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Field effects of ivermectin and fenbendazole on earthworm populations and the disappearance of dung pats from bolus-treated cattle
Applied Soil Ecology 24 (2003) 207–218
Field effects of ivermectin and fenbendazole on earthworm populations and the disappearance of dung pats from bolus-treated cattle Tina S. Svendsen a,∗ , Jørn Grønvold a , Peter Holter b , Christian Sommer c a
Section of Zoology, Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Department of Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark c Fiskedamsgade 16, DK-2100 Copenhagen Ø, Denmark Received 12 December 2002; received in revised form 23 June 2003; accepted 25 June 2003
Abstract The disappearance of artificially formed dung pats of grazing cattle treated with sustained-release boluses containing either ivermectin or fenbendazole was compared with the disappearance of pats of untreated cattle through two successive grazing seasons. As earthworms play a major part in dung pat disappearance in northern temperate pastures, possible long-term effects of dung from treated cattle on worm populations were also examined. In a permanent pasture, the earthworm fauna was quantified within 50 m2 enclosed plots provided with control dung pats, ivermectin pats or fenbendazole pats. In each plot, worms were extracted from the soil at dung-free locations and from 6-week-old dung pats + the underlying soil. Numbers, biomass and species composition of earthworms thus obtained were unaffected by the drug treatments. Whether the treatments had an impact on dung pat disappearance depended on season, weather and local differences between plots at the experimental site. The disappearance of dung with ivermectin was significantly delayed throughout the first grazing season, but this effect was only seen in spring of the following year. The disappearance of dung with fenbendazole did not show any consistent pattern compared to control pats. © 2003 Elsevier B.V. All rights reserved. Keywords: Antiparasitic drug; Sustained-release bolus; Cow pat; Pasture; Lumbricidae; Coprophagous fauna
1. Introduction Rapid disappearance and mineralisation of cattle dung pats in pastures is essential in a sustainable grazing system with limited fouling and efficient nutrient cycling. In northern temperate regions such as ∗ Corresponding author. Tel.: +45-35-28-26-52; fax: +45-35-28-26-70. E-mail address: [email protected] (T.S. Svendsen).
Denmark, weather, insect activity and trampling are all involved in the recycling process, but earthworms are by far the most important single biological factor. Earthworms not only account for most of the invertebrate biomass under grassland (Curry, 1987), but are also the main agents responsible for removal and transport of dung into the soil. Hence they facilitate the action of soil bacteria and fungi (Holter, 1979; Putman, 1983), which is vital to mineralisation.
0929-1393/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0929-1393(03)00096-9
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The processes and organisms involved in dung disappearance may be affected by several factors, one of which is the treatment of grazing cattle with antiparasitic drugs, particularly the avermectins. Residues (parent drug + metabolites) of these drugs are excreted in dung and can have adverse effects on the development and survival of the associated fauna. Furthermore, the treatment may change the dung attractiveness to the fauna, which may not only be attributable to the excreted drug residues, but also to changes in the dung microflora (Wardhaugh and Mahon, 1991; Holter et al., 1993; Strong et al., 1996). A number of studies have concluded that avermectin residues have detrimental effects on several species of dung-dwelling Diptera and Coleoptera (e.g. Wall and Strong, 1987; Madsen et al., 1990; Wardhaugh and Mahon, 1991; Sommer et al., 1992; Barth et al., 1993; Wratten et al., 1993), but findings in respect to the rate of dung disappearance have been highly variable and reveal no common trend. Regional differences in the composition of the dung fauna, differences in weather conditions, the methodology used, or the mode of drug administration (injection, pour-on formulation or sustained-release boluses) might explain some of this variation. Several authors consider sustained-release delivery systems potentially more harmful to the pasture ecosystem than the other formulations due to the constant and prolonged excretion of drug residues from treated cattle (Sommer and Nielsen, 1992; Barth et al., 1993; Herd et al., 1993; Strong et al., 1996; McKellar, 1997; Alvinerie et al., 1998). However, long-term surveys are few in spite of the extensive use of sustained-release boluses for prophylaxis against parasitic nematodes in ruminants (McKellar, 1997). Furthermore, the earthworm fauna must be taken into account in relation to dung pat disappearance under northern temperate conditions. The importance of worms for pastureland ecology is unquestionable, but only few studies have considered the effect of ivermectin on this fauna (Wall and Strong, 1987; Madsen et al., 1990; Sommer et al., 1992; Wratten et al., 1993). There is a need for longer-term studies in view of the longevity and slow reproduction rate of these worms compared to the coprophagous insect fauna. Another commonly used group of antiparasitic agents is the benzimidazoles (McKellar, 1997). One of these compounds, fenbendazole, was introduced
in the 1970s and has been established worldwide as an anthelmintic in various preparations, including a sustained-release bolus (Tiefenbach, 1993). Fenbendazole and its metabolites, some of which have high antiparasitic activity, are excreted in the dung of treated animals (Short et al., 1987; McKellar, 1997). However, fenbendazole residues have not been found to be toxic to dung-colonising insects (Strong et al., 1996). The effect of fenbendazole on earthworms is unknown. Other benzimidazoles, however, are used as fungicides and are highly toxic to earthworms (Stringer and Wright, 1973; Edwards and Brown, 1982). Therefore, the earthworm fauna and hence the disappearance rate of dung pats may be affected by fenbendazole treatment of grazing cattle. This paper reports the results of a 2-year Danish field study designed (1) to test whether treatment of cattle with ivermectin or fenbendazole, administered in sustained-release boluses, affected the disappearance of dung pats; and (2) to ascertain whether exposure to dung from treated cattle over more than 4 months each year led to any detectable long-term effects on the earthworm populations. The discussion includes an assessment of the methodology used in this and earlier related field studies. 2. Materials and methods 2.1. General The experiment was carried out over two successive grazing seasons, from 28 April to 25 October 1999 and from 10 May to 11 November 2000, in an old permanent pasture on a private farm near Skibby in North Zealand (55◦ 45 N, 12◦ 14 E), Denmark. Soil temperatures were measured with two data loggers (Gemini TINYTAG PLUS, Metric A/S Denmark) placed at a depth of 15 cm in two locations at the experimental site. Data on rainfall were obtained from two weather stations (belonging to the Danish Meteorological Institute) about 20 km north and south of Skibby. The weather data during the experiment are shown in Fig. 1. 2.2. Livestock treatments At spring turnout in 1999, sustained-release boluses of ivermectin (Ivomec® vet. SR Bolus, MSD Agvet)
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25
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Fig. 1. Soil temperature measured at 15 cm depth and mean monthly rainfall measured at two weather stations situated approximately 20 km north and south of the experimental site. The asterisk (∗) signifies only the cumulative amount of rain until the experiment ended in each grazing season.
