Ring-testing and Field-validation of a Terrestrial Model Ecosystem (TME) –

An Instrument for Testing Potentially Harmful Substances: Effects of

Carbendazim on Nematodes

THOMAS MOSER,l,* HANS-JOACHIM SCHALLNAß,1 SUSAN E. JONES,2 CORNELIS A.M. VANGESTEL,3 JOSEE E. KOOLHAAS,3 JOSE M.L. RODRIGUES4 AND JORG ROMBKE1

1ECT Oekotoxikologie GmbH, Bottgerstr. 2-14, D-65439 Florsheim am Main, Germany2University of Wales, School of Agricultural and Forestry Sciences, Bangor, Gwynedd LL57 2UW, Wales, UK3Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

4Departamento de Biologia, Universidade de Aveiro, Aveiro, Portugal

Accepted 24 October 2002

Abstract. The effects of the fungicide carbendazim (applied in the formulation Derosal�) on nematodeswas determined in Terrestrial Model Ecosystem (TME) tests and field-validation studies. TMEs consistedof intact soil columns (diameter 17.5 cm; length 40 cm) taken from a grassland or, in one case, from anarable site. The TMEs were taken from the same site where the respective field study was performed. Thetests were performed in Amsterdam (The Netherlands), Bangor (Wales, England), Coimbra (Portugal) andFlorsheim (Germany). Differences concerning nematode overall abundance, the number of nematodefamilies, the trophical structure of the nematode cenosis and the maturity index (MI) were not foundbetween the controls of TME tests and the respective field-validation studies. Effects caused by the chemicaltreatment, however, were observed on the number of nematode families, on the trophical structure of thenematode cenosis and on the maturity index (MI). Effects on the relative abundance of the omnivorousnematodes were most pronounced, whereas the overall nematode abundance was not affected. The ob-served effects appear not to differ between the TME tests and the respective field-validation studies. Allmeasurement endpoints in both TMEs and field, showed rather large variations. Therefore, NOEC-valueswere often equal or higher than the highest treatment level and EC50-values were calculated only for theomnivorous nematodes. NOEC- and EC50-values derived from the TME ring-test and the field-validationstudy indicate that the reproducibility (i.e. the variation between the partners) was reasonable, althoughdifferent soils from different sites were used. The EC50-values determined for the effect of carbendazim onthe relative abundance of the omnivorous nematodes ranged between 0.93 and 7.24 kg a.i./ha (1.24–9.63 mg/kg). Due to the higher sensitivity of the relative abundance of the omnivorous nematodes com-pared to the other measurement endpoints it is recommended to use this parameter as the main endpoint.

Keywords: carbendazim; Nematodes; soil mesocosms; community effects

Introduction

Risk assessment of chemicals usually is based onthe results of single species toxicity tests, which areperformed in the laboratory (Van Leeuwen and

*To whom correspondence should be addressed:

Tel.: +49-6145-956430; Fax: +49-6145-956499;

E-mail: th-moser@ect.de

Ecotoxicology, 13, 61–74, 2004

� 2004 Kluwer Academic Publishers. Manufactured in The Netherlands.

Hermens, 1995). It is, however, realised that suchsingle species tests may not be sufficient to predicteffects on the level of ecosystem structure andfunctioning (Cairns, 1984). In the past, such effectshave been studied in the field but the results aredifficult to assess and the efforts are high. There-fore, mesocosm tests have been advocated, whichmay close the gap between laboratory and fieldstudies. For the soil environment, this has resultedin the development of several types of model eco-systems (Morgan and Knacker, 1994; Sheppard,1997; Edwards et al., 1998; Knacker et al., 2004).Such model ecosystems may be applied in a highertier of risk assessment, when results of single spe-cies laboratory toxicity tests give matter of concernwith regard to the potential risk of a chemical inthe soil environment (see Weyers et al., 2004).

Free-living terrestrial nematodes occur world-wide everywhere even in deserts. They are the mostabundant metazoa in soils, reaching densities of 2–20 millions per square meter. They interact closelywith other soil organisms and contribute consi-derably to soil biological processes (e.g. decom-position of organic matter) which constitute themas important members of the soil fauna (Freck-man, 1982; Andrassy, 1984). The nematode faunapossesses a high potential to serve as an instrumentfor environmental studies and soil quality assess-ment because of its high density and diversity, highcolonisation rate, slight active movement and easeof isolation. Additionally, the partly permeablecuticule facilitates that nematodes are in directcontact with soil pore water and dissolved xeno-biotics. Moreover, nematodes represent a tro-phically heterogeneous group (Bongers, 1988;Traunspurger et al., 1995).

