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Ring-Testing and Field-Validation of a Terrestrial Model Ecosystem (TME) - An Instrument for Testing Potentially Harmful Substances: Effects of Carbendazim on

Article in Ecotoxicology · February 2004 DOI: 10.1023/B:ECTX.0000012408.58017.08 · Source: PubMed

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Ring-Testing and Field-Validation of a Terrestrial Model Ecosystem (TME) – An Instrument for Testing Potentially Harmful Substances: Effects of Carbendazim on Earthworms

JO¨ RG RO¨ MBKE,1,* CORNELIS A.M. VAN GESTEL,2 SUSAN E. JONES,3 JOSE´ E E. KOOLHAAS,2 JOSE´ M.L. RODRIGUES4 AND THOMAS MOSER1 1ECT Oekotoxikologie GmbH, Bo¨ttgerstr. 2-14, D-65439 Flo¨rsheim am Main, Germany 2Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands 3University of Wales, School of Agricultural and Forestry Sciences, Bangor, Gwynedd LL57 2UW, Wales, UK 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 earthworms () was determined in Terrestrial Model Ecosystem (TME) tests and field-validation studies. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) taken from a grassland or, in one case, from an arable site. The TMEs were taken from the same site where the respective field-validation study was performed. The tests were performed in Amsterdam (The Netherlands), Bangor (Wales, UK), Coimbra (Portugal) and Flo¨ rsheim (Germany). The sites selected had an coenosis representative of the different land use types and regions. In addition, the differences between the coenosis found in the TMEs and the respective field sites were in general low. A high variability was found between the replicate samples, which reduces the probability of determining significant differences by the statistical evaluation of the data. Similar effects of the chemical treatment were observed on abundance as well as on biomass. Effects were most pronounced 16 weeks after application of the test chemical. The observed effects on earthworm abundance and biomass did not differ between the TME tests and the respective field-validation studies. Effects on earthworm diversity were difficult to assess since the number of individuals per species was low in general. However, the genus Lumbricus and in particular L. terrestris and L. rubellus seemed to be more affected by the chemical treatment than others. NOEC and EC50-values derived from the TME pre-test, the TME ring-test and the field-validation study indicate that the TMEs of the different partners delivered comparable results although different soils were used. Due to the high variability NOECs could often not be determined. The EC50-values for the effect of carbendazim on earthworm abundance ranged between 2.04 and 48.8 kg a.i./ha (2.71–65.2 mg/kg soil) and on earthworm biomass from 1.02 to 34.6 kg a.i./ha (1.36–46.0 mg/kg soil). These results indicate that the abundance and biomass of earth- worms are suitable endpoints in ecotoxicological studies with TMEs.

Keywords: carbendazim; Lumbricidae; earthworms; soil mesocosms; community effects

*To whom correspondence should be addressed: Tel.: +49-6145-9564-20; Fax: +49-6145-9564-99; E-mail: [email protected] 106 Ro¨mbke et al.

