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Effects of ivermectin in dung pats on earthworm (Megascolecidae) populations and pat degradation in Japanese grassland
Applied Soil Ecology 31 (2006) 280–285 www.elsevier.com/locate/apsoil
Effects of ivermectin in dung pats on earthworm (Megascolecidae) populations and pat degradation in Japanese grassland S. Kaneda a, N. Yamashita a,*, T. Uchida a, S. Shimano a, N. Miyoshi a, M. Sasaki b, Y. Enami c a
Department of Upland Farming, National Agricultural Research Center for Tohoku Region, Arai, Fukushima-city 960-2156, Japan b Fukushima University, Kanayagawa, Fukushima-city 960-1293, Japan c Shiga Prefecture Agricultural Reserch Center, Agricultural Experiment Station Dainaka 516 Azuchi-cho, Gamou-gunn, Shiga 521-1301, Japan Received 18 January 2005; received in revised form 27 April 2005; accepted 3 May 2005
Abstract The effects of residual ivermectin in dung pats on earthworm activity and dung decomposition in Japanese grassland, where Megascolecidae are the dominant group of dung decomposers, were studied. Artificial cowpats containing 0, 0.1, and 1 mg ivermectin kg1 dung were prepared and deposited on grassland in October 2003. Pats were collected again for analysis 1, 3, 5, and 7 weeks after deposition. Earthworms were collected from the soil around pats at a depth of 0–10 cm. The Megascolecidae, Pheretima (Amynthas) heteropoda and Pheretima (Amynthas) divergens, together accounted for more than 90 and 99% of earthworm individuals and biomass, respectively. Earthworms aggregated around the pats regardless of the ivermectin treatment. Dung-degradation rate was also unaffected by the ivermectin treatments. Dung decomposition appeared to be due mainly to earthworm activity, as dung beetles were rare at this site. These results suggest that ivermectin may have no adverse effects on Megascolecidae activity and on the degradation of cowpats in pastureland sites where earthworms dominate the dung decomposer community. # 2005 Elsevier B.V. All rights reserved. Keywords: Anthelmintic; Dung pats; Megascolecidae; Pat degradation; Ivermectin; Earthworms
1. Introduction Ivermectin efficiently controls parasitic gastrointestinal and pulmonary nematodes, mites, lice, warble flies and ticks at a low dosage (Campbell et al., 1983). * Corresponding author. Tel.: +81 24 593 6173; fax: +81 24 593 2155. E-mail address: [email protected] (N. Yamashita).
The drug is also a potent insecticide against dungbreeding parasitic insects (Strong, 1992). An outstanding feature of the drug is its persistent activity. Most of the ivermectin is ultimately excreted in the feces of livestock as a mixture of the original drug and its metabolites. Thus, the use of ivermectin as an anthelmintic in free-range livestock might have negative effects on non-target invertebrates, such as dung beetles and earthworms, which feed on dung and
0929-1393/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2005.05.001
S. Kaneda et al. / Applied Soil Ecology 31 (2006) 280–285
contribute to pat degradation in grassland ecosystems (Strong, 1992; Herd, 1995). A delay in dung decomposition may lead to fouling of pastureland and affect nutrient cycling. Earthworms are important dung decomposers in pastureland (Holter, 1979; Hendriksen, 1991). Several studies conducted in Europe focused on effects of the residues and metabolites of ivermectin on earthworm populations around dung pats in the field. Earthworms were equally abundant in control and treated dung (Wall and Strong, 1987; Madsen et al., 1990; Sommer et al., 1992; Wratten et al., 1993; Svendsen et al., 2003). Other studies were concerned with ivermectin’s toxicity under controlled laboratory conditions; earthworm fecundity and mortality were investigated at different concentrations of ivermectin in dung provided as food (Madsen et al., 1988; Svendsen et al., 2002) and in the soil (Halley et al., 1989; Gunn and Sadd, 1994). Ivermectin appeared toxic at high concentrations in the artificial soils but showed no adverse effects on earthworm growth and survival at the low levels typically observed on pastures (Madsen et al., 1988; Svendsen et al., 2002). However, effects of ivermectin on earthworms and on dung decomposition have not been studied until now in Japan, where the Megascolecidae are the dominant family of earthworms. The effects of ivermectin on earthworm populations, therefore, may differ between Japan and Europe where lumbricids dominate earthworm communities. As ivermectin usage has become more widespread recently in Japan due to its simple application procedures, we investigated the effects of this drug on dung decomposition and on the populations of earthworms in the field.