Recent interest in soil biology and increasingsoil pollution has raised the number of studies onterrestrial non-plant parasitic nematodes. Severallaboratory test methods to investigate effects ofchemicals on nematodes have been proposed (VanKessel et al., 1989; Donkin and Dusenbery, 1993;Kammenga and Riksen, 1996; Kammenga et al.,1996; Niemann and Debus, 1996; ASTM, 2001).From numerous studies nematodes are known tobe affected by a wide variety of chemicals in soil(Yeates et al., 1983; Jaffee and McInnis, 1990;Weiss and Larink, 1991; Parmelee et al., 1993;Parmelee et al., 1997; Salminen and Haimi, 1997).To measure effects of soil pollution not only on the

species level but also on the nematode communitylevel, the maturity index (MI), originally deve-loped to monitor environmental disturbancesbased on the soil nematode species composition,is a useful instrument (Korthals et al., 1996).Therefore, these organisms were chosen as an in-dicator to investigate potential effects of chemicalson the soil biocoenosis.

In this paper, results on the nematode abun-dance, the number of families, the trophic struc-ture and the MI obtained from ecotoxicologicaltests using Terrestrial Model Ecosystems (TMEs)are described. TMEs are defined as controlled,reproducible systems that attempt to simulate theprocesses and interactions of components in aportion of the terrestrial environment (Gillett andWitt, 1980; Sheppard, 1997). These tests wereperformed within the frame of the TME-projectsponsored by the European Union (Contract No.:ENV4-CT97-0470). The aim of the TME projectwas to improve and field validate the TME testsystem, first described by Van Voris et al. (1985)and used in studies reported by Knacker et al.(1989), Frederickson et al. (1991) and Chekai et al.(1993). For that purpose, the TME tests wereperformed at four different European sites withdifferent soils, but using the same equipment, testchemical, test design, and the same endpoints. Thetests were designed in a way which allowed acomparison of NOEC- and EC50-values for severalendpoints. In order to investigate the ecologicalrelevance of this laboratory TME test system, afield-validation study was performed. Carbenda-zim was chosen as the model chemical for theTME-tests and the field-validation study. Car-bendazim is a fungicide that is used at large scalein agriculture throughout Europe (Cuppen et al.,2000; Frampton and Wratten, 2000).

Materials and methods

Experimental set-up

Three types of tests were performed: in the firstyear a TME pre-test, in the second year, based onthe experience gained during the pre-test, the TMEring-test and in parallel to the ring-test a field-validation study. The TME-project was conductedat four sites throughout Europe by the following

62 Moser et al.

project partners: ECT Oekotoxikologie GmbH,Florsheim, Germany (1), Vrije Universiteit Am-sterdam, Institute of Ecological Science, TheNetherlands (2), University of Wales, School ofAgricultural and Forestry Sciences, UK. (3) andUniverisdade de Coimbra, Instituto Ambientee Vida, Portugal (4). In Coimbra, TME tests andthe field-validation study were performed using anarable site, whereas the respective work at thethree other sites was done using a grassland. Theproperties of the soils from these four sites areshown in Table 1. The TME tests were startedwith the extraction of the TME soil cores. TMEswere taken by means of a soil core extractor,containing a HDPE tube (diameter 17.5 cm;length 40 cm), which served as a soil core encase-ment. Grass was cut just before TME extraction.TMEs were either placed in temperature-con-trolled carts in a climatic chamber (Amsterdam,Florsheim, Coimbra) or in a greenhouse (Bangor).TMEs were watered up to three times per weekusing artificial rainwater slightly modified accord-ing to Velthorst (1993). The model chemicalcarbendazim was applied after an acclimatisationperiod of 2–4 weeks.

For the field-validation study, 30 field plots,each 25 m2, were marked out by each partner atthe same site where the TMEs were extracted. Sixplots served as controls, and 24 plots were treatedwith the model chemical (4 plots for each of the6 treatment levels; the plots were completelyrandomised). Before spraying the model chemical,the grass cover was cut on the grassland sites(Amsterdam, Bangor, Florsheim) or the soil wasploughed at the arable site (Coimbra).