Introduction throughout Europe (Cuppen et al., 2000; Framp- ton and Wratten, 2000). This chemical was also Risk assessment of chemicals is usually based on chosen as a reference substance in the earthworm the results of single species toxicity tests, which are chronic reproduction test (ISO 1998; OECD 2001), performed in the laboratory (Van Leeuwen and since it is known for its high toxicity to earth- Hermens, 1995). It is, however, realised that such worms. single species tests may not be sufficient to predict effects on the level of ecosystem structure and functioning (Cairns, 1984). In the past, such effects Materials and methods have been studied in the field but the results are difficult to assess and the efforts are high. There- Experimental set-up fore, mesocosm tests have been advocated, which may close the gap between laboratory and field Three types of tests were performed: in the first studies. For the soil environment, this has resulted year a TME pre-test, in the second year, based on in the development of several types of model eco- the experience gained during the pre-test, the TME systems (Morgan and Knacker, 1994; Sheppard, ring-test and in parallel to the ring-test a field- 1997; Edwards et al., 1998; Knacker et al., 2004). validation study. The TME-project was conducted Such model ecosystems may be applied in a higher at four sites throughout Europe by the following tier of risk assessment, when results of single spe- project partners: ECT Oekotoxikologie GmbH, cies laboratory toxicity tests provide reasons for Flo¨ rsheim, Germany (1), Vrije Universiteit Am- concern with regard to the potential risk of a sterdam (Institute of Ecological Science, The chemical in the soil environment (see Weyers et al., Netherlands) (2), University of Wales, Bangor 2004). (School of Agricultural and Forestry Sciences, In this paper, results on earthworm abundance, UK) (3) and Universidade de Coimbra (Instituto biomass and diversity gained by tests using Ter- Ambiente e Vida, Portugal) (4). In Coimbra, TME restrial Model Ecosystems (TMEs) are described. tests and the field-validation study were performed TMEs are defined as controlled, reproducible at an arable site, whereas the respective work at systems that attempt to simulate the processes and the three other sites was done on a grassland. The interactions of components in a portion of the properties of the soils from these four sites were terrestrial environment (Gillett and Witt, 1980; measured using ISO guidelines (ISO 1993; ISO Sheppard, 1997). These tests were performed 1994; ISO 1995; Table 1). The TME tests started within the framework of the TME-project spon- with the extraction of the TME soil cores. TMEs sored by the European Union (Contract No.: were taken by means of a soil core extractor, ENV4-CT97-0470). The aim of this project was containing a HDPE tube (diameter 17.5 cm; the improvement and validation of the TME test length 40 cm), which served as a soil core encase- system, first described by Van Voris et al. (1985) ment. Grass was cut just before TME extraction. and used in studies reported by Knacker et al. The TMEs were either placed in temperature- (1989), Frederickson et al. (1991) and Chekai et al. controlled carts in a climatic chamber (Amster- (1993). For that purpose, the TME tests were dam, Flo¨ rsheim, Coimbra) or in a greenhouse performed at four different European sites with (Bangor). TMEs were irrigated up to three times different soils, but using similar equipment, test per week using artificial rainwater slightly modi- chemical, test design, and endpoints. The tests fied according to Velthorst (1993). The model were designed in a way which should allow a chemical carbendazim was applied after an accli- comparison of NOEC- and EC50-values for several matisation period of two to four weeks. endpoints. In order to investigate the ecological For the field-validation study, 30 field plots, realism of this laboratory TME test system, a field- each 25 m2, were marked out by each partner at validation study was performed. Carbendazim was the same site where the TMEs were extracted. Six chosen as the model chemical for the TME tests plots served as controls, and 24 plots were treated and the field-validation study. Carbendazim is a with the model chemical (four plots for each of the fungicide that is used on a large scale in agriculture six treatment levels; the plots were completely TME – Effects of Carbendazim on Earthworms 107

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 = organic matter

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

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 Flo¨ rsheim D Silty clay loam 36.5 5.2 5.3–5.9 Meadow

randomised). Before spraying the model chemical tailed description of the set-up of the TME- the grass cover was cut on the grassland sites project, see Knacker et al. (2004). (Amsterdam, Bangor, Flo¨ rsheim) or the soil was ploughed at the arable site (Coimbra). Earthworm sampling Carbendazim was applied to the TMEs and field plots as Derosal, containing 360 g carbendazim/l. In the TME pre-test, earthworm samples were In the TME pre-tests, carbendazim was applied at taken 1, 8 and 16 weeks after application. After dosages of 0 (T0), 0.36 (T1), 2.16 (T2), 13.0 (T3) taking the samples for all other endpoints, the re- and 77.8 (T4) kg a.i./ha. In the TME ring-test and maining soil of the TME cores was sorted by hand the field-validation study at dosages of 0 (T0), 0.36 for earthworms. At each sampling point six un- (T1), 1.08 (T2), 3.24 (T3), 9.72 (T4), 29.2 (T5) and treated TMEs (control) and three TMEs for each 87.5 (T6) kg a.i./ha. In the laboratory, carbenda- treatment level were sampled. In the TME ring- zim (dissolved in 50 ml of water per soil core test, samples were taken 1, 8 and 16 weeks after surface (=222 cm2)) was applied using a pipette. application of the test chemical. After 1 week, only The TMEs were irrigated immediately after treat- the control, the lowest (T1) and the highest (T6) ment. In the field-validation study, carbendazim treatment were sampled. Six control TMEs and was applied by a plot sprayer, commonly used in four TMEs per treatment level were used in the agricultural practice, using a 3 l spray solution (6 l TME ring-test. In the field, sampling was done for highest dosage) per plot (@1200 l/ha). Imme- only at the end of the test; i.e. after 16 weeks. In diately after spraying, a volume of 30 l of water the field a combination of methods was used to was sprayed onto the plot to wash off the Derosal achieve a complete assessment of the earthworm from the plants onto the soil. The control plots biocoenosis (Dunger and Fiedler 1997): firstly, a were also sprayed with 30 l of water. square of 50 50 cm (0.25 m2) soil was dug out up Various measurement endpoints were chosen to to a depth of 15 cm. This excavated topsoil layer determine the fate and effect of the model chemical was sorted by hand. Additionally, 10 l of a 0.5% as well as the structure and function of the ter- formaldehyde solution was sprinkled in the same restrial compartment. The fate endpoints included plot from which the topsoil was removed to extract the measurement of residues of the model chemical the remaining worms from the soil. Earthworms in the upper soil layer (0–5 cm), in the soil layer were col1ected in 70% ethanol, then fixed in 4% from 5 to 15 cm, and in the leachate. The effect formaldehyde for 1–2 weeks. Afterwards, they endpoints were classified into functional endpoints were finally preserved in 70% ethanol. Shortly af- (nutrients in leachate and soil which describe as- ter fixation the adult worms were counted and pects of nutrient cycling; soil enzyme activity; mi- determined to the species level while juveniles were crobial substrate induced respiration; bacterial identified at the genus level (Graff 1953; Støp- growth; feeding activity of soil organisms; organic Bowitz 1969; Bouche´ 1972; Sims and Gerard matter decomposition) and structural endpoints 1985). Site-specific keys for three sites (Amster- (abundance as well as diversity and community dam, Bangor and Flo¨ rsheim) were developed by structure of microarthropods, nematodes, enchyt- Partner (1). Directly after determination the bio- raeids, lumbricids and plant biomass. For a de- mass was measured as wet mass and converted by 108 Ro¨mbke et al. a factor of ten to dry mass. All values are given per test with those in the respective field-validation square meter (i.e. the conversion factor between study a U-test according to Mann–Whitney (2- individuals per TME soil core and per square sided, p £ 0.05) was used (Sparks, 2000; Sachs, meter was 41.5). 1999; Norusis, 1998).