2. Materials and methods 2.1. General Experiments were carried out between October 15 and December 03, 2003, in a grassland area located in Fukushima-city (378430 N and 1408230 E), Japan. The soil was an Umbric Andosol, according to the FAO classification. Soil temperature was measured at a depth of 5 cm at the experimental site. Precipitation was monitored near the experimental field. The weather data collected during the experimental period are shown in Fig. 1.
281
Fig. 1. Soil temperature (continuous line) measured at 5 cm depth and daily precipitation (bars) at the experimental field. Precipitation data for November 7–12 not available.
2.2. Experimental design Freshly voided dung was collected from cattle (Japanese shorthorn) feeding mainly on orchard grass, and stored at 30 8C until use. Samples containing 0, 0.1, or 1 mg ivermectin kg1 dung were prepared using acetone as a solvent. A concentration of 0.1 mg kg1 wet weight corresponds to the average level detected in the feces of Japanese shorthorns at 1, 3, and 7 days after application of the recommended ivermectin dose of 0.05 mg kg1 bw (Yamashita et al., 2004). The maximum concentration in the excrements of cattle treated with this dose did not exceed 1 mg kg1 wet weight (Yamashita et al., 2004). A dung-free control treatment was included out to determine whether earthworms gathered around dung. Artificial pats of 700 g wet weight and ca. 15 cm diameter were deposited in the test site on October 15. The grass was cut to a height of about 5 cm, immediately before placement of the dung. Spots for deposition including empty spaces for the dungfree control were spaced evenly at 1.5 m intervals in an ordered, alternating sequence across the field. Four replicates of each dung type including the dung-free control were sampled 1, 3, 5, and 7 weeks after deposition. Each treatment comprised 16 pats. 2.3. Measurements Dung which had remained moist during the experimental period was collected easily by hand because pats
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S. Kaneda et al. / Applied Soil Ecology 31 (2006) 280–285
Fig. 2. Time courses of ivermectin concentrations per unit wet (a) and dry weight (b) of dung and soil, respectively. Ivermectin had been added to the dung at 0.1 mg kg1 wet weight at the start of the experiment. (*) Dung; (~) soil from 0 to 5 cm depth; and (!) soil from 5 to 10 cm depth. Values shown are means S.E.; n = 3. Dry-weight data for soil at time 0 are missing.
in order to normalize the counts and to stabilize variances. All statistical analyses were performed using SPSS II for Windows, SPSS Inc., Chicago, USA.
had not been disturbed by birds or small invertebrates which were rare at the location. The pats collected were dried in an oven at 105 8C and weighed to calculate the decomposition rate. Earthworms were collected by digging and hand-sorting from a 25 cm 25 cm underneath each pat to a depth of 10 cm. Animals were fixed in 10% formalin, identified according to Easton (1981) and Ishizuka (2001), and weighed. The concentrations of ivermectin in dung and in the soil underneath dung pats at depths of 0–5 cm and 5–10 cm were determined by a high-performance liquid chromatographic analysis of fluorescent derivatives prepared by reaction of the isolates with methylimidazole and trifluoroacetic acid (Payne et al., 1995).
3. Results During the period of observation, the ivermectin concentration in artificial pats prepared with 0.1 mg kg1 of the drug was relatively stable around 0.1 and 0.5 mg kg1 on a wet weight and dry weight basis, respectively (Fig. 2). The concentration in the soil remained below 0.02 mg kg1 with regard to wet as well as dry weight during the experimental period. In this field, Megascolecidae accounted for more than 90% of the individuals and 99% of the biomass of earthworms. Pheretima (Amynthas) heteropoda (Goto and Hatai, 1898) and Pheretima (Amynthas) divergens (Michaelsen, 1892) were the dominant species. Earthworms were attracted to the dung; their density became significantly higher in dung treatments than in dung-free controls after 3 weeks, and remained at increased levels until the end of the experiment ( p < 0.05; Table 1). The biomass of earthworms underneath the pats tended to increase with time
2.4. Statistical analysis To analyze the effects of ivermectin on earthworm number and biomass, and the decomposition rates of pats, the GLM procedure was used with ivermectin treatment as a main factor and time as a covariate. Differences in earthworm number between dung without ivermectin and dung-free controls were tested using the Mann–Whitney U-test. The earthworm number were transformed log 10 prior to the analysis
Table 1 Number of earthworms per litre of dung-free and ivermectin-free dung plots, determined in soil (25 cm 25 cm) at a depth of 0–10 cm Weeks after start of experiment
Dung-free (n = 4; S.E.) Dung (n = 4; S.E.)