Carbendazim was applied to the TMEs and fieldplots as Derosal�, containing 360 g carbendazim/l.In the TME ring-test and the field-validation studythe dosages were 0 (T0), 0.36 (T1), 1.08 (T2), 3.24(T3), 9.72 (T4), 29.2 (T5) and 87.5 (T6) kg car-

bendazim/ha. In the laboratory, carbendazim wasapplied to the soil using a pipette. The TMEs wereirrigated immediately after treatment. In the field-validation study, carbendazim was applied by aplot sprayer, commonly used in agricultural prac-tice, using a 3 l (6 l for highest dosage) spray so-lution per plot (ffi1200 l/ha). Immediately afterspraying, a volume of 30 l of water was sprayedonto the plot to wash off the Derosal� from theplants onto the soil. The control plots were alsosprayed with 30 l of water.

Various measurement endpoints were chosen todetermine the fate and effect of the model chemicalas well as the structure and function of the ter-restrial compartment. The fate endpoints includedthe measurement of residues of the model chemicalin the upper soil layer (0–5 cm), in the soil layerfrom 5 to 15 cm and in the leachate. The effectendpoints were classified into functional endpoints(nutrients in leachate and soil which describe as-pects of nutrient cycling; soil enzyme activity; mi-crobial substrate induced respiration; bacterialgrowth; feeding activity of soil organisms; organicmatter decomposition) and structural endpoints(abundance as well as diversity and communitystructure of microarthropods, nematodes, enchy-traeids, lumbricids and plant biomass. For adetailed description of the set-up of the TME-project, it is referred to Knacker et al. (2004).

Nematode sampling

Nematodes were investigated in the TME ring-testand the field-validation study sixteen weeks afterapplication of the test chemical. Samples weretaken from six untreated TMEs or field plots(control) and from four TMEs or field plots foreach treatment level. Soil samples for the extrac-tion of nematodes were taken from the top 5 cm

Table 1. Soil properties and site use of the four experimental fields from which the soil cores for the TME tests were extracted and the

respective field-validation studies were performed (for details see Knacker et al. (2004); OM ¼ organic matter)

Participant/

location Country code Texture Clay (%) OM (%) pH (KCl) Land use

Amsterdam NL Silty loam 7.9 4.5 4.8–5.1 Meadow

Bangor UK Loam 20.5 6.1 5.8–6.6 Pasture

Coimbra P Silty loam 24.7 3.4 6.4–7.1 Arable field

Florsheim D Silty clay loam 36.5 5.2 5.3–5.9 Meadow

TME – Effects of Carbendazim on Nematodes 63

soil by means of a cork borer with a diameter of1.5–3 cm. TME and field soil samples from Am-sterdam and Coimbra were sent to Florsheimwithin 24 h after sampling and stored at approxi-mately 4–6 �C for no longer than 1 week. Thepartner in Bangor extracted, counted and deter-mined the nematodes themselves. The extractionof nematodes from the soil sample followedCobb’s modified decanting and sieving method(s’Jacob and Van Bezooijen, 1984; Southey, 1986).Each sample (¼soil cylinder) was steeped in400 ml tap water (1 l beaker) for approximately15 min, then soil and water were stirred for 60 s,and after 15 s of sedimentation the supernatantwas carefully decanted in a collecting plastic bowl.This procedure was repeated twice, each time with400 ml tap water. The remaining sediment wasdiscarded, and the combined nematode suspensionwas poured through a cascade of several sieveswith decreasing mesh sizes (1000, 350, 175, 100and 45 lm). To improve the cleaning, the nema-tode suspension was poured five times through the45 lm sieve. The nematodes on the 45 lm sievewere rinsed with tap water and transferred in aseparate plastic bowl. The collected supernatant ofthe nematode suspension was carefully decanted.The remaining nematodes were poured into anextraction sieve containing a cotton-wool filter.The extraction sieve was placed in a shallow trayfilled with tap water. Within the following 24 h thenematodes actively moved through the cotton-wool into the tray. This nematode suspension waspoured through a small 20 lm sieve to separatethe nematodes from the water. The nematodeswere carefully rinsed from the 20 lm sieve into acounting dish. Thereafter the nematodes werecounted alive under an inverse microscope (mag-nification 160·). The counted samples weretransferred into 5 ml polypropylene-tubes. For thetaxonomical identification of the nematodes, thesamples were heated up to 60 �C and conserved informaldehyde (4%). Until identification the sam-ples were stored in a refrigerator at 4–6 �C. Themicroscopic identification was performed 3–6 months after sampling. Per sample a totalnumber of 100 nematodes was determined. Thetaxonomic composition and abundance of these100 individuals were used to extrapolate on thetotal number of nematodes in the respectivesample.