Statistical evaluation Results NOEC/LOEC and EC50-values were determined for earthworm abundance and biomass. NOEC/ Earthworm abundance and biomass: controls LOEC-values were determined from single repli- cate values. Firstly, the data were tested for homo- In the TME pre-test (Fig. 1) the mean number geneity by applying Cochran’s test. In case of (sd, n ¼ 4–6) of earthworms in the controls was variance homogeneity, NOEC/LOEC-values were 218 ± 179 ind/m2 in Amsterdam, 382 ± 123 ind/ determined by analysis of variance (ANOVA) m2 in Bangor and 374 ± 243 ind/m2 in Flo¨ rsheim followed by a Dunnett’s t-test (l-sided; p £ 0.05). after 1 week. In Coimbra no earthworm sampling In case of inhomogeneity, NOEC/LOEC-values took place. After 8 weeks, the mean number of were determined using a Bonferroni-U-test ac- earthworms decreased in Amsterdam and increased cording to Holm (l-sided; p £ 0.05). The EC50- slightly in Bangor and Flo¨ rsheim. Towards the end values were calculated applying a logistic model of the TME pre-test (after 16 weeks) an increase in according to Haanstra et al. (1985) using the the number of earthworms was observed in Am- treatment mean values. To compare the earth- sterdam whereas in Bangor and Flo¨ rsheim it re- worm abundance and biomass in the TME ring- turned to the level observed after 1 weeks.

Amsterdam Bangor Coimbra Flörsheim 800 800 800 800 Pre-test Pre-test Pre-test Pre-test

600 600 600 600

400 400 400 400

200 200 200 200

0 0 0 0 +1 +8 +16 +1 +8 +16 +1 +8 +16 +1 +8 +16 800 800 800 800 Ring-test Ring-test Ring-test Ring-test

600 600 600 600

400 400 400 400

200 200 200 200

0 0 0 0

Abundance [ind/m²] +1 +8 +16 +1 +8 +16 +1 +8 +16 +1 +8 +16 800 800 800 800 Field Field Field Field

600 600 600 600

400 400 400 400

200 200 200 200

0 0 0 0 +1 +8 +16 +1 +8 +16 +1 +8 +16 +1 +8 +16 Sampling Point

Figure 1. Abundance of earthworms in control TMEs and field plot, 1, 8 and 16 after application. Data are given for the TME pre-test, TME ring test and field-validation study at Amsterdam, Bangor, Coimbra and Flo¨ rsheim. TME – Effects of Carbendazim on Earthworms 109