1
3
5
7
2.28 0.51 4.56 0.86
1.48 0.50* 3.68 0.21
1.04 0.10* 4.80 0.82
1.72 0.57* 5.08 1.54
* Indicate statistically significant differences between treatments at p < 0.05 (Mann–Whitney U-test).
S. Kaneda et al. / Applied Soil Ecology 31 (2006) 280–285
283
Fig. 3. Earthworm density around dung pats containing ivermectin. Number of individuals (a) and total earthworm biomass (b) per litre of soil determined underneath pats containing (*) 0 mg kg1, (~) 0.1 mg kg1, and (&) 1 mg kg1 (wet weight) ivermectin initially. Earthworms were collected by hand from 25 cm 25 cm at a depth of 0–10 cm. No earthworms were found within the dung. Values are means S.E.; n = 4.
( p < 0.05; Fig. 3). There was no significant difference between ivermectin treatments in the number and the biomass of earthworms ( p > 0.05; Fig. 3). The decomposition rate of the artificial pats seemed constant until the end of observation in week 7, when the pats had lost almost half of their weight (Fig. 4). There was no significant difference in the decomposition rates between the treatments (P > 0.05).
4. Discussion Generally, the decomposition of dung depends to a great extent on the dung fauna population. The fauna is
Fig. 4. Weight loss of artificial dung pats deposited in the field at time 0. Pats contained (*) 0 mg kg1, (~) 0.1 mg kg1, and (&) 1 mg kg1 (wet weight) ivermectin initially. Values shown are means S.E.; n = 4. Data for the 0 mg kg1 treatment at 1 week after setting pats are missing.
affected by weather conditions (Herd, 1995) and might also respond to ivermectin concentrations in the dung. However, ivermectin treatments did not affect earthworm accumulation around dung pats and pat decomposition in our experiments. The weather conditions at the experimental site deviated slightly from the longterm mean. According to the weather station of the Meteorological Agency of Japan (http://www.data.kishou.go.jp/index.htm), the mean temperature and precipitation in Fukushima-city (37845.50 N and 140828.20 E) were somewhat higher in 2004 than usual. In this location, the Megascolecidae including the particularly abundant species P. (A.) heteropoda and P. (A.) divergens, dominated the earthworm fauna. According to the classification of Uchida (2004) and Ishizuka (2001), P. (A.). heteropoda is a polyhumicendogeic species. P. (A.). divergens might be included in the same group on the basis of its microhabitat preference and of its internal and external morphology (Ishizuka, 2001). Ten worms collected 3 weeks after deposition of the artificial pats were dissected to examine their gut contents. The intestines of individuals taken from sites of pat deposition contained numerous particles of organic matter, irrespective of the drug treatment. Whereas contents of animals from dung-free control locations included mainly mineral particles and only small amounts of organic matter. Uchida and Yamashita (unpublished data) observed P. (A.). heteropoda feeding on dung. In this study, earthworms accumulated around pats, and it appears likely that they came into contact with ivermectin by feeding on dung.
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The concentration of ivermectin in the dung decreased only slightly during the course of our experiments, which is in line with previous reports (Sommer et al., 1992; Sommer and Steffansen, 1993). Ivermectin levels in feces depend on the microbial degradation of the drug and on leaching. Sommer et al. (1992) and Sommer and Steffansen (1993) found that ivermectin concentrations did not decrease in pats under field conditions during 45 days after deposition. Halley et al. (1989) studied degradation half-lives of ivermectin under various conditions. In mixtures of feces and soil kept at room temperature in the dark, half-life periods varied between 93 and 240 days, while under outdoor conditions, they were 7–14 days in summer and 91–217 days in winter. On the other hand, the half-life of ivermectin was reduced to 3 h when thin films of it were exposed to direct summer sunlight. Soil-column leaching experiments revealed that ivermectin bound tightly to soils and organic matter, while its metabolites leached out (Halley et al., 1989). As the results of Halley et al. (1989) were obtained with mixtures of feces and soil, they appear applicable to ivermectin decomposition in earthworm casts rather than dung pats deposited on the soil surface. Although ivermectin appears susceptible to photodegradation, this process did not seem to occur in the present study. We conclude that ivermectin might be taken up by dung feeding organisms until the complete disappearance of the dung pat. Our results accord with previous studies reporting the absence of adverse effects of