were administered to eight heifers (mean weight 415 kg). Eighteen heifers (366 kg) were given fenbendazole boluses (Panacur® vet. SR Bolus, Hoechst Roussel vet.) and four heifers (500 kg) remained untreated and served as a control group. The three groups of heifers were pastured on three separate paddocks close to the experimental site. In 2000, 7 heifers (478 kg) received ivermectin boluses and 20 (423 kg) were given fenbendazole boluses. A group of four animals (512 kg) served as control, and the three groups were kept in the same paddocks as in the previous year. In both years, none of the cattle used had been treated with antiparasitics for at least 6 months prior to the bolus treatments. Dung collection started about 2 weeks after bolus-treatment to allow drug excretion rates to stabilise (see Herd et al., 1996). 2.3. Dung collection In both years, about 25 l of freshly voided dung was collected every 2 weeks from each group of cattle.
Each batch of dung was thoroughly mixed before use in one of three field experiments, designed to monitor changes in earthworm numbers and dung disappearance in the presence or absence of parasiticide residues. 2.4. Experimental design Experiments were conducted at 8-week intervals (spring, summer and autumn, Table 1) in 1999 and 2000. A 22 m ×22 m area of reasonably homogeneous pasture was fenced to keep cattle out. The central 450 m2 area was divided into nine 50 m2 plots (about 7.1 m × 7.1 m), enclosed by barriers that extended 20 cm above and below ground level. The barriers prevented substantial earthworm movement between plots, and between plots and the adjoining pasture. Artificial pats of freshly voided dung were exposed to earthworm colonisation in the plots according to a 3 × 3 Latin square design (3 plots × 3 treatments (control, ivermectin, fenbendazole)). The experimental arrangement in terms of the allocation of different
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Table 1 Characteristics of experimental dung pats at the time of placement in the field (n = 6) Year of deposition and dung type
% water content ± S.E.
C/N ratio ± S.E.
1999 Control Ivermectin Fenbendazole
87.5 ± 0.30 86.1 ± 0.44 86.7 ± 0.24
18.0 ± 0.12 16.7 ± 0.11 16.9 ± 0.06
2000 Control Ivermectin Fenbendazole
87.9 ± 0.36 87.7 ± 0.80 87.4 ± 0.36
17.6 ± 1.05 16.7 ± 0.68 17.4 ± 0.34
The estimates of C/N ratios are based on the assumption that 50% of the dung weight-loss by ignition is organic C (Nelson and Sommers, 1982).
dung types between plots remained the same between experiments and years. Each plot was allocated eight pats of a standard size (1 kg wet weight and 16 cm in diameter) and known physical characteristics (Table 1). Pats were placed on squares of nylon netting (mesh size 1 cm) to facilitate later retrieval and were covered by wire netting (2 cm mesh) to prevent bird damage. They were placed randomly, but positions previously used for experimental pats and earthworm sampling (involving formaldehyde) were avoided. A numbered plastic tag identified the location of each pat. At weeks 2, 4 and 6 after the placement of the experimental pats, each plot received a further six dung pats collected from the same group of cattle as the pats already in the plot. The additional pats were placed randomly, without any nylon netting, and were intended to simulate dung deposition at normal stocking rates, i.e. eight cattle per hectare each depositing 10 kg of dung per day. These pats were left undisturbed and were, therefore, available as food for the earthworm population throughout the duration of each experiment.The grass was cut to ca. 3 cm and removed from the experimental area every 2 weeks, but was allowed to grow higher around the dung pats to simulate the natural grazing habit of cattle. 2.5. Dung pat sampling and analysis At each plot, two experimental pats (i.e. those on nylon netting) were sampled for analysis every 2
weeks, and hence six pats of each type of dung were removed on each sampling occasion. Prior to each grazing season, the order of pats to be sampled was randomly chosen. The wet weight of each pat was recorded in the field and the pats were brought to the laboratory in plastic bags stored in cool boxes. After storage at 5 ◦ C overnight, invertebrates and vegetation were removed from the pats by hand sorting. Of the invertebrates found, only earthworms were kept (in 70% ethanol) for later counting, weighing and identification. Each pat was carefully disintegrated and homogenised before two sub-samples were dried to constant weight at 105 ◦ C. The dried samples were ground and samples of known weight were re-dried overnight at 105 ◦ C and subsequently incinerated at 550 ◦ C for 2 h for determination of their organic matter content. To determine the proportion of organic matter that had disappeared from the pats in the field, samples of fresh dung (i.e. the mixed dung prior to the formation of artificial pats) were processed similarly. In addition, the total nitrogen content of the dried, pulverised fresh dung was determined by a micro-Kjeldahl procedure as described in Hesse (1971). The percentage of the initial amount of organic matter remaining in the ivermectin and the fenbendazole pats after 2, 4, 6 and 8 weeks of field exposure was tested separately against control pats using an analysis of variance with a Random Plot Effect which deals with two pats per plot separately, but nested (PROC MIXED, SAS system V8) (classification variables: treatment, season, time; two variables referring to the position of the dung pats in the Latin square: column and row). By inspection of residual plots, it was concluded that transformation of the response variable was unnecessary. 2.6. Earthworm sampling In spring, before any dung pats were deposited, the initial earthworm population was estimated. In each of the nine plots, four circular 0.1 m2 frames were laid out and the grass within these circles was cut to ground level and removed. The locations used for earthworm sampling within each plot were randomly chosen before the experiment started. Two or three lots of approximately 2.5 l of 0.3% formalin solution (1.3 dl of 23.4% formaldehyde solution in 10 l of
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water) were applied to each encircled area at 10 min intervals and the emerging earthworms were collected (formalin was applied until no more worms appeared on the soil surface). The earthworm population was then assessed every 8 weeks throughout each grazing season. On these occasions, worms in each plot were sampled in each of four 0.1 m2 circles. Two of these were located at dung-free plot locations (i.e. areas in each experimental plot that had no previous history of dung deposition) and two were centred on 6-week-old experimental pats; 6-week-old pats were chosen because earlier observations (Holter, 1983) suggested that this was the age when maximum numbers of worms might be present. The dung pats were removed immediately before formalin extraction. Collected earthworms were stored in containers with moist tissue paper and brought to the laboratory in cool boxes. The following day worms from each 0.1 m2 plot were counted, weighed and preserved in 70% alcohol before identification. Adult and, when possible, sub-adult worms were identified to species whereas juveniles were separated at the genus level (using descriptions and nomenclature in Sims and Gerard, 1985). The relative contribution of each species to the total fresh biomass was estimated from the weight of preserved specimens (after Holter, 1979). Worms hand-sorted from the dung itself were processed similarly in the laboratory. The mean biomass of earthworms collected under pats or in dung-free locations in the ivermectin and fenbendazole plots were tested separately against the corresponding biomass in the control plots. The SAS procedure PROC MIXED was used as in the analyses of dung pat disappearance (no transformations used). Possible effects of ivermectin or fenbendazole on the occurrence (total biomass) of the most abundant earthworm species and juvenile groups were tested against controls in a linear model (SAS procedure PROC GLM), which included the effect of time. Means were separated by Tukey’s multiple comparisons. Finally, earthworm species were separated into ecological groups and the relative contributions (percentage of the total biomass) of these groups were tested in a linear model similar to the one used for the analysis of single species.