The earthworm biomass in the TME pre-test Earthworm abundance and biomass: effects of the was lowest in Amsterdam (4.2 ± 3.3 g dw/m2), model chemical carbendazim followed by Bangor with 7.7 ± 2.8 g dw/m2 and highest in Flo¨ rsheim (18.44 ± 9.37 g dw/m2). The One week after carbendazim treatment, slight to change in biomass with time showed a similar moderate (up to 50%) reductions in earthworm pattern as the abundance. numbers were seen at the three highest (Flo¨ rsheim In the TME ring-test after 1 week, the mean TME pre-test), two highest (Amsterdam TME pre- number of earthworms in the controls was in the test) or highest treatment level only (Amsterdam same order of magnitude for all sites (between TME ring-test, Bangor TME pre-test and TME 280 ± 58.8 and 334 ± 68.4 ind/m2) except for ring-test). These effects never differed significantly Coimbra, where the mean number of earthworms from the control. In Coimbra no earthworms were was low (72.2 ± 60.5 ind/m2) (Fig. 1). With time, collected in the TME pre-test. In the TME ring- the number of earthworms at all sites remained test only a very low number of earthworms was more constant than in the TME pre-test. Earth- found. The data of the TME ring-test after 1 week worm biomass was slightly higher in Amsterdam were not evaluated statistically since only T1 and (13.1 ± 7.7 g dw/m2) than in Bangor (8.9 ± 1.8 g T6 were sampled. dw/m2), but it was highest for the Flo¨ rsheim Eight weeks after carbendazim treatment, mean TMEs (18.6 ± 9.6 g dw/m2). In the Coimbra earthworm numbers in the Amsterdam TME pre- TME ring-test the earthworm biomass was low test were significantly reduced compared to the (2.2 ± 1.8 g dw/m2) corresponding to the ob- control at the two highest treatment levels (Fig. 2). served low earthworm numbers. The fluctuation of In the TME ring-test, earthworm numbers were earthworm biomass with time was low and similar reduced at several treatment levels, but this re- to the pattern observed for the number of earth- duction was only statistically significant at the worms (data not shown). highest treatment level. In the Bangor TME pre- In the field-validation study, the mean number of test and TME ring-test, earthworm abundance earthworms found in the control plots in Amster- decreased at the two or three highest treatment dam, Bangor and Flo¨ rsheim after 16 weeks did not levels, but effects found were not statistically sig- differ markedly from those in the respective TMEs nificant in comparison to the controls. In Coim- in the ring-test at the same sampling point (Fig. 1). bra, a reduction in earthworm numbers was found In Coimbra the mean number of earthworms was at all treatment levels in the TME ring-test, but nearly twice as high in the field than in the TMEs. due to the low number of earthworms, a statistical The differences between earthworm numbers evaluation of the data was not applicable. In the found in the field-validation study and in the Flo¨ rsheim TME pre-test, earthworm numbers respective TMEs at all sites were not statistically showed a dose-related reduction, which was sta- significant. In general, a high variability was tistically significant at the highest treatment level. found between the replicate samples. That was In the TME ring-test, earthworm numbers were observed for all partners at all sampling points in reduced at several treatment levels, but effects were the TME pre-test and the TME ring-test. In the not statistically significant in comparison to the field-validation study, variability was still high but controls. in all cases lower than in the respective TME ring- The mean number of earthworms was signifi- tests. cantly reduced at the two highest treatment levels In the field-validation study control earthworm in the Amsterdam and Flo¨ rsheim pre-tests biomass did not differ from that in the TMEs in 16 weeks after treatment with carbendazim. Amsterdam, whereas for Bangor and Coimbra a (Fig. 3). In the Bangor TME pre-test, a similar significantly higher biomass was found in the field trend was seen, but due to the high variability compared to the TMEs. In Flo¨ rsheim earthworm between the replicate samples and the absence of biomass in the control field plots was more than earthworms at the highest treatment level, statis- 50% below that in the TMEs. This difference was tical evaluation of the data was not applicable. In not statistically significant, probably due to the the TME ring-test, a dose-related decrease high variability between the samples. in earthworm numbers was found, which was 110 Ro¨mbke et al.

Amsterdam Bangor Coimbra Flörsheim 800 800 800 800 Pre-test Pre-test Pre-test Pre-test +8 +8 +8 +8 600 600 600 600

400 400 400 400

*

200 200 200 200

* * 0 0 0 0 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4

800 800 800 800 Ring-test Ring-test Ring-test Ring-test +8 +8 +8 +8 600 600 600 600 Abundance [ind./m²]