ivermectin on earthworm activity (Wall and Strong, 1987; Madsen et al., 1990; Sommer et al., 1992; Wratten et al., 1993; Svendsen et al., 2003). Svendsen et al. (2003) investigated the long term effects of ivermectin and fenbendazole on earthworm populations and dung pat decomposition in two grazing seasons in Denmark. Ivermectin excreted by heifers to which it had been administered as sustained release boluses had no negative impact on earthworm population size, biomass, and species composition. In the present study, we have shown that these results from European earthworm communities in which the Lumbricidae are predominant also hold for the Megascolecidae-dominated Japanese soils. From observations of earthworm abundance at their experimental site, Svendsen et al. (2003) concluded that ivermectin did not affect dung decomposition. In the present study, earthworms also
were abundant and accumulated around the dung pats during the entire period of observation regardless of ivermectin treatments. The small population size of dung beetles at our site also points to an important role of earthworms in dung decomposition. Moreover, the slightly higher than average precipitation recorded during our field study probably promoted earthworm activity which is known to be restricted by dry conditions (Holter, 1979; Madsen et al., 1990). We conclude that ivermectin has no adverse effects on Megascolecidae activity, and does not affect cowpat decomposition in pastureland sites where earthworms dominate in the dung decomposer community. In this study, ivermectin was added to artificial dung pats which did not contain metabolites that may occur in the dung of ivermectin-treated cattle. At present, it cannot be ruled out that ivermectin metabolites could influence earthworm behaviour. Further studies into the effects of ivermectin and its metabolites on earthworms using voided dung from ivermectin-treated cattle will help to further elucidate potential impacts of ivermectin on Megascolecidae populations in pasturelands. Acknowledgements We thank Mr. Tanji, Y. and Mr. Izawa, N., National Agricultural Research Center for Tohoku Region, for assistance in the field work. We also thank Dr. Ito, M.T. and Mr. Sakai, H., Yokohama National University, for identification of earthworms. We acknowledge the members of Lab Soil Management, National Agricultural Research Center for Tohoku Region for helpful discussion. We thank the anonymous referees for their useful comments on the paper. This study was supported by Budgets for Environmental Research allocated to National Research Institutes. S.K. is currently supported by JSPS Research Fellowships for Young Scientists. References Campbell, W.C., Fisher, M.H., Stapley, E.O., Albers-Scho¨nberg, G., Jacob, T.A., 1983. Ivermectin: a potent new antiparasitic agent. Science 221, 823–828. Easton, E.G., 1981. Japanese earthworms: a synopsis of the Megadrile species (Oligochaeta). Bull. Br. Museum (Natural history) Zool. Ser. 40, 33–65.
S. Kaneda et al. / Applied Soil Ecology 31 (2006) 280–285 Gunn, A., Sadd, J.W., 1994. The effect of ivermectin on the survival, behaviour and cocoon production of the earthworm Eisenia fetida. Pedobiologia 38, 327–333. Halley, B.A., Jacob, T.A., Lu, A.Y.H., 1989. The environmental impact of the use of ivermectin: environmental effects and fate. Chemosphere 18, 1543–1563. Herd, R.P., 1995. Endectocidal drugs: ecological risks and countermeasures. Int. J. Parasitol. 25, 875–885. Hendriksen, N.B., 1991. The effects of earthworms on the disappearance of particles from cattle dung pats during decay. Pedobiologia 35, 139–146. Holter, P., 1979. Effect of dung-beetles (Aphodius spp.) and earthworms on the disappearance of cattle dung. Oikos 32, 393–402. Ishizuka, K., 2001. Taxonomic study of the genus Pheretima s. lat. (Oligochaeta Megascolecidae) from Japan. Bull. Seikei Univ. 3 (33), 1–125 (in Japanese). Madsen, M., Grønvold, J., Nansen, P., Holter, P., 1988. Effect of treatment of cattle with some anthelmintics on the subsequent degradation of their dung. Acta Vet. Scand. 29, 515–517. Madsen, M., Overgaard Nielsen, B., Holter, P., Pedersen, O.C., Brøchner Jespersen, J., Vagn Jensen, K.-M., Nansen, P., Grønvold, J., 1990. Treating cattle with ivermectin: effects on the fauna and decomposition of dung pats. J. Appl. Ecol. 27, 1–15. Payne, L.D., Hicks, M.B., Wehner, T.A., 1995. Determination of abamectin and/or ivermectin in cattle feces at low parts per billion levels using HPLC with fluorescence detection. J. Agric. Food Chem. 43, 1233–1237. Sommer, C., Steffansen, B., 1993. Changes with time after treatment in the concentrations of ivermectin in fresh cow dung and in cow pats aged in the field. Vet. Parasitol. 48, 67–73.