The following measurement endpoints wereused for the evaluation of the effects of carben-dazim on nematodes in the TME ring-tests and inthe field-validation studies: abundance (number ofindividuals per cm3), number of families, trophicstructure, MI and plant parasite index (PPI). Theidentification of nematodes was conducted eitheron the family or genus level. The identification andtaxonomical classification was performed by usingthe key of Bongers (1988). Classification of thenematodes into trophic groups was done accord-ing to Yeates et al. (1993). In total the followingtrophical groups were distinguished:• Bacterivores (BAC) use Procaryota as a foodsource. It is possible that some members of thisgroup with a broader oral cavity are able toconsume other food sources as well.

• Fungivores (FUN) include taxa which piercefungal hyphae either by using a tylenchidal stylet(Stomatostyl) or a dorylaimidal spear (Odonto-styl).

• Omnivores (OMN) are a few representatives ofthe Dorylimida, which are able to pierce fungalhyphae or animal nutrition by using a spear.

• Predators (PRE) either pierce and suck theirprey by using a spear or swallow it by using abig mouth cavity.

• Phytophages feed on higher plants by using astylet or a spear. This group is divided in realplant parasites (PP) and epidermal and root hairfeeders (WEP).

The MI (Bongers 1990) uses the assemblage ofnematode communities as an instrument to assessthe condition of soil or sediment ecosystems.Based on their ability to colonise new habitats,nematode families are classified on a colonizer–persister (c–p) scale ranging from 1 to 5. Nema-tode families comprising species that rapidlyincrease in number in early stages of successionwere considered as colonisers and receive a low c–pvalue. They have similar characteristics as r-stra-tegists. The persisters among the nematodes arecomparable with K-strategists and they generallylive in habitats with a long durational stability.With increasing c–p values the egg size, the ge-neration time and the sensitivity against distur-bances of the habitat increase, while the size of theuterus, the number of eggs and the reproductionrate decreases. Nematodes with a low c–p valueoften greatly fluctuate in population density, are

64 Moser et al.

viviparous or have the ability to produce perma-nent larvae. In contrast, nematodes with a highc–p value have a lower colonisation ability thatmeans a lower ability to colonise new, oftenephemeric habitats than those with a lower c–pvalue.

Phytophagous nematodes differ in their reactionto soil pollutions from non-parasitic nematodes(Bongers, 1990; Yeates, 1994; Bongers et al.,1997). Therefore, they were excluded from thecalculation of the MI and were combined to cal-culate a PPI. The MI and the PPI were calculatedas the weighted mean of the single c–p values:

MI or PPI ¼Xn

i¼1

mðiÞ � f ðiÞ;

where v(i) is the c–p value assigned to taxon i andf(i) is the abundance of the taxon i.

Statistical evaluation

NOEC/LOEC-values were determined for thenematode overall abundance, the number of fam-ilies, the MI and the PPI as well as for the relativeabundance of the different trophic groups. EC50-values were determined for the relative abundanceof the omnivores only. All indices (MI and PPI)were transformed by log 10(x + 3/8) prior tostatistical testing. The data for the trophic groupswere transformed by arcsin(

pðxþ 3=8Þ= ð100þ3=4Þ). NOEC/LOEC-values were determinedfrom single replicate values. Firstly, the datawere tested for homogeneity by applying Coch-ran’s test. In case of variance homogeneity,NOEC/LOEC-values were determined by analysisof variance (ANOVA) followed by a Dunnett’s t-test (1-sided; p £ 0.05). In case of inhomogeneity,NOEC/LOEC-values were determined using aBonferroni-U-test according to Holm (1-sided;p £ 0.05). The tests with the data for the PPI, thePP and the root hair and epidermal feeders (WEP)were performed 2-sided. The EC50-value was cal-culated applying a logistic model according toHaanstra et al. (1985) using the treatment meanvalues. To compare the nematode overall abun-dance, the number of families, the MI and the PPIas well as the relative abundance of the differ-ent trophic groups in the TME ring-test with those

in the respective field-validation study a U-testaccording to Mann–Whitney (2-sided, p £ 0.05)was used (Norusis, 1998; Sachs; 1999; Sparks,2000).

Results

Nematode abundance and number of families

In the TME ring-test the mean number of nema-todes (Fig. 1) in the controls was highest in Ban-gor (204 ± 98.2 ind/cm3) followed by Florsheim(64.9 ± 30.2 ind/cm3) and Amsterdam (38.6 ±22.6 ind/cm3). In Coimbra nematode abundancewas very low (4.3 ± 1.3 ind/cm3). In the field-validation study the mean number of nematodesin the controls was in the same order of magni-tude as in the TMEs in Amsterdam. In Bangorand Florsheim fields, nematode abundancereached only half the TME level, whereas inCoimbra it was twice as high as in the respectiveTMEs.