400 400 400 400

200 * 200 200 200

0 0 0 0 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 Treatment Level

Figure 2. Effect of carbendazim on the abundance of earthworms (ind/m2). Data are given for the TME pre-test and the TME ring test, after 8 weeks, performed in Amsterdam, Bangor, Coimbra and Flo¨ rsheim. Significant differences compared to the control are indicated by an asterisk. Treatment levels: TME pre-test: T0 ¼ Control, T1 ¼ 0.36, T2 ¼ 2.16, T3 ¼ 12.96, T4 ¼ 77.76 kg a.i./ha; TME ring test: 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. statistically significant at the three highest treat- effects on abundance in particular in the ring-test ment levels in Amsterdam (at the highest treat- and the field-validation study. ment level no earthworms were found) and Flo¨ rsheim, but not in Bangor. In the Coimbra TME ring-test, no effects on earthworm numbers NOEC and EC50-values were seen. In the field-validation studies a dose- related decrease in earthworm numbers was found, Due to the high variability between the replicate which was statistically significant at the three samples of each treatment level and the control, it highest treatment levels in Amsterdam, at the two was not possible in many cases to detect significant highest treatment levels in Bangor and at the differences between the different treatment levels highest treatment level only in Flo¨ rsheim (Fig. 3). and the control by parametric or non-parametric In the field-validation study of Coimbra, earth- tests although an effect on earthworm abundance worm numbers were reduced at the four highest or biomass seems to be obvious. Therefore, very treatment levels, but effects were not statistically often the NOEC was greater or equal to the significant in comparison to the controls. highest treatment level (Tables 2 and 4). Despite Effects of carbendazim on earthworms biomass the high variability, the calculation of EC50-values showed a similar pattern as on the earthworm was possible, but the 95% confidence intervals abundance (Fig. 4). This was observed at all were sometimes high (Tables 3 and 5). The NOE- sampling points in the TME pre-test, the TME Cs for the effect of carbendazim on earthworm ring-test and the field-validation study in Amster- abundance ranged from 2.16 to ‡87.5 kg a.i./ha dam, Bangor and Coimbra. In Flo¨ rsheim effects and for earthworm biomass from 1.08 to on biomass seemed to be more pronounced than ‡87.5 kg a.i./ha. The EC50-values ranged from TME – Effects of Carbendazim on Earthworms 111

Amsterdam Bangor Coimbra Flörsheim 800 800 800 800 Pre-test Pre-test Pre-test Pre-test +16 600 +16 600 +16 600 +16 600

400 400 400 400 * 200 200 200 200 * * * 0 0 0 0 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4 800 800 800 800 Ring-test Ring-test Ring-test Ring-test 600 +16 600 +16 600 +16 600 +16

400 400 400 400 * 200 * 200 200 200 * * * * 0 0 0 0 Abundance [ind/m²] T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 800 800 800 800 Field Field Field Field +16 600+16 600 +16 600 600 +16

* 400 * 400 400 400 * * * 200 * 200 200 200 * 0 0 0 0 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 Treatment Level

Figure 3. Effect of carbendazim on the abundance of earthworms (ind/m2). Data are given for the TME pre-test and the TME ring test and the field-validation study, after 16 weeks, performed in Amsterdam, Bangor, Coimbra and Flo¨ rsheim. Significant differences compared to the control are indicated by an asterisk. Treatment levels: see Fig. 2.

2.04 to 49.0 (earthworm abundance) and from 1.02 Amsterdam (4 species). The most common species to 34.6 kg a.i./ha (earthworm biomass). Interest- caliginosa was found at all sites. The ingly enough the EC50-values for the TME pre-test Amsterdam earthworm community was domina- and TME ring-test are often below the respective ted by A. caliginosa and Lumbricus rubellus. NOECs, whereas the EC50-values for the field- L. castaneus and Allolobophora chlorotica were validation study (with a lower variability com- only found in one or two TME or field samples. In pared to the TME tests) were higher than the Bangor three Aporrectodea species (A. caliginosa, NOECs. A comparison of the EC50-values indi- A. longa and A. rosea) were most abundant. In cated that the variation between the results of the Flo¨ rsheim earthworm community was dominated different partners was low. Additionally, the EC50- by juvenile Aporrectodea individuals (probably values derived from the TME tests are comparable mainly A. caliginosa) A. antipae, Octolasion sp. and to those derived from the field-validation study Lumbricus terrestris, which occurred only in the although those from the TME tests seemed to be soils of Bangor and Flo¨ rsheim. Murchieona mi- somewhat lower. nuscula was only found in Bangor and Aporrecto- dea antipae only in Flo¨ rsheim. The earthworm community of Coimbra differed clearly from the Earthworm diversity: controls and chemical other investigated sites because 3 of the 7 species treatments found in Coimbra were determined only for this partner. One of these 3 species did occur only once The earthworm biocoenosis (Table 6) was most in the samples. Probably it is Allolobophora fer- divers in Flo¨ rsheim and Coimbra with 7 species nandae (Graff, 1957), but the taxonomic status of determined, followed by Bangor (6 species) and this rare species is not clear (Zicsi, 1976). 112 Ro¨mbke et al.