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Sommer, C., Steffansen, B., Overgaard Nielsen, B., Grønvold, J., Vagn Jensen, K.-M., Brøchner Jespersen, J., Springborg, J., Nansen, P., 1992. Ivermectin excreted in cattle dung after subcutaneous injection or pour-on treatment: concentrations and impact on dung fauna. Bull. Entomol. Res. 82, 257– 264. Strong, L., 1992. Avermectins: a review of their impact on insects of cattle dung. Bull. Entomol. Res. 82, 265–274. Svendsen, T.S., Sommer, C., Holter, P., Grønvold, J., 2002. Survival and growth of Lumbricus terrestris (Lumbricidae) fed on dung from cattle given sustained-release boluses of ivermectin or fenbendazole. Eur. J. Soil Biol. 38, 389– 392. Svendsen, T.S., Grønvold, J., Holter, P., Sommer, C., 2003. Field effects of ivermectin and fenbendazole on earthworm populations and the disappearance of dung pats from bolus-treated cattle. Appl. Soil Ecol. 24, 207–218. Uchida, T., 2004. Feeding strategies on earthworms (Megascolecidae, Oligochaeta) in Japan. Ph.D. Thesis. Yokohama National University. Wall, R., Strong, L., 1987. Environmental consequences of treating cattle with the antiparasitic drug ivermectin. Nature 327, 418– 421. Wratten, S.D., Mead-Briggs, M., Gettinby, G., Ericsson, G., Baggott, D.C., 1993. An evaluation of the potential effects of ivermectin on the decomposition of cattle dung pats. Vet. Record 133, 365–371. Yamashita, N., Yoshida, N., Watanabe, A., Mikami, A., 2004. Effects of the anthelmintics on development of the dung degrading insects. Tohoku Agric. Res. 57, 119–120 (in Japanese).
Effects of ivermectin in dung pats on earthworm (Megascolecidae) populations and pat degradation in Japanese grassland S. Kaneda a, N. Yamashita a,*, T. Uchida a, S. Shimano a, N. Miyoshi a, M. Sasaki b, Y. Enami c a
Department of Upland Farming, National Agricultural Research Center for Tohoku Region, Arai, Fukushima-city 960-2156, Japan b Fukushima University, Kanayagawa, Fukushima-city 960-1293, Japan c Shiga Prefecture Agricultural Reserch Center, Agricultural Experiment Station Dainaka 516 Azuchi-cho, Gamou-gunn, Shiga 521-1301, Japan Received 18 January 2005; received in revised form 27 April 2005; accepted 3 May 2005
Abstract The effects of residual ivermectin in dung pats on earthworm activity and dung decomposition in Japanese grassland, where Megascolecidae are the dominant group of dung decomposers, were studied. Artificial cowpats containing 0, 0.1, and 1 mg ivermectin kg1 dung were prepared and deposited on grassland in October 2003. Pats were collected again for analysis 1, 3, 5, and 7 weeks after deposition. Earthworms were collected from the soil around pats at a depth of 0–10 cm. The Megascolecidae, Pheretima (Amynthas) heteropoda and Pheretima (Amynthas) divergens, together accounted for more than 90 and 99% of earthworm individuals and biomass, respectively. Earthworms aggregated around the pats regardless of the ivermectin treatment. Dung-degradation rate was also unaffected by the ivermectin treatments. Dung decomposition appeared to be due mainly to earthworm activity, as dung beetles were rare at this site. These results suggest that ivermectin may have no adverse effects on Megascolecidae activity and on the degradation of cowpats in pastureland sites where earthworms dominate the dung decomposer community. # 2005 Elsevier B.V. All rights reserved. Keywords: Anthelmintic; Dung pats; Megascolecidae; Pat degradation; Ivermectin; Earthworms
1. Introduction Ivermectin efficiently controls parasitic gastrointestinal and pulmonary nematodes, mites, lice, warble flies and ticks at a low dosage (Campbell et al., 1983). * Corresponding author. Tel.: +81 24 593 6173; fax: +81 24 593 2155. E-mail address: [email protected] (N. Yamashita).