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3. Results 3.1. Disappearance of dung pats The residual amount of organic matter in ivermectin or fenbendazole pats in relation to the control pats varied throughout the different periods (Fig. 2), but the overall effects each year are given in Table 2. Throughout most of 1999 and in the spring/early summer of 2000, significantly higher amounts of organic matter disappeared from the control pats than from pats containing ivermectin. However, in the summer and autumn of 2000, there was a tendency for ivermectin pats to disappear faster. Likewise, the effects of fenbendazole were inconsistent across seasons and years (Fig. 2). Throughout 1999, the disappearance rate of fenbendazole pats was similar to, or lower than that of the control dung, whereas in the following year fenbendazole pats disappeared faster than, or at the same rate as the control pats. Furthermore, positional effects were highly significant in the first season, but absent in 2000 (Table 2). 3.2. Earthworm biomass The mean biomass of earthworms at dung-free plot locations was about 50 g m−2 throughout the spring Table 2 F values of analyses of variance with random plot effects (PROC MIXED, classification variables: season and treatment, as well as row and column (positions) in the Latin square) on the mass of remaining organic matter in dung pats from treated cattle (ivermectin or fenbendazole sustained-release boluses) versus untreated controls Season
Treatment
Season × treatment
Position
1999 Ivermectin Fenbendazole
97.97∗∗∗ 22.58∗∗∗
40.08a,∗∗∗ 30.62a,∗∗∗
1.36 ns 19.98∗∗∗
11.66∗∗∗ 18.64∗∗∗
2000 Ivermectin Fenbendazole
423.97∗∗∗ 334.59∗∗∗
1.06 ns 30.72b,∗∗∗
12.68∗∗∗ 12.90∗∗∗
7.68∗∗ 3.24 ns
ns: not significant. a More organic matter left in the ivermectin and fenbendazole pats than in the control pats. b Less organic matter left in the fenbendazole pats than in the controls. ∗∗ P < 0.01. ∗∗∗ P < 0.001.
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% of initial organic matter +SE
Grazing season 1999 100 90 80 70 60 50 40 30 20 10 0 2
4
6
8
2
May-July
4
6
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4
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% of initial organic matter +SE
Grazing season 2000 100 90 80 70 60 50 40 30 20 10 0 2
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ivermectin
fenbendazole
Fig. 2. Disappearance of dung pats formed from the dung of untreated cattle or cattle treated with either ivermectin or fenbendazole expressed as the percentage of initial organic matter remaining after 2, 4, 6 and 8 weeks on pasture. Vertical bars indicate S.E. of 6 sampled dung pats per dung type per week.
and summer, increasing slightly in August (Fig. 3). In both years there was a significant seasonal effect (P < 0.001): the biomass was highest in autumn, especially in 2000 when the mean biomass values in all dung-free plot locations were about twice those recorded in 1999. Also, worms were clearly attracted to dung pats in all plots (Fig. 3). The biomass—with or without dung—
in the ivermectin plots did not differ significantly (P > 0.05) from corresponding values in the control plots. In 1999, fenbendazole plots had a lower worm biomass than did the control plots (dung-free locations (P < 0.001) and dung pats (P < 0.05)). However, inexplicable positional effects (P < 0.05) were also significant. By contrast, no significant effects of
T.S. Svendsen et al. / Applied Soil Ecology 24 (2003) 207–218 Worms under dung-free plot locations
Worms under dung-free plot locations 40 Biomass (g) +SE
Biomass (g) +SE
40 30 20 10 0
30 20 10 0
APR
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Worms in and under dung pats 40 Biomass (g) +SE
40 Biomass (g) +SE
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30 20 10 0
30 20 10 0
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JUL
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control
ivermectin
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control
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fenbendazole
Fig. 3. Mean biomass of earthworms (per 0.1 m2 ) in experimental plots with dung pats formed from untreated dung or dung containing residues of ivermectin or fenbendazole. Worms were collected by formalin extraction from either dung-free plot locations or from artificial pats exposed to earthworm colonisation for 6 weeks. Vertical bars indicate S.E. of six samples per dung type per time (12 samples per dung type in spring samplings at dung-free plot locations).
fenbendazole treatment or position were detectable in 2000. 3.3. Species composition In both grazing seasons, the earthworm community consisted of 11 species belonging to the following ecological groups (Bouché, 1977). Anecic worms: Lumbricus terrestris (mean adult wet weight 1.81 g) and Aporrectodea longa (1.32 g); endogeic species: Allolobophora chlorotica (0.19 g), Aporrectodea caliginosa (0.33 g), A. rosea (0.15 g), Octolasion cyaneum (1.09 g) and O. tyrtaeum (0.52 g); epigeic worms: L. castaneus (0.12 g), L. rubellus (0.46 g), Eiseniella tetraedra (0.06 g) and Dendrodrilus rubidus (0.06 g). The juvenile worms were separated into two groups: juvenile Lumbricus spp. (mean wet weight 0.10 g) and ‘other juveniles’ (0.13 g). Figs. 4 and 5 show the relative contribution of each group to
the total biomass under dung-free plot locations and in/under dung pats. Sub-adult specimens of L. terrestris (mean wet weight 0.80 g) and A. longa (0.66 g) are included in the anecic group. There were no negative effects of ivermectin or fenbendazole on the biomass of the five most abundant species L. terrestris, A. longa, A. chlorotica, A. caliginosa and L. castaneus when tested separately (data not shown). Generally, the biomass of L. terrestris, A. longa and L. castaneus was significantly higher in the presence of dung pats, whereas the two endogeic species, A. chlorotica and A. caliginosa, were less attracted to dung pats. The remaining species were not tested separately. The abundance of juveniles and adults depended significantly on time of the year. In and under dung pats, the biomass of juvenile Lumbricus spp. was significantly (P < 0.001) higher than under dung-free locations, whereas this was seen for ‘other juveniles’ in 1999 only (P < 0.005). There
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Fig. 4. The relative contribution of ecological groups and juveniles to the total biomass of earthworms extracted in 1999 from plots receiving C: control, I: ivermectin or F: fenbendazole dung.