In the TME ring-test the number of familiesdetermined at the different sites did not differmarkedly (Table 2). In the control TMEs of Am-sterdam, Bangor, Coimbra, Florsheim, 20, 15, 14and 16 nematode families were determined, re-spectively (Fig. 2). In the field-validation study,the number of families in the controls was com-parable to that in the respective TMEs in Am-sterdam (18) and Bangor (16) but higher inCoimbra (19) and Florsheim (22).

No consistent effects of carbendazim on theabundance of nematodes were found at the dif-ferent sites (Fig. 1). The abundance slightly de-creased at the two highest treatment levels (T5 andT6) in the TME ring-test at Amsterdam andFlorsheim and was not affected in Bangor andCoimbra. In the field-validation studies also nodose–response relationship was found for thenumber of nematodes; in Bangor the abundanceincreased with increasing treatment levels underfield conditions. Effects at the different treatmentlevels were not statistically significant except forthe field-validation study of Bangor. In general, ahigh variability was found between the replicatesamples. This was observed for all partners in theTME ring-test and the field-validation study,which reduces the probability of determining

TME – Effects of Carbendazim on Nematodes 65

significant differences by the statistical evaluationof the data.

In contrast to nematode abundance, a treatmentdependent decrease in the number of nematodefamilies was determined in the TME ring-test at allsites (Fig. 2). Significant reduction in the numberof families occurred in Amsterdam (T4–T6), Co-imbra (T2, T3, T5 and T6) and Florsheim (T6).Similar effects were observed in all respective field-validation studies with statistically significant ef-fects at the highest treatment levels in Amsterdam(T5 and T6), Bangor (T6) and Florsheim (T6).

Trophical structure

The trophical structure of the nematode commu-nity in the control TMEs was dominated by PPand bacterivores (BAC) in Amsterdam and Ban-gor (Fig. 3a), whereas in Coimbra and Florsheim(Fig. 3b) root hair and epidermal feeders (WEP) aswell as omnivores (OMN) were more abundant.At all sites the number of fungivores (FUN) and,in particular, the number of predators (PRE) were

lowest. In the field-validation study the trophicalstructure of the nematode community in the con-trol field plots was comparable to that in the re-spective TMEs.

Carbendazim treatment changed the trophicalstructure of the nematode communities in theTMEs and the field plots at all investigated sites(Fig. 3a and b). The relative abundance of PPincreased with increasing carbendazim concentra-tion at all sites except for the Bangor field-validation study and the Florsheim TME tests.Differences between the controls and the differenttreatment levels concerning the PP were statisti-cally significant in the TME ring-tests of Amster-dam (T6), Bangor (T6) and Coimbra (T3–T6) andin the respective field-validation studies in Am-sterdam (T4, T6) and Coimbra (T4) but not inFlorsheim. Root hair and epidermal feeders(WEP) seemed to be not affected by carbendazimin the TMEs and the field plots at nearly all sites.In Bangor, TMEs revealed an increased relativeabundance for WEP compared to the control atthe treatment levels T3 and T4. The strongest de-crease in the relative abundance was observed for

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Figure 1. Effect of carbendazim on the abundance of nematodes (ind/cm3). Data are given for the TME ring test and the field-

validation study, after 16 weeks, performed in Amsterdam, Bangor, Coimbra and Florsheim. Significant (Dunnett-t-test, 1-sided;

p £ 0.05) differences compared to the control are indicated by an asterisk. Treatment levels: T0 ¼ Control, T1 ¼ 0.36, T2 ¼ 1.08,

T3 ¼ 3.24, T4 ¼ 9.72, T5 ¼ 29.16, T6 ¼ 87.48 kg a.i./ha.