The drug is also a potent insecticide against dungbreeding parasitic insects (Strong, 1992). An outstanding feature of the drug is its persistent activity. Most of the ivermectin is ultimately excreted in the feces of livestock as a mixture of the original drug and its metabolites. Thus, the use of ivermectin as an anthelmintic in free-range livestock might have negative effects on non-target invertebrates, such as dung beetles and earthworms, which feed on dung and
0929-1393/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2005.05.001
S. Kaneda et al. / Applied Soil Ecology 31 (2006) 280–285
contribute to pat degradation in grassland ecosystems (Strong, 1992; Herd, 1995). A delay in dung decomposition may lead to fouling of pastureland and affect nutrient cycling. Earthworms are important dung decomposers in pastureland (Holter, 1979; Hendriksen, 1991). Several studies conducted in Europe focused on effects of the residues and metabolites of ivermectin on earthworm populations around dung pats in the field. Earthworms were equally abundant in control and treated dung (Wall and Strong, 1987; Madsen et al., 1990; Sommer et al., 1992; Wratten et al., 1993; Svendsen et al., 2003). Other studies were concerned with ivermectin’s toxicity under controlled laboratory conditions; earthworm fecundity and mortality were investigated at different concentrations of ivermectin in dung provided as food (Madsen et al., 1988; Svendsen et al., 2002) and in the soil (Halley et al., 1989; Gunn and Sadd, 1994). Ivermectin appeared toxic at high concentrations in the artificial soils but showed no adverse effects on earthworm growth and survival at the low levels typically observed on pastures (Madsen et al., 1988; Svendsen et al., 2002). However, effects of ivermectin on earthworms and on dung decomposition have not been studied until now in Japan, where the Megascolecidae are the dominant family of earthworms. The effects of ivermectin on earthworm populations, therefore, may differ between Japan and Europe where lumbricids dominate earthworm communities. As ivermectin usage has become more widespread recently in Japan due to its simple application procedures, we investigated the effects of this drug on dung decomposition and on the populations of earthworms in the field.
2. Materials and methods 2.1. General Experiments were carried out between October 15 and December 03, 2003, in a grassland area located in Fukushima-city (378430 N and 1408230 E), Japan. The soil was an Umbric Andosol, according to the FAO classification. Soil temperature was measured at a depth of 5 cm at the experimental site. Precipitation was monitored near the experimental field. The weather data collected during the experimental period are shown in Fig. 1.
281
Fig. 1. Soil temperature (continuous line) measured at 5 cm depth and daily precipitation (bars) at the experimental field. Precipitation data for November 7–12 not available.
2.2. Experimental design Freshly voided dung was collected from cattle (Japanese shorthorn) feeding mainly on orchard grass, and stored at 30 8C until use. Samples containing 0, 0.1, or 1 mg ivermectin kg1 dung were prepared using acetone as a solvent. A concentration of 0.1 mg kg1 wet weight corresponds to the average level detected in the feces of Japanese shorthorns at 1, 3, and 7 days after application of the recommended ivermectin dose of 0.05 mg kg1 bw (Yamashita et al., 2004). The maximum concentration in the excrements of cattle treated with this dose did not exceed 1 mg kg1 wet weight (Yamashita et al., 2004). A dung-free control treatment was included out to determine whether earthworms gathered around dung. Artificial pats of 700 g wet weight and ca. 15 cm diameter were deposited in the test site on October 15. The grass was cut to a height of about 5 cm, immediately before placement of the dung. Spots for deposition including empty spaces for the dungfree control were spaced evenly at 1.5 m intervals in an ordered, alternating sequence across the field. Four replicates of each dung type including the dung-free control were sampled 1, 3, 5, and 7 weeks after deposition. Each treatment comprised 16 pats. 2.3. Measurements Dung which had remained moist during the experimental period was collected easily by hand because pats
282
S. Kaneda et al. / Applied Soil Ecology 31 (2006) 280–285
Fig. 2. Time courses of ivermectin concentrations per unit wet (a) and dry weight (b) of dung and soil, respectively. Ivermectin had been added to the dung at 0.1 mg kg1 wet weight at the start of the experiment. (*) Dung; (~) soil from 0 to 5 cm depth; and (!) soil from 5 to 10 cm depth. Values shown are means S.E.; n = 3. Dry-weight data for soil at time 0 are missing.
in order to normalize the counts and to stabilize variances. All statistical analyses were performed using SPSS II for Windows, SPSS Inc., Chicago, USA.
had not been disturbed by birds or small invertebrates which were rare at the location. The pats collected were dried in an oven at 105 8C and weighed to calculate the decomposition rate. Earthworms were collected by digging and hand-sorting from a 25 cm 25 cm underneath each pat to a depth of 10 cm. Animals were fixed in 10% formalin, identified according to Easton (1981) and Ishizuka (2001), and weighed. The concentrations of ivermectin in dung and in the soil underneath dung pats at depths of 0–5 cm and 5–10 cm were determined by a high-performance liquid chromatographic analysis of fluorescent derivatives prepared by reaction of the isolates with methylimidazole and trifluoroacetic acid (Payne et al., 1995).