were no effects of drug treatments on the biomass of juvenile worms.
4. Discussion 4.1. Methodology The concentration of ivermectin or fenbendazole in the dung pats were not determined by chemical analysis. However, the experimental pats (as well as the additional pats) consisted of a mixture of dung from at least seven treated animals. This ensured that these pats did contain residues of parasiticide, even if a bo-
lus in one of the animals had failed to work properly. In both years we observed (visual judgement) a much smaller number of dung-beetle larvae (Aphodius spp.) in the ivermectin pats compared with controls, and their development was clearly stunted. In contrast, we found that the number and development of dung beetle-larvae in pats containing fenbendazole and in control pats were similar. This is in accordance with the findings of Strong et al. (1996) and confirms that ivermectin residues were indeed present in the ‘ivermectin pats’. Any differences in dung pat disappearance due to variations in size and surface area were reduced by the use of standardised, artificially formed pats in contrast
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Fig. 5. Relative contribution of ecological groups and juveniles to the total biomass of earthworms extracted in 2000 from plots receiving C: control, I: ivermectin or F: fenbendazole dung.
to naturally voided pats studied by Barth et al. (1993, 1995) and Wratten et al. (1993). The dung pats were deposited in a cattle-free enclosure and covered by netting to prevent any differences due to trampling by cattle or disturbance by birds. Finally, because the dung pats were placed on pieces of net, the sampling was standardised as the material above the net was defined as remaining dung, whereas everything below the net was regarded as soil. Contamination with soil, probably due to earthworm activity, may strongly increase the dry weight of dung pats and hence invalidate studies where the degradation of dung pats has been measured as simple dry weight loss (Wall and Strong, 1987; Wratten et al., 1993). To overcome this
difficulty, at least in part, we quantified dung disappearance in terms of decline of the amount of organic matter in the pats as recommended by Holter (1979) and Herd et al. (1993). The initial moisture content of dung pats has been seen as an important factor affecting the rate of dung disappearance. Barth et al. (1993, 1995) have pointed out the lack of this information in several papers dealing with the effect of ivermectin on decomposition, as dung from untreated cattle might have higher moisture content as a result of infection with parasitic nematodes. According to Barth et al. (1993, 1995), even small differences in moisture content of 1–2% may have an effect on dung disappearance, i.e. the
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higher water content leads to an increase in dung disappearance. However, we found no correlation (r = 0.07–0.15) between initial moisture content of the dung (which did not differ much between paddocks, cf. Table 1) and the subsequent pat degradation at any time after deposition. Furthermore, judged from our observations on C/N ratios (Table 1), the dung from the three paddocks did not differ substantially as regards other basic characteristics. Hence, it seems unlikely that the present results have been seriously affected by the necessary use of three, apparently similar, paddocks that were managed in the same way. 4.2. Dung pat disappearance and abundance of earthworms The present study confirms that dung disappearance is a result of several factors of which drug residues is only one. In this comparison of ivermectin, fenbendazole and control pats, differences in the disappearance could not be ascribed to drug treatment alone. The effect of season was highly significant, and so was the location of dung pats within the experimental area. On the other hand, there were no strong toxic effects of faecally excreted ivermectin or fenbendazole on pastureland earthworm populations following bolus implantation in grazing cattle. Likewise, there was no indication of important negative, indirect effects such as lower attractiveness of dung from treated animals. These results are supported by laboratory experiments (Svendsen et al., 2002; Svendsen et al., unpublished) in which long-term exposure of ivermectin or fenbendazole in dung had no lethal or adverse sub-lethal effects on L. terrestris. The effect of season on the disappearance of dung is probably a result of the influence of weather on earthworm activity (Holter, 1979). In both years, the highest disappearance rate of dung occurred in autumn, with high soil humidity and low temperatures (Fig. 1) and hence a high biomass of (active) earthworms (Figs. 2 and 3). Moreover, weather may also have affected the activity of insects, including their impact on dung disappearance. Thus, the faster disappearance of fenbendazole pats compared with those containing ivermectin in the summers of 1999 and 2000, i.e. during periods of high soil temperatures (Fig. 1) and low earthworm activity, might be attributed to the fact that ivermectin is toxic to insects
whereas fenbendazole is not. Reduced insect activity in the ivermectin dung could explain part of the delay in pat disappearance compared with the controls, as there were no differences in earthworm abundance between treatments. Furthermore, compared with 1999, dung pats in all treatments disappeared faster in the autumn of 2000 which experienced heavy rainfall soon after the placement of experimental pats in September (Fig. 1). The associated weathering of these newly exposed pats may have facilitated the dung dispersal by earthworms and thus made up for any missing insect activity (due to the ivermectin treatment) that might otherwise have delayed the disappearance of pats. Earlier studies in Denmark showed delayed disappearance of dung from cattle given ivermectin either as a subcutaneous injection of 200 g kg−1 or as a 0.5 mg kg−1 pour-on treatment (Madsen et al., 1990; Sommer et al., 1992). These studies followed the disappearance of pats in the field from August and throughout the autumn in a single year. The same result was seen in the present study in late summer and autumn in 1999 but not in 2000, which emphasises the importance of season and weather for dung disappearance, and the value of longer-term studies. Herd et al. (1996) measured steady-state ivermectin concentrations of 2.9–4.0 g g−1 dry weight and Alvinerie et al. (1998) measured steady-state concentrations of about 9 g g−1 dry weight in dung from cattle administered with the same sized bolus as that used in the current study. The concentrations of ivermectin in the dung used by Madsen et al. (1990) and Sommer et al. (1992) were up to 9.0 g g−1 dry weight (at 1–2 days after pour-on treatment, maximum concentration after injection was 3.9 g g−1 dry weight). However, Madsen et al. (1990) and Sommer et al. (1992) observed a reduced disappearance rate of dung produced up to 20 days after ivermectin administration (pour-on or injection), when the concentration in the dung was probably below 0.3 g g−1 dry weight (Sommer et al., 1992; Sommer and Steffansen, 1993; Herd et al., 1996). Hence, the lack of consistent effects of ivermectin on dung disappearance in the current study is unlikely to be a result of low residual concentrations. In our study, all dung pats, including the untreated controls, disappeared at a higher rate than the control pats in the earlier Danish field studies, indicating a more abundant earthworm fauna in our study and thus a less pronounced effect on dung disappearance due to missing
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insect activity. The high activity of earthworms may be a result of the regular addition of dung pats in each plot (i.e. the addition of dung pats every second week besides the experimental pats), which is an improvement in comparison with the earlier studies, and essential to investigate long-term effects on the earthworm population. 4.3. Concluding remarks The results reported here suggest that under temperate field conditions with a high level of earthworm activity, disappearance of dung pats from cattle administered sustained-release boluses of ivermectin and fenbendazole is not significantly affected by the faecally-excreted drug residues. Earthworms were equally attracted to dung pats irrespective of drug treatment and no long-term effects on the studied population were observed. Faecally-excreted ivermectin has detrimental effects on the activity of insect larvae in dung pats, but the impact on dung disappearance may be overridden by the effect of earthworms, when these are favoured by suitable weather. This partly explains the different findings in earlier field trials under varying, albeit temperate climatic conditions.
