriand Bollag (1991). This adverse effect was not permanentsince a significant increase in the activity of this enzymewas observed on the 90th day of incubation in both soils(Fig. 3). A significant increase in the two phosphatase ac-tivities and in ß-glucosidase activity were observed on theaddition of F-metalaxyl and P-metalaxyl to both soils(Figs. 4, 5). This increase was more pronounced in theCameroonian soil. Dehydrogenase activity was stimulat-ed on the 90th and on the 30th and 90th day of incubationby F-metalaxyl and P-metalaxyl, respectively, in the Ger-man soil (Figs. 4b, 5b). P-metalaxyl, however, signifi-cantly decreased the activity of dehydrogenase in theCameroonian soil (Fig. 5a). As P-metalaxyl and F-met-alaxyl caused delays of >75 days of recovery of the dehy-drogenase activity in the Cameroonian soil (Figs. 4a, 5a),their effects on the activity of this enzyme were, thus,critical in this soil (Domsch et al. 1983).

In general, dehydrogenase activity appeared moresensitive to all fungicides in both soils, though to vary-ing degrees. This is in agreement with many reports onthe adverse effects of pesticides including fungicides ondehydrogenase activity (Schuster and Schroeder 1990).Dehydrogenase occurs intracellularly in all living micro-bial cells and it is linked with microbial respiratory pro-cesses. Its rapid degradation in soils could be followedby cell death and, thus, it does not accumulate in soils(Somerville et al. 1987). Dehydrogenase activity hasbeen reported to reflect the microbial activity of soil(Thalman 1968; Nannipieri et al. 1990; Tabatabai 1994).

The fungicides, in general, stimulated the activities ofphosphatases and ß-glucosidase. Being extracellular en-zymes, they are generally protected from degradation byadsorption on clays or humic substances and, thus, mayaccumulate (Skujins 1976; Boyd and Mortland 1990;Nannipieri et al. 1990). Moreover, the extracellular en-zymes, immobilized by soil colloids, may not be as sen-sitive to fungicides as those associated with microbialcells (Nannipieri 1994).

In conclusion, the application of the investigated phe-nylamide fungicides at their maximum recommendedfield rates had positive and negative effects on soilchemical and biochemical properties. The positive effect

397

Fig. 4 Effect of F-metalaxyl on enzyme activities in the Camer-oonian (a) and German (b) soils. *P<0.05 between fungicide addi-tion and no fungicide addition (paired Student t-test test)

Fig. 5 Effect of P-metalaxyl on enzyme activities in the Camer-oonian (a) and German (b) soils. *P<0.05 between fungicide addi-tion and no fungicide addition (paired Student t-test test)

on phosphatases and ß-glucosidase activities was proba-bly due to the microbial growth stimulated by the addi-tion of these fungicides which served as sources of ener-gy. In general, mefenoxam like F-metalaxyl exerted anegative influence on biochemical parameters of soil asmanifested by the observed decrease in available N, es-pecially NO3

– in German soil, and altered enzymatic ac-tivities, especially that of dehydrogenase. The effects ofP-metalaxyl and F-metalaxyl on the content of availableN were normal in the Cameroonian soil and critical inthe German soil. These chemicals also exerted a criticaleffect on the activity of dehydrogenase in the Cameroon-ian soil. The stimulatory effect of mefenoxam on avail-able N, phosphatases and ß-glucosidase activities was, ingeneral, greater than that of F-metalaxyl. F-metalaxyland P-metalaxyl, in general, exerted similar effects onsoil properties.

Acknowledgements We are grateful to the Alexander von Humboldt Foundation for periods of research spent by the first author in Germany, under the Georg Forster Research Fellowship.We thank Novartis Agro (Frankfurt) for kindly supplying the me-fenoxam and F-metalaxyl employed, and Mr. A. Achermann fromNovartis Crop Protection (Basel) for the interest shown in thisstudy. We acknowledge the technical assistance of Mr. JürgenStorp.

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