3. Results During the period of observation, the ivermectin concentration in artificial pats prepared with 0.1 mg kg1 of the drug was relatively stable around 0.1 and 0.5 mg kg1 on a wet weight and dry weight basis, respectively (Fig. 2). The concentration in the soil remained below 0.02 mg kg1 with regard to wet as well as dry weight during the experimental period. In this field, Megascolecidae accounted for more than 90% of the individuals and 99% of the biomass of earthworms. Pheretima (Amynthas) heteropoda (Goto and Hatai, 1898) and Pheretima (Amynthas) divergens (Michaelsen, 1892) were the dominant species. Earthworms were attracted to the dung; their density became significantly higher in dung treatments than in dung-free controls after 3 weeks, and remained at increased levels until the end of the experiment ( p < 0.05; Table 1). The biomass of earthworms underneath the pats tended to increase with time
2.4. Statistical analysis To analyze the effects of ivermectin on earthworm number and biomass, and the decomposition rates of pats, the GLM procedure was used with ivermectin treatment as a main factor and time as a covariate. Differences in earthworm number between dung without ivermectin and dung-free controls were tested using the Mann–Whitney U-test. The earthworm number were transformed log 10 prior to the analysis
Table 1 Number of earthworms per litre of dung-free and ivermectin-free dung plots, determined in soil (25 cm 25 cm) at a depth of 0–10 cm Weeks after start of experiment
Dung-free (n = 4; S.E.) Dung (n = 4; S.E.)
1
3
5
7
2.28 0.51 4.56 0.86
1.48 0.50* 3.68 0.21
1.04 0.10* 4.80 0.82
1.72 0.57* 5.08 1.54
* Indicate statistically significant differences between treatments at p < 0.05 (Mann–Whitney U-test).
S. Kaneda et al. / Applied Soil Ecology 31 (2006) 280–285
283
Fig. 3. Earthworm density around dung pats containing ivermectin. Number of individuals (a) and total earthworm biomass (b) per litre of soil determined underneath pats containing (*) 0 mg kg1, (~) 0.1 mg kg1, and (&) 1 mg kg1 (wet weight) ivermectin initially. Earthworms were collected by hand from 25 cm 25 cm at a depth of 0–10 cm. No earthworms were found within the dung. Values are means S.E.; n = 4.
( p < 0.05; Fig. 3). There was no significant difference between ivermectin treatments in the number and the biomass of earthworms ( p > 0.05; Fig. 3). The decomposition rate of the artificial pats seemed constant until the end of observation in week 7, when the pats had lost almost half of their weight (Fig. 4). There was no significant difference in the decomposition rates between the treatments (P > 0.05).
4. Discussion Generally, the decomposition of dung depends to a great extent on the dung fauna population. The fauna is
Fig. 4. Weight loss of artificial dung pats deposited in the field at time 0. Pats contained (*) 0 mg kg1, (~) 0.1 mg kg1, and (&) 1 mg kg1 (wet weight) ivermectin initially. Values shown are means S.E.; n = 4. Data for the 0 mg kg1 treatment at 1 week after setting pats are missing.
affected by weather conditions (Herd, 1995) and might also respond to ivermectin concentrations in the dung. However, ivermectin treatments did not affect earthworm accumulation around dung pats and pat decomposition in our experiments. The weather conditions at the experimental site deviated slightly from the longterm mean. According to the weather station of the Meteorological Agency of Japan (http://www.data.kishou.go.jp/index.htm), the mean temperature and precipitation in Fukushima-city (37845.50 N and 140828.20 E) were somewhat higher in 2004 than usual. In this location, the Megascolecidae including the particularly abundant species P. (A.) heteropoda and P. (A.) divergens, dominated the earthworm fauna. According to the classification of Uchida (2004) and Ishizuka (2001), P. (A.). heteropoda is a polyhumicendogeic species. P. (A.). divergens might be included in the same group on the basis of its microhabitat preference and of its internal and external morphology (Ishizuka, 2001). Ten worms collected 3 weeks after deposition of the artificial pats were dissected to examine their gut contents. The intestines of individuals taken from sites of pat deposition contained numerous particles of organic matter, irrespective of the drug treatment. Whereas contents of animals from dung-free control locations included mainly mineral particles and only small amounts of organic matter. Uchida and Yamashita (unpublished data) observed P. (A.). heteropoda feeding on dung. In this study, earthworms accumulated around pats, and it appears likely that they came into contact with ivermectin by feeding on dung.