Acknowledgements We are grateful to Lise and Jørgen Due for access to cattle and pastureland. Malene Michel, Jan Tækker Christiansen, Hanne Lynge Christiansen, Hanne Rawat, Annette Spangenberg, Leif Stausholm Jensen and Torben Frost are thanked for their excellent technical assistance. Thanks to Torben Martinussen and Ib Skovgaard for statistical advice and to Keith Wardhaugh for improving the manuscript. Valuable comments from John Finn and an anonymous reviewer are also gratefully acknowledged. The Danish Agricultural and Veterinary Research Council financially supported this study. References Alvinerie, M., Sutra, J.F., Galtier, P., Lifschitz, A., Virkel, G., Sallovitz, J., Lanusse, C., 1998. Persistence of ivermectin in plasma and faeces following administration of a sustainedrelease bolus to cattle. Res. Vet. Sci. 66, 57–61.
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Barth, D., Heinze-Mutz, E.M., Roncalli, R.A., Schlüter, D., Gross, S.J., 1993. The degradation of dung produced by cattle treated with an ivermectin slow-release bolus. Vet. Parasitol. 48, 215– 227. Barth, D., Karrer, M., Heinze-Mutz, E.M., 1995. Significance of moisture content of dung pats for colonisation and degradation of cattle dung. Appl. Parasitol. 36, 11–21. Bouché, M.B., 1977. Stratégies lombriciennes. Ecol. Bull. 25, 122–132. Curry, J.P., 1987. The invertebrate fauna of grassland and its influence on productivity. 1. The composition of the fauna. Grass Forage Sci. 42, 103–120. Edwards, P.J., Brown, S.M., 1982. Use of grassland plots to study the effects of pesticides on earthworms. Pedobiologia 24, 145– 150. Herd, R., Strong, L., Wardhaugh, K., 1993. Research recommendations. Vet. Parasitol. 48, 337–340. Herd, R.P., Sams, R.A., Ashcraft, S.M., 1996. Persistence of ivermectin in plasma and faeces following treatment of cows with ivermectin sustained-release, pour-on or injectable formulations. Int. J. Parasitol. 26, 1087–1093. Hesse, P.R., 1971. A Textbook of Soil Chemical Analysis. John Murray, London. Holter, P., 1979. Effect of dung-beetles (Aphodius spp.) and earthworms on the disappearance of cattle dung. Oikos 32, 393–402. Holter, P., 1983. Effect of earthworms on the disappearance rate of cattle droppings. In: Satchell, J.E. (Ed.), Earthworm Ecology: From Darwin to vermiculture. Chapman & Hall, London, pp. 49–57. Holter, P., Sommer, C., Grønvold, J., Madsen, M., 1993. Effects of ivermectin treatment on the attraction of dung beetles (Coleoptera: Scarabaeidae and Hydrophilidae) to cow pats. Bull. Entomol. Res. 83, 53–58. Madsen, M., Nielsen, B.O., Holter, P., Pedersen, O.C., Jespersen, J.B., 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. McKellar, Q.A., 1997. Ecotoxicology and residues of anthelmintic compounds. Vet. Parasitol. 72, 413–435. Nelson, D.W., Sommers. L.E., 1982. Total carbon, organic carbon, and organic matter. In: Page, A.L., Miller, R.H., Keeney, D.R. (Eds.), Methods of soil analysis. Part 2. Chemical and microbiogical properties, second ed. American Society of Agronomy, Inc. & Soil Science Society of America, Inc., Madison, Wisconsin, USA, pp. 539–579. Putman, R.J., 1983. Carrion and Dung: The Decomposition of Animal Wastes. The Institute of Biology’s Studies in Biology, no. 156, Edward Arnold, London, 29 pp. Short, C.R., Barker, S.A., Hsieh, L.C., Ou, S.-P., McDowell, T., Davis, L.E., Neff-Davis, C.A., Koritz, G., Bevill, R.F., Munsiff, I.J., 1987. Disposition of fenbendazole in cattle. Am J. Vet. Res. 6, 958–961. Sims, R.W., Gerard, B.M., 1985. Earthworms. Synopsis of the British Fauna, no. 31, Linnaean Society London, London, 171 pp. Sommer, C., Nielsen, B.O., 1992. Larvae of the dung beetle Onthophagus gazella F. (Col., Scarabaeidae) exposed to lethal
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and sublethal ivermectin concentrations. J. Appl. Entomol. 114, 502–509. 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. Sommer, C., Steffansen, B., Nielsen, B.O., Grønvold, J., Vagn Jensen, K.-M., Jespersen, J.B., 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. Stringer, A., Wright, M.A., 1973. The effect of benomyl and some related compounds on Lumbricus terrestris and other earthworms. Pestic. Sci. 4, 165–170. Strong, L., Wall, R., Woolford, A., Djeddour, D., 1996. The effect of faecally excreted ivermectin and fenbendazole on the insect colonisation of cattle dung following the oral administration of sustained-release boluses. Vet. Parasitol. 62, 253–266. 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, 319–322. Tiefenbach, B., 1993. The Panacur SR Bolus. A new formulation for the control of gastrointestinal and lung nematodes in grazing cattle. Papers given at the 14th International Conference of the World Association for the Advancement of Veterinary Parasitology in Cambridge, 9 August, 1993. Wall, R., Strong, L., 1987. Environmental consequences of treating cattle with the antiparasitic drug ivermectin. Nature 327, 418– 421. Wardhaugh, K.G., Mahon, R.J., 1991. Avermectin residues in sheep and cattle dung and their effects on dung-beetle (Coleoptera:Scarabaeidae) colonization and dung burial. Bull. Entomol. Res. 81, 333–339. Wratten, S.D., Mead-Briggs, M., Gettinby, G., Ericsson, G., Baggott, D.G., 1993. An evaluation of the potential effects of ivermectin on the decomposition of cattle dung pats. Vet. Record 133, 365–371.