66 Moser et al.

the fungivores and, in particular, for the omni-vores in the TME ring-tests as well as in the field-validation studies at all sites. The effects on therelative abundance of the omnivores were statis-tically significant in the TMEs of Amsterdam (T3–T6) and Coimbra (T3, T5-T6). In the respective

field-validation studies, statistically significantdifferences were determined for Amsterdam (T2–T6), Bangor (T3–T6) and Florsheim (T5–T6). Thenumber of predators was low in general andtherefore, statistical evaluation of the data wasnot performed. However, when predators were

Table 2. Nematode families determined in all samples of the TME ring tests (T) and the respective field-validation studies (F) in

Amsterdam, Bangor, Coimbra and Florsheim

Amsterdam Bangor Coimbra Florsheim

Bacterial feeding

Alaimidae T/F T/F T/F T/F

Bastianidae T/F F

Bunonematidae T/F F

Cephalobidae T/F T/F T/F T/F

Diplogasteridae T/F

Monhysteridae F

Panagrolaimidae F F T/F

Plectidae T/F T/F T/F T/F

Prismatolaimidae T/F T/F F F

Rhabditidae T/F T/F T/F T/F

Teratocephalidae T/F T/F T/F

Hyphal feeding

Anguinidae F

Aphelenchidae T T/F T/F

Aphelenchoididae T/F T/F T/F T/F

Diphterophoridae T/F T/F T/F T/F

Leptonchidae T/F T/F T

Omnivores

Aporcelaimidae T/F T/F T/F T/F

Belondiridae T F T

Dorylaimidae T/F

Nordiidae T T

Qudsianematidae T/F T/F T/F

Thornematidae T/F T/F T/F

Plant feeding

Criconematidae F F F

Dolichodoridae T/F T/F T/F T/F

Hemicycliophoridae F

Hoplolaimidae T/F T/F T/F T/F

Longidoridae T T/F T/F

Paratylenchidae T/F T/F T/F

Pratylenchidae T/F T/F T/F

Trichodoridae T/F

Predation

Anatonchidae F

Mononchidae T T T/F T

Tripylidae F T F F

Epidermal and root hair

feeders

Tylenchidae T/F T/F T/F T/F

Total 24/22 17/16 18/24 22/25

TME – Effects of Carbendazim on Nematodes 67

observed in the controls they disappeared in thehigher treatment levels.

Maturity index

The MI (Fig. 4) in the control TMEs and in thecontrol field plots were similar in Amsterdam (2.94versus 2.78) and Bangor (2.21 versus 2.15),whereas a higher MI value was calculated for thefield in Coimbra (3.02 versus 3.78) and a signifi-cantly higher MI value for the Florsheim field(2.66 versus 3.34). The PPI did not differ betweenthe control TMEs and the respective field-valida-tion study at all sites except for Bangor where thevalue determined for the TMEs was higher com-pared to the one for the field.

The MI decreased with increasing treatmentlevels in the TME ring-tests as well as in the re-spective field-validation studies at all sites. Thiseffect was statistically significant for the threehighest treatment levels in the TMEs of Amster-dam. In the field-validation studies significantdifferences in the different treatments versus thecontrol were observed in Amsterdam (T5), Bangor

(T3 and T4), Coimbra (T4 and T5) and Florsheim(T6). The replicate values showed a high variabil-ity and a clear dose–response relationship washardly observed. The PPI was not influenced bythe model chemical neither in the TMEs nor in therespective field-validation studies at all sites.

NOEC and EC50-values

Due to the high variability of the data it was onlypossible in a few cases to detect significant diffe-rences between the different treatment levels andthe control when applying parametric or non-parametric statistical tests although dose-relatedeffects on the measurement endpoints were obvi-ous. Therefore, in many cases the NOEC wasequal or higher than the highest treatment level(Table 3). The calculation of EC50-values was alsoinfluenced by the high variability of the data whichoften did not allow to establish a clear dose–re-sponse relationship for most of the parameters.The sole exception was the relative abundance ofthe omnivorous nematodes. The EC50-values cal-

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Figure 2. Effect of carbendazim on the number of nematode families (total number of families in all replicates of each treatment). Data

are given for the TME ring test and the field-validation study, after 16 weeks, performed in Amsterdam, Bangor, Coimbra and

Florsheim. Significant (Dunnett-t-test, 1-sided; p £ 0.05) differences compared to the control are indicated by an asterisk. Treatment

levels: see Figure 1.

68 Moser et al.

(A)

(B)

Figure 3. Effect of carbendazim on the relative distribution pattern of trophic groups of the nematodes (% of overall abundance).

Data are given for the TME ring test and the field-validation study, after 16 weeks, performed in Amsterdam and Bangor (3a) and in

Coimbra and Florsheim (3b). ¼ bacterivores, h ¼ fungivores, j ¼ omnivores, ¼ plant parasites, ¼ predators, ¼ root hair

and epidermal feeders. Significant (Dunnett-t-test, 1-sided; p £ 0.05) differences compared to the control are indicated by an asterisk.