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The concentration of ivermectin in the dung decreased only slightly during the course of our experiments, which is in line with previous reports (Sommer et al., 1992; Sommer and Steffansen, 1993). Ivermectin levels in feces depend on the microbial degradation of the drug and on leaching. Sommer et al. (1992) and Sommer and Steffansen (1993) found that ivermectin concentrations did not decrease in pats under field conditions during 45 days after deposition. Halley et al. (1989) studied degradation half-lives of ivermectin under various conditions. In mixtures of feces and soil kept at room temperature in the dark, half-life periods varied between 93 and 240 days, while under outdoor conditions, they were 7–14 days in summer and 91–217 days in winter. On the other hand, the half-life of ivermectin was reduced to 3 h when thin films of it were exposed to direct summer sunlight. Soil-column leaching experiments revealed that ivermectin bound tightly to soils and organic matter, while its metabolites leached out (Halley et al., 1989). As the results of Halley et al. (1989) were obtained with mixtures of feces and soil, they appear applicable to ivermectin decomposition in earthworm casts rather than dung pats deposited on the soil surface. Although ivermectin appears susceptible to photodegradation, this process did not seem to occur in the present study. We conclude that ivermectin might be taken up by dung feeding organisms until the complete disappearance of the dung pat. Our results accord with previous studies reporting the absence of adverse effects of ivermectin on earthworm activity (Wall and Strong, 1987; Madsen et al., 1990; Sommer et al., 1992; Wratten et al., 1993; Svendsen et al., 2003). Svendsen et al. (2003) investigated the long term effects of ivermectin and fenbendazole on earthworm populations and dung pat decomposition in two grazing seasons in Denmark. Ivermectin excreted by heifers to which it had been administered as sustained release boluses had no negative impact on earthworm population size, biomass, and species composition. In the present study, we have shown that these results from European earthworm communities in which the Lumbricidae are predominant also hold for the Megascolecidae-dominated Japanese soils. From observations of earthworm abundance at their experimental site, Svendsen et al. (2003) concluded that ivermectin did not affect dung decomposition. In the present study, earthworms also
were abundant and accumulated around the dung pats during the entire period of observation regardless of ivermectin treatments. The small population size of dung beetles at our site also points to an important role of earthworms in dung decomposition. Moreover, the slightly higher than average precipitation recorded during our field study probably promoted earthworm activity which is known to be restricted by dry conditions (Holter, 1979; Madsen et al., 1990). We conclude that ivermectin has no adverse effects on Megascolecidae activity, and does not affect cowpat decomposition in pastureland sites where earthworms dominate in the dung decomposer community. In this study, ivermectin was added to artificial dung pats which did not contain metabolites that may occur in the dung of ivermectin-treated cattle. At present, it cannot be ruled out that ivermectin metabolites could influence earthworm behaviour. Further studies into the effects of ivermectin and its metabolites on earthworms using voided dung from ivermectin-treated cattle will help to further elucidate potential impacts of ivermectin on Megascolecidae populations in pasturelands. Acknowledgements We thank Mr. Tanji, Y. and Mr. Izawa, N., National Agricultural Research Center for Tohoku Region, for assistance in the field work. We also thank Dr. Ito, M.T. and Mr. Sakai, H., Yokohama National University, for identification of earthworms. We acknowledge the members of Lab Soil Management, National Agricultural Research Center for Tohoku Region for helpful discussion. We thank the anonymous referees for their useful comments on the paper. This study was supported by Budgets for Environmental Research allocated to National Research Institutes. S.K. is currently supported by JSPS Research Fellowships for Young Scientists. References Campbell, W.C., Fisher, M.H., Stapley, E.O., Albers-Scho¨nberg, G., Jacob, T.A., 1983. Ivermectin: a potent new antiparasitic agent. Science 221, 823–828. Easton, E.G., 1981. Japanese earthworms: a synopsis of the Megadrile species (Oligochaeta). Bull. Br. Museum (Natural history) Zool. Ser. 40, 33–65.
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