Field effects of ivermectin and fenbendazole on earthworm populations and the disappearance of dung pats from bolus-treated cattle Tina S. Svendsen a,∗ , Jørn Grønvold a , Peter Holter b , Christian Sommer c a
Section of Zoology, Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Department of Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark c Fiskedamsgade 16, DK-2100 Copenhagen Ø, Denmark Received 12 December 2002; received in revised form 23 June 2003; accepted 25 June 2003
Abstract The disappearance of artificially formed dung pats of grazing cattle treated with sustained-release boluses containing either ivermectin or fenbendazole was compared with the disappearance of pats of untreated cattle through two successive grazing seasons. As earthworms play a major part in dung pat disappearance in northern temperate pastures, possible long-term effects of dung from treated cattle on worm populations were also examined. In a permanent pasture, the earthworm fauna was quantified within 50 m2 enclosed plots provided with control dung pats, ivermectin pats or fenbendazole pats. In each plot, worms were extracted from the soil at dung-free locations and from 6-week-old dung pats + the underlying soil. Numbers, biomass and species composition of earthworms thus obtained were unaffected by the drug treatments. Whether the treatments had an impact on dung pat disappearance depended on season, weather and local differences between plots at the experimental site. The disappearance of dung with ivermectin was significantly delayed throughout the first grazing season, but this effect was only seen in spring of the following year. The disappearance of dung with fenbendazole did not show any consistent pattern compared to control pats. © 2003 Elsevier B.V. All rights reserved. Keywords: Antiparasitic drug; Sustained-release bolus; Cow pat; Pasture; Lumbricidae; Coprophagous fauna
1. Introduction Rapid disappearance and mineralisation of cattle dung pats in pastures is essential in a sustainable grazing system with limited fouling and efficient nutrient cycling. In northern temperate regions such as ∗ Corresponding author. Tel.: +45-35-28-26-52; fax: +45-35-28-26-70. E-mail address: [email protected] (T.S. Svendsen).
Denmark, weather, insect activity and trampling are all involved in the recycling process, but earthworms are by far the most important single biological factor. Earthworms not only account for most of the invertebrate biomass under grassland (Curry, 1987), but are also the main agents responsible for removal and transport of dung into the soil. Hence they facilitate the action of soil bacteria and fungi (Holter, 1979; Putman, 1983), which is vital to mineralisation.
0929-1393/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0929-1393(03)00096-9
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The processes and organisms involved in dung disappearance may be affected by several factors, one of which is the treatment of grazing cattle with antiparasitic drugs, particularly the avermectins. Residues (parent drug + metabolites) of these drugs are excreted in dung and can have adverse effects on the development and survival of the associated fauna. Furthermore, the treatment may change the dung attractiveness to the fauna, which may not only be attributable to the excreted drug residues, but also to changes in the dung microflora (Wardhaugh and Mahon, 1991; Holter et al., 1993; Strong et al., 1996). A number of studies have concluded that avermectin residues have detrimental effects on several species of dung-dwelling Diptera and Coleoptera (e.g. Wall and Strong, 1987; Madsen et al., 1990; Wardhaugh and Mahon, 1991; Sommer et al., 1992; Barth et al., 1993; Wratten et al., 1993), but findings in respect to the rate of dung disappearance have been highly variable and reveal no common trend. Regional differences in the composition of the dung fauna, differences in weather conditions, the methodology used, or the mode of drug administration (injection, pour-on formulation or sustained-release boluses) might explain some of this variation. Several authors consider sustained-release delivery systems potentially more harmful to the pasture ecosystem than the other formulations due to the constant and prolonged excretion of drug residues from treated cattle (Sommer and Nielsen, 1992; Barth et al., 1993; Herd et al., 1993; Strong et al., 1996; McKellar, 1997; Alvinerie et al., 1998). However, long-term surveys are few in spite of the extensive use of sustained-release boluses for prophylaxis against parasitic nematodes in ruminants (McKellar, 1997). Furthermore, the earthworm fauna must be taken into account in relation to dung pat disappearance under northern temperate conditions. The importance of worms for pastureland ecology is unquestionable, but only few studies have considered the effect of ivermectin on this fauna (Wall and Strong, 1987; Madsen et al., 1990; Sommer et al., 1992; Wratten et al., 1993). There is a need for longer-term studies in view of the longevity and slow reproduction rate of these worms compared to the coprophagous insect fauna. Another commonly used group of antiparasitic agents is the benzimidazoles (McKellar, 1997). One of these compounds, fenbendazole, was introduced
in the 1970s and has been established worldwide as an anthelmintic in various preparations, including a sustained-release bolus (Tiefenbach, 1993). Fenbendazole and its metabolites, some of which have high antiparasitic activity, are excreted in the dung of treated animals (Short et al., 1987; McKellar, 1997). However, fenbendazole residues have not been found to be toxic to dung-colonising insects (Strong et al., 1996). The effect of fenbendazole on earthworms is unknown. Other benzimidazoles, however, are used as fungicides and are highly toxic to earthworms (Stringer and Wright, 1973; Edwards and Brown, 1982). Therefore, the earthworm fauna and hence the disappearance rate of dung pats may be affected by fenbendazole treatment of grazing cattle. This paper reports the results of a 2-year Danish field study designed (1) to test whether treatment of cattle with ivermectin or fenbendazole, administered in sustained-release boluses, affected the disappearance of dung pats; and (2) to ascertain whether exposure to dung from treated cattle over more than 4 months each year led to any detectable long-term effects on the earthworm populations. The discussion includes an assessment of the methodology used in this and earlier related field studies. 2. Materials and methods 2.1. General The experiment was carried out over two successive grazing seasons, from 28 April to 25 October 1999 and from 10 May to 11 November 2000, in an old permanent pasture on a private farm near Skibby in North Zealand (55◦ 45 N, 12◦ 14 E), Denmark. Soil temperatures were measured with two data loggers (Gemini TINYTAG PLUS, Metric A/S Denmark) placed at a depth of 15 cm in two locations at the experimental site. Data on rainfall were obtained from two weather stations (belonging to the Danish Meteorological Institute) about 20 km north and south of Skibby. The weather data during the experiment are shown in Fig. 1. 2.2. Livestock treatments At spring turnout in 1999, sustained-release boluses of ivermectin (Ivomec® vet. SR Bolus, MSD Agvet)
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209
25
20 Temperature (C)
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25 15 10 5
15 10 5 0
120
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80
80
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0
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20
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AUG
SEP
OCT*
MAY
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1999
monthly rainfall
AUG
SEP
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NOV*
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soil temperature
monthly rainfall
soil temperature
Fig. 1. Soil temperature measured at 15 cm depth and mean monthly rainfall measured at two weather stations situated approximately 20 km north and south of the experimental site. The asterisk (∗) signifies only the cumulative amount of rain until the experiment ended in each grazing season.