Treatment levels: see Figure 1.

TME – Effects of Carbendazim on Nematodes 69

culated for the relative abundance of the omni-vorous nematodes ranged from 0.9 to 7.2 kg

carbendazim/ha and were in four of six cases lowerthan the respective NOECs (Table 4).

Figure 4. Effect of carbendazim on the maturity index of nematodes. Data are given for the TME ring test and the field-validation

study, after 16 weeks, performed in Amsterdam, Bangor, Coimbra and Florsheim. Significant (Dunnett-t-test, l-sided;

p £ 0.05) differences compared to the control are indicated by an asterisk. Treatment levels: see Figure 1.

Table 3. NOEC-values (in kg carbendazim/ha) determined for all measurement endpoints investigated for the nematodes in the TME

ring tests and field-validation studies performed in Amsterdam (A), Bangor (B), Coimbra (C) and Florsheim (F)

TME Field

A B C F A B C F

Abundance ‡87.5 ‡87.5 ‡87.5 ‡87.5 ‡87.5 29.2# ‡87.5 ‡87.5No. of

families

3.24 ‡87.5 9.72 29.2 9.72 29.2 ‡87.5 29.2

MI 3.24 ‡87.5 ‡87.5 ‡87.5 * * * 29.2

PPI ‡87.5 ‡87.5 1.08+ ‡87.5 ‡87.5 ‡87.5 ‡87.5 ‡87.5Bacterivores ‡87.5 29.2 * 29.2 29.2 ‡87.5 ‡87.5 ‡87.5Fungivores 29.2 ‡87.5 ‡87.5 ‡87.5 ‡87.5 ‡87.5 ‡87.5 ‡87.5Omnivores 1.08 ‡87.5 9.72 ‡87.5 0.36 1.08 ‡87.5 9.72

Plant

Parasites

29.2+ 29.2+ 1.08+ ‡87.5 29.2+ ‡87.5 * ‡87.5

Predators ‡87.5 ‡87.5 * ‡87.5 ‡87.5 * ‡87.5 ‡87.5Epidermal

and root hair

feeders

‡87.5 * ‡87.5 ‡87.5 ‡87.5 ‡87.5 ‡87.5 ‡87.5

* = not applicable; # = increase.

70 Moser et al.

Discussion

Controls

The abundances and the number of families ofnematodes determined for the grasslands in Am-sterdam, Bangor and Florsheim as well as for thearable land in Coimbra correspond to the valuesfound for these types of fields in the literature(Sohlenius, 1980). Differences between the controlsof the TME ring-test and the controls of the field-validation study were low with regard to thenematode abundance and the number of nematodefamilies. The variability of the data is high, but it iswell known from other field studies (Wasileweska,1974; Rombke and Dreher, 1999) and must be seenas a consequence of a heterogeneous distributionof nematodes in soils. This heterogeneous distri-bution can be explained by the formation ofclusters, which depend on several factors like soilmoisture, soil texture, temperature, location offood, intra- or interspecific attraction or repellen-cy, and competition (Twinn, 1974; Ettema, 1998).A more extended sampling scheme proposed byFreckman (1982), which might include pooling ofsamples and the investigation of sample aliquots,could reduce the variability between samples.

The trophical structure of the nematode popu-lation in the control TMEs did not differ from thatfound in the control field plots. Similar to findingsfrom Freckman and Caswell (1985), the trophicalstructure was dominated by the PP and the bac-terivores whereas the numbers of fungivores and,in particular, of predators were low.

The maturity indices determined for the controlsin this study were always above 2. Surprisingly thehighest MI values were calculated for the agricul-tural (disturbed) field site in Coimbra. According

to Bongers and Ferris (1999) MI values below2 indicate eutrophication and disturbance whilevalues close to 4 can be expected in undisturbedand stable soils.

Effects of the model chemical carbendazim

Carbendazim strongly adsorbs to soil organicmaterial and remains in the soil for up to 3 years(World Health Organization, 1993). Effects ofcarbendazim and related compounds on nema-todes have been reported by Hoestra (1976),Friedmann and Platzer (1980) and Jaffee andMcInnis (1990). The sensitivity of nematodes to-wards carbendazim is confirmed by the results ofthe present study.