Amsterdam Bangor Coimbra Flörsheim 30 30 30 30 Pre-test Pre-test Pre-test Pre-test 25 25 25 25 +16 +16 +16 +16 20 20 20 20 15 15 15 15 10 10 10 10 5 5 5 5 * * * * 0 0 0 0 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4 T0 T1 T2 T3 T4 30 30 30 30 Ring-test Ring-test Ring-test Ring-test 25 25 25 25 +16 +16 +16 +16 20 20 20 20 15 15 15 15

10 * 10 10 10 * 5 5 5 5 * * * * * 0 0 0 0 Biomass [g dw/m²] T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 30 30 30 30 Field Field Field Field 25 25 25 25 +16 +16 +16 +16 20 20 20 20 15 15 15 15 * 10 * * 10 10 * 10 5 * 5 5 5 * * * 0 0 0 0 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 T0 T1 T2 T3 T4 T5 T6 Treatment Level

Figure 4. Effect of carbendazim on the biomass of earthworms (g dw/m2). Data are given for the TME pre-test, the TME ring test and the field-validation study, after 16 weeks, performed in Amsterdam, Bangor, Coimbra and Flo¨ rsheim. Significant (Dunnett-t-test, l- sided; p £ 0.05) differences compared to the control are indicated by an asterisk. Treatment levels: see Fig. 2.

Table 2. NOEC-values (in kg a.i./ha) for the effect of bricus was more affected by the chemical treatment carbendazim on earthworm abundance in the TME pre-test, than other genera. In particular in Flo¨ rsheim, and the TME ring test and the field-validation studie´ d, 8 and 16 weeks after application of the model chemical in Amsterdam to a lesser extend in Bangor the number of (A), Bangor (B), Coimbra (C) and Flo¨ rsheim (F) L. terrestris decreased clearly with the treatment, while the number of individuals belonging to the NOEC genus Aporrectodea remained constant. A similar Weeks A B C F observation was made in Amsterdam, where the species L. rubellus was affected more than the two TME pre-test þ8 2.16 ‡77.8 – 13.0 Aporrectodea species. TME ring test 29.2 29.2 a ‡87.5 TME pre-test þ16 2.16 a – 2.16 TME ring test 3.24 ‡87.5 a 3.24 Field 3.24 9.72 ‡87.5 29.2 Discussion – = No sampling. a Not applicable. Controls

The abundance and biomass of the earthworms Despite the fact that effects of carbendazim on determined for the grasslands in Amsterdam, the number of earthworm species were visible, the Bangor and Flo¨ rsheim, as well as for the arable absolute number of specimen belonging to one land in Coimbra, corresponded to the values found particular species was too low (except for Flo¨ rs- for similar field types referred to in the literature heim) to use this parameter as a measurement (Satchell, 1983; Ro¨ mbke et al., 1997). Fluctuations endpoint. However, it seems that the genus Lum- between the sampling points and differences be- TME – Effects of Carbendazim on Earthworms 113

Table 3. EC50-values (in kg a.i./ha) and the 95% confidence limits (CL) calculated for the effect of carbendazim on earthworm abundance in the TME pre-test, the TME ring test and the field-validation study, 8 and 16 weeks after application of the model chemical at Amsterdam (A), Bangor (B), Coimbra (C) and Flo¨ rsheim (F)

EC50 lower–upper 95% CL

Time, week A B C F

TME pre-test þ8 10.9 a 2.04 0.02–230 – – 4.01 0.2–83.8 TME ring test 12.9 1.4–117 6.02 0.5–67.7 bb26.5 0.8–858 TME pre-test þ16 2.80 0.2–42.7 2.35 1.2–4.6 – – 7.41 1.5–37.7 TME ring test 3.00 1.5–5.9 3.91 1.1 bb4.33 1.3–14.4 Field 11.5 0.4–324 48.8 13.3–179 bb49.0 30.1–79.6

– = no sampling. a )3 3 b Lower CL < 10 · EC50 and upper CL > 10 · EC50; Not applicable.

Table 4. NOEC (in kg a.i./ha) for the effect of carbendazim on earthworm biomass in the TME pre-test, the TME ring test and the field-validation study, 8 and 16 weeks after application of the model chemical in Amsterdam (A), Bangor (B), Coimbra (C) and Flo¨ rsheim (F)

NOEC

Week A B C F

TME pre-test þ8 ‡77.8 ‡77.8 – 13.0 TME ring test ‡87.5 ‡87.5 a 9.72 TME pre-test þ16 13.0 2.16 – 13.0 TME ring test 3.24 ‡87.5 a 1.08 Field 3.24 9.72 ‡87.5 3.24

– = No sampling. a Not applicable.