were administered to eight heifers (mean weight 415 kg). Eighteen heifers (366 kg) were given fenbendazole boluses (Panacur® vet. SR Bolus, Hoechst Roussel vet.) and four heifers (500 kg) remained untreated and served as a control group. The three groups of heifers were pastured on three separate paddocks close to the experimental site. In 2000, 7 heifers (478 kg) received ivermectin boluses and 20 (423 kg) were given fenbendazole boluses. A group of four animals (512 kg) served as control, and the three groups were kept in the same paddocks as in the previous year. In both years, none of the cattle used had been treated with antiparasitics for at least 6 months prior to the bolus treatments. Dung collection started about 2 weeks after bolus-treatment to allow drug excretion rates to stabilise (see Herd et al., 1996). 2.3. Dung collection In both years, about 25 l of freshly voided dung was collected every 2 weeks from each group of cattle.
Each batch of dung was thoroughly mixed before use in one of three field experiments, designed to monitor changes in earthworm numbers and dung disappearance in the presence or absence of parasiticide residues. 2.4. Experimental design Experiments were conducted at 8-week intervals (spring, summer and autumn, Table 1) in 1999 and 2000. A 22 m ×22 m area of reasonably homogeneous pasture was fenced to keep cattle out. The central 450 m2 area was divided into nine 50 m2 plots (about 7.1 m × 7.1 m), enclosed by barriers that extended 20 cm above and below ground level. The barriers prevented substantial earthworm movement between plots, and between plots and the adjoining pasture. Artificial pats of freshly voided dung were exposed to earthworm colonisation in the plots according to a 3 × 3 Latin square design (3 plots × 3 treatments (control, ivermectin, fenbendazole)). The experimental arrangement in terms of the allocation of different
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Table 1 Characteristics of experimental dung pats at the time of placement in the field (n = 6) Year of deposition and dung type
% water content ± S.E.
C/N ratio ± S.E.
1999 Control Ivermectin Fenbendazole
87.5 ± 0.30 86.1 ± 0.44 86.7 ± 0.24
18.0 ± 0.12 16.7 ± 0.11 16.9 ± 0.06
2000 Control Ivermectin Fenbendazole
87.9 ± 0.36 87.7 ± 0.80 87.4 ± 0.36
17.6 ± 1.05 16.7 ± 0.68 17.4 ± 0.34
The estimates of C/N ratios are based on the assumption that 50% of the dung weight-loss by ignition is organic C (Nelson and Sommers, 1982).
dung types between plots remained the same between experiments and years. Each plot was allocated eight pats of a standard size (1 kg wet weight and 16 cm in diameter) and known physical characteristics (Table 1). Pats were placed on squares of nylon netting (mesh size 1 cm) to facilitate later retrieval and were covered by wire netting (2 cm mesh) to prevent bird damage. They were placed randomly, but positions previously used for experimental pats and earthworm sampling (involving formaldehyde) were avoided. A numbered plastic tag identified the location of each pat. At weeks 2, 4 and 6 after the placement of the experimental pats, each plot received a further six dung pats collected from the same group of cattle as the pats already in the plot. The additional pats were placed randomly, without any nylon netting, and were intended to simulate dung deposition at normal stocking rates, i.e. eight cattle per hectare each depositing 10 kg of dung per day. These pats were left undisturbed and were, therefore, available as food for the earthworm population throughout the duration of each experiment.The grass was cut to ca. 3 cm and removed from the experimental area every 2 weeks, but was allowed to grow higher around the dung pats to simulate the natural grazing habit of cattle. 2.5. Dung pat sampling and analysis At each plot, two experimental pats (i.e. those on nylon netting) were sampled for analysis every 2
weeks, and hence six pats of each type of dung were removed on each sampling occasion. Prior to each grazing season, the order of pats to be sampled was randomly chosen. The wet weight of each pat was recorded in the field and the pats were brought to the laboratory in plastic bags stored in cool boxes. After storage at 5 ◦ C overnight, invertebrates and vegetation were removed from the pats by hand sorting. Of the invertebrates found, only earthworms were kept (in 70% ethanol) for later counting, weighing and identification. Each pat was carefully disintegrated and homogenised before two sub-samples were dried to constant weight at 105 ◦ C. The dried samples were ground and samples of known weight were re-dried overnight at 105 ◦ C and subsequently incinerated at 550 ◦ C for 2 h for determination of their organic matter content. To determine the proportion of organic matter that had disappeared from the pats in the field, samples of fresh dung (i.e. the mixed dung prior to the formation of artificial pats) were processed similarly. In addition, the total nitrogen content of the dried, pulverised fresh dung was determined by a micro-Kjeldahl procedure as described in Hesse (1971). The percentage of the initial amount of organic matter remaining in the ivermectin and the fenbendazole pats after 2, 4, 6 and 8 weeks of field exposure was tested separately against control pats using an analysis of variance with a Random Plot Effect which deals with two pats per plot separately, but nested (PROC MIXED, SAS system V8) (classification variables: treatment, season, time; two variables referring to the position of the dung pats in the Latin square: column and row). By inspection of residual plots, it was concluded that transformation of the response variable was unnecessary. 2.6. Earthworm sampling In spring, before any dung pats were deposited, the initial earthworm population was estimated. In each of the nine plots, four circular 0.1 m2 frames were laid out and the grass within these circles was cut to ground level and removed. The locations used for earthworm sampling within each plot were randomly chosen before the experiment started. Two or three lots of approximately 2.5 l of 0.3% formalin solution (1.3 dl of 23.4% formaldehyde solution in 10 l of