According to Bongers (1990) and Korthals et al.(1996) both eutrophication and soil contaminationcan induce a reduction of the nematode MI. In thecase of eutrophication the number of individualsof taxa with low c–p values is increased, whereas inthe case of soil contamination the number of in-dividuals, in particular of taxa with high c–p val-ues, as well as the number of taxa is decreased. Inthis study the MI was reduced by carbendazim atall study sites. In Amsterdam and Florsheim, thedecrease of the MI was correlated with a reductionof the overall nematode abundance. According toBongers (1990) this is an indication for the impactof a chemical stressor on the MI. In the TME ring-tests in Bangor and Coimbra as well as in the field-validation studies at all sites the reduction of theMI was accompanied by a reduction of the totalnumber of nematode taxa. Even in the field plotsin Bangor, where the overall abundance of nema-todes increased by carbendazim treatment, thenumber of nematode taxa was reduced. The ob-served decrease in the number of nematode taxa

Table 4. EC50-values (in kg carbendazim/ha) and the 95% confidence limits (CL) calculated for the effect on omnivorous nematodes in

the TME ring tests and field-validation studies in Amsterdam (A), Bangor (B), Coimbra (C) and Florsheim (F)

A B C F

EC50 Lower–upper

95% CL

EC50 Lower–upper

95% CL

EC50 Lower–upper

95% CL

EC50 Lower–upper

95% CL

TME ring

test

0.93 0.5–1.9 * * * * 5.42 1.0–28.1

Field 2.33 0.04–131.9 2.41 0.2–23.6 6.79 0.2–206.0 7.24 7.2–18.1

* = not applicable.

TME – Effects of Carbendazim on Nematodes 71

was mainly caused by a reduction in the number ofbacterivorous, fungivorous and omnivorous fam-ilies. These trophic groups are known to be mostsensitive for environmental stressors (Korthalset al., 1996; Parmelee et al., 1997) and, in partic-ular, the omnivores are considered as indicatorsof ecosystem disturbances (Thomas, 1978). Thereduction of the relative abundance of the om-nivorous nematodes was strongly correlated with adecrease of the MI at all study sites. Even fluctu-ations in the response of this trophic group atdifferent treatment levels were reflected by the MI.In general it seems that the model chemical re-duced the number of nematodes among most taxa;in particular, the taxa with high c–p values wereaffected. The effects on the MI as well as on the‘indicator’ groups (omnivores) and the unchangedPPI reveal that the nematode coenosis was dis-turbed by the chemical stressor rather than byeutrophication.

The comparison of the NOEC- and EC50-valuesderived from the TME ring-test and the field-vali-dation study indicate that the reproducibility (i.e.the variation between the partners) was reason-able, although different soils from different siteswere used. Furthermore, the NOEC- and EC50-values determined in the TME ring-tests appear tobe comparable to the values derived from the re-spective field-validation studies. The NOEC- andEC50-values calculated for the effect of carbenda-zim on the relative abundance of omnivores weretransformed from kg carbendazim/ha to mgcarbendazim/kg by a conversion factor of 1.33according to EPPO (2001). The converted NOEC-values calculated for the TMEs and the field-vali-dation studies ranged from 0.48 to 12.93 mgcarbendazim/kg (NOECs equal or higher than thehighest treatment level were excluded). EC50-val-ues calculated for the relative abundance of om-nivores of all TME and field-validation studiesranged between 1.24 and 9.63 mg carbendazim/kg. The range of effects measured for omnivorousnematodes is similar to EC50-values determinedfor the abundance of the enchytraeid genus Fri-dericia in the same TME and field soils, whichranged between 0.9 and 24.7 mg carbendazim/kg(Moser et al., 2003, this issue). In a laboratoryring-test with the enchytraeid Enchytraeus albidusEC50-values for the measurement endpoint repro-duction of 0.20–2.36 mg carbendazim/kg were

calculated (Rombke and Moser, 2002). Further-more, LC50-values for E. albidus of 5.5 and 7 mgcarbendazim/kg were reported by Rombke (1989)and Federschmidt (1994) respectively.

In summary, at all study sites investigated andindependent from the soil characteristics, carben-dazim altered the relative abundance of the om-nivorous nematodes. It is recommended to use thisgroup as indicator for effects of chemical stressorson the nematode community. The similarity of theresponses between the TME ring-tests and thefield-validation studies supports the assumptionthat TMEs represents the field situation for nem-atodes realistically.

Acknowledgements

This research was financially supported by theEuropean Union (Project No. ENV4-CT97-0470).The support by Ralf Lenz who performed anematode training course at ECT and confirmedthe nematode identifications is gratefully ac-knowledged. Hans-Joachim Schallnass providedexcellent advice to solve statistical problems.

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