Table 5. EC50-values (in kg a.i./ha) and the 95% confidence limits (CL) calculated for the effect of carbendazim on earthworm biomass in the TME pre-test, the TME ring test and the field-validation study, 8 and 16 weeks after application of the model chemical in Amsterdam (A), Bangor (B), Coimbra (C) and Flo¨ rsheim (F)

Time, week EC50 lower–upper 95% CL

ABCF

TME pre-test þ8 aa aa ––aa TME ring test 3.44 b 7.83 0.3–230.0 aa3.16 b TME pre-test þ16 3.29 0.6–18.3 2.16 b – – 1.02 b TME ring test 4.42 1.8–10.6 8.57 baa1.07 0.7–1.6 Field 8.69 0.02–4842 34.6 4.8–248.4 9.84 b 9.13 b

– = No sampling. a )3 3 b Lower CL < 10 · EC50 and upper CL > 10 · EC50; Not applicable. tween the TME pre-test, TME ring-test and field- Soil moisture content and the amount of food validation study were within the ranges expected available are the most important factors influen- from publications cited above. The variability be- cing distribution patterns. In addition, Satchell tween replicate control samples is well known from (1955) assumed that aggregations might occur many field studies since earthworms are not evenly when earthworms are reproducing more rapidly distributed in soil (Edwards and Bohlen, 1996). than the offspring can disperse from the breeding 114 Ro¨mbke et al.

Table 6. Earthworm species found in the TME pre-test, the TME ring test and the field-validation study at Amsterdam, Bangor, Coimbra and Flo¨ rsheim