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water) were applied to each encircled area at 10 min intervals and the emerging earthworms were collected (formalin was applied until no more worms appeared on the soil surface). The earthworm population was then assessed every 8 weeks throughout each grazing season. On these occasions, worms in each plot were sampled in each of four 0.1 m2 circles. Two of these were located at dung-free plot locations (i.e. areas in each experimental plot that had no previous history of dung deposition) and two were centred on 6-week-old experimental pats; 6-week-old pats were chosen because earlier observations (Holter, 1983) suggested that this was the age when maximum numbers of worms might be present. The dung pats were removed immediately before formalin extraction. Collected earthworms were stored in containers with moist tissue paper and brought to the laboratory in cool boxes. The following day worms from each 0.1 m2 plot were counted, weighed and preserved in 70% alcohol before identification. Adult and, when possible, sub-adult worms were identified to species whereas juveniles were separated at the genus level (using descriptions and nomenclature in Sims and Gerard, 1985). The relative contribution of each species to the total fresh biomass was estimated from the weight of preserved specimens (after Holter, 1979). Worms hand-sorted from the dung itself were processed similarly in the laboratory. The mean biomass of earthworms collected under pats or in dung-free locations in the ivermectin and fenbendazole plots were tested separately against the corresponding biomass in the control plots. The SAS procedure PROC MIXED was used as in the analyses of dung pat disappearance (no transformations used). Possible effects of ivermectin or fenbendazole on the occurrence (total biomass) of the most abundant earthworm species and juvenile groups were tested against controls in a linear model (SAS procedure PROC GLM), which included the effect of time. Means were separated by Tukey’s multiple comparisons. Finally, earthworm species were separated into ecological groups and the relative contributions (percentage of the total biomass) of these groups were tested in a linear model similar to the one used for the analysis of single species.
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3. Results 3.1. Disappearance of dung pats The residual amount of organic matter in ivermectin or fenbendazole pats in relation to the control pats varied throughout the different periods (Fig. 2), but the overall effects each year are given in Table 2. Throughout most of 1999 and in the spring/early summer of 2000, significantly higher amounts of organic matter disappeared from the control pats than from pats containing ivermectin. However, in the summer and autumn of 2000, there was a tendency for ivermectin pats to disappear faster. Likewise, the effects of fenbendazole were inconsistent across seasons and years (Fig. 2). Throughout 1999, the disappearance rate of fenbendazole pats was similar to, or lower than that of the control dung, whereas in the following year fenbendazole pats disappeared faster than, or at the same rate as the control pats. Furthermore, positional effects were highly significant in the first season, but absent in 2000 (Table 2). 3.2. Earthworm biomass The mean biomass of earthworms at dung-free plot locations was about 50 g m−2 throughout the spring Table 2 F values of analyses of variance with random plot effects (PROC MIXED, classification variables: season and treatment, as well as row and column (positions) in the Latin square) on the mass of remaining organic matter in dung pats from treated cattle (ivermectin or fenbendazole sustained-release boluses) versus untreated controls Season
Treatment
Season × treatment
Position
1999 Ivermectin Fenbendazole
97.97∗∗∗ 22.58∗∗∗
40.08a,∗∗∗ 30.62a,∗∗∗
1.36 ns 19.98∗∗∗
11.66∗∗∗ 18.64∗∗∗
2000 Ivermectin Fenbendazole
423.97∗∗∗ 334.59∗∗∗
1.06 ns 30.72b,∗∗∗
12.68∗∗∗ 12.90∗∗∗
7.68∗∗ 3.24 ns
ns: not significant. a More organic matter left in the ivermectin and fenbendazole pats than in the control pats. b Less organic matter left in the fenbendazole pats than in the controls. ∗∗ P < 0.01. ∗∗∗ P < 0.001.
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% of initial organic matter +SE
Grazing season 1999 100 90 80 70 60 50 40 30 20 10 0 2
4
6
8
2
May-July
4
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July-Sep.
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% of initial organic matter +SE
Grazing season 2000 100 90 80 70 60 50 40 30 20 10 0 2
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2
June-Aug.
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ivermectin
fenbendazole
Fig. 2. Disappearance of dung pats formed from the dung of untreated cattle or cattle treated with either ivermectin or fenbendazole expressed as the percentage of initial organic matter remaining after 2, 4, 6 and 8 weeks on pasture. Vertical bars indicate S.E. of 6 sampled dung pats per dung type per week.
and summer, increasing slightly in August (Fig. 3). In both years there was a significant seasonal effect (P < 0.001): the biomass was highest in autumn, especially in 2000 when the mean biomass values in all dung-free plot locations were about twice those recorded in 1999. Also, worms were clearly attracted to dung pats in all plots (Fig. 3). The biomass—with or without dung—
in the ivermectin plots did not differ significantly (P > 0.05) from corresponding values in the control plots. In 1999, fenbendazole plots had a lower worm biomass than did the control plots (dung-free locations (P < 0.001) and dung pats (P < 0.05)). However, inexplicable positional effects (P < 0.05) were also significant. By contrast, no significant effects of
T.S. Svendsen et al. / Applied Soil Ecology 24 (2003) 207–218 Worms under dung-free plot locations
Worms under dung-free plot locations 40 Biomass (g) +SE
Biomass (g) +SE
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30 20 10 0
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control
ivermectin
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Fig. 3. Mean biomass of earthworms (per 0.1 m2 ) in experimental plots with dung pats formed from untreated dung or dung containing residues of ivermectin or fenbendazole. Worms were collected by formalin extraction from either dung-free plot locations or from artificial pats exposed to earthworm colonisation for 6 weeks. Vertical bars indicate S.E. of six samples per dung type per time (12 samples per dung type in spring samplings at dung