Amsterdam Bangor Coimbra Flo¨ rsheim

Allolobophora chlorotica Aporrectodea caliginosa Allolobophora chlorotica Aporrectodea caliginosa Aporrectodea caliginosa Aporrectodea longa Allolobophora fernandaea Aporrectodea rosea Lumbricus castaneus Aporrectodea rosea Aporrectodea caliginosa Aporrectodea antipae Lumbricus rubellus Lumbricus rubellus Aporrectodea rosea Lumbricus castaneus Lumbricus terrestris Criodrilus lacuum Lumbricus terrestris Murchieona minuscula Eiseniella tetraeda Octolasion cyaneum Octolasion lacteum Octolasion lacteum a Taxonomical revision necessary. site. The inhomogeneity of the earthworm distri- laboratory studies it is known to be highly toxic to bution in the field seems to have been realistically earthworms (Adema et al., 1985; Vonk et al. 1986, reflected by the TMEs. The large sampling area of Van Gestel 1992). Acute effects (mainly mortality) each field sample (0.25 m2) compared to the small have been found at soil concentrations between 0.9 TMEs (approx. 0.024 m2) can explain the generally and 5.7 mg carbendazim/kg, using different lower variability obtained for worm numbers and earthworm species, test durations and test sub- biomass in the field. strates. Chronic effects occurred at concentrations The low earthworm biomass in the TMEs of between 0.6 and 1.9 mg carbendazim/kg. The Bangor compared to the earthworm biomass in the toxicity of carbendazim to earthworms is con- field, can be explained by the absence of the species firmed by the results of the presented work. At all Lumbricus terrestris in the TMEs. As L. terrestris sites, independent of soil properties, effects on is the biggest middle European earthworm species, earthworms have been observed. These effects the presence of a single specimen can contribute seemed to be most pronounced 16 weeks after considerably to the overall biomass, in particular if application of the model chemical (Fig. 3). At this the total number of earthworms is low. In Coim- sampling point a clear dose–response relationship bra the field was ploughed before extracting the was observed in the TME pre-test, the TME ring- soil cores. Therefore, the initial earthworm popu- test and the field-validation study at Amsterdam, lation was already disturbed when the experiment Bangor and Flo¨ rsheim. started. As a consequence the number of earth- Effects on single species could not be statistically worms found was very low, making the interpre- evaluated since the absolute numbers were too low tation of the Coimbra data difficult. Compared to at the different sites and sampling points. How- the field the biomass of earthworms was high in ever, at least in Flo¨ rsheim L. terrestris and in the TMEs of Flo¨ rsheim. This was probably due to Amsterdam L. rubellus seemed to be more affected a long period of drought in the field before sam- than e.g. A. caliginosa. This was also reported by pling. Drought causes migration of L. terrestris to other authors (Federschmidt, 1994; Lofs-Holmin, deep soil layers. From there L. terrestris could ei- 1981). One reason for the more sensitive reaction ther not be extracted; because the formaldehyde of L. terrestris and L. rubellus might be the feeding solution did not penetrate deep enough into the behaviour of these species, which feed on the soil soil, or the earthworms were in a state of quies- surface where carbendazim is adsorbed. In addi- cence and thus not reacting to formaldehyde tion, referring to the results of laboratory studies (Edwards and Bohlen, 1996). with carbendazim (Federschmidt, 1994) and benomyl (Heimbach, 1988), the LC50 values for Effects of carbendazim L. terrestris are lower in comparison to those for other species such as Eisenia fetida. Carbendazim strongly adsorbs to soil organic The NOEC and EC50-values derived from the material and remains in the soil for up to 3 years TME pre-test, TME ring-test and field-validation (World Health Organization, 1993). From various study (Tables 2–5) indicate that the TME tests of TME – Effects of Carbendazim on Earthworms 115 the different partners delivered comparable results, centration level in the uppermost soil layer for although different soils from different sites were about 1 year. used. Additionally, the NOEC and EC50-values In summary, effects of carbendazim on the derived from the TME tests are comparable to earthworms on European meadow and crop sites those derived from the field-validation study. Since were found starting at estimated soil concentra- it was often not possible to determine NOEC-val- tions of approximately 0.5 mg a.i./kg. Indepen- ues due to the high variability of the data, only the dently from the site and soil characteristics as well EC50-values will be discussed in detail here (Landis as the respective earthworm biocoenosis, direct et al., 1997). However, when calculating the EC50- and indirect effects were found in narrow concen- values, this variability results in high 95% confi- tration ranges (for earthworm biomass mainly dence intervals. The EC50-values were transformed between 1 and 15 mg carbendazim/kg). The com- from kg carbendazim/ha to mg carbendazim/kg by parison of the EC50-values indicated that the re- a conversion factor of 1.33 according to EPPO producibility was higher than, for example, in the (2001). The converted EC50-values for earthworm laboratory ring-test of the acute earthworm test abundance appear to range from 2.71 to 65.2 mg (Edwards, 1984). carbendazim/kg and for earthworm biomass from It is recommended that earthworm abundance 1.36 to 46.2 mg carbendazim/kg for all TME tests and biomass can be used as the main endpoints. and the field-validation study. The lower range of However, if large species, especially L. terrestris, the biomass EC50-values can be explained by the occur at a specific site, biomass is preferred, since it reaction of L. terrestris, which is more affected than is more sensitive than the abundance of earth- other species (Federschmidt, 1994). This difference worms. No distinct differences between the three between the two measurement endpoints was only investigation levels (laboratory, TME and field) observed in Bangor and Flo¨ rsheim (Figs. 3 and 4). were found. Thus, theoretically the investigation of The range of effect concentrations is in agreement one level would have been sufficient for the model with data from the literature; for example, LC50- chemical carbendazim, but the ecological relevance values for E. fetida or E. andrei of 5.7 and 9.3 mg of the results gained in laboratory tests could only carbendazim/kg (Vonk et al., 1986, Van Gestel be proven by performing TME or field tests. et al., 1992) or an EC50-value of 2.9 mg carben- dazim/kg for E. andrei (cocoon production) (Van Earthworm diversity Gestel et al., 1992) have been reported. Federsch- midt (1994) determined LC50-values for L. terres- The species composition in the Bangor and Flo¨ rs- tris of 0.9 mg carbendazim/kg after 14 days and heim sites is as expected (Sims and Gerard, 1985); 2.6 mg carbendazim/kg after 28 days. only the relatively rarely found species M. minus- Carbendazim applied in various field studies has cula is exceptional for Bangor. Probably this worm caused effects on earthworms independently from is more widely distributed than documented in the site use and soil characteristics at concentrations literature because it is difficult to identify due to ranging between 0.4 and 1.6 mg carbendazim/kg external taxonomic characteristics, which are often (Van Gestel, 1992). Under field conditions species not well developed. With only four species (and in specific increases or decreases in the number of fact only two dominant species), the site near earthworms can be induced by carbendazim (Lofs- Amsterdam showed the lowest diversity of all sites Holmin 1981), which may result in effects on litter investigated. Compared to the other sites, the soil decomposition (Cook and Swait, 1975; Keogh and of Amsterdam had the highest content of sand and Whitehead, 1975). On a grassland near Frankfurt/ the lowest water holding capacity, which might be Main, Germany, which showed similar soil char- the reason for the low number of earthworm spe- acteristics as the field site of Flo¨ rsheim, the number cies. Maybe the former use of the site as an agri- of earthworms decreased and litter decomposition cultural field is also partly responsible for this was delayed at concentrations of 0.48 and 4.8 mg result. The diversity at the site of Coimbra is diffi- carbendazim/kg (Knacker et al., 1994). In the cult to assess since similar sites have rarely been latter study carbendazim was sprayed six times studied in Portugal (Trigo et al., 1988). Eiseniella bimonthly, to achieve an almost constant con- tetraeda and Criodrilus lacuum, found at the 116 Ro¨mbke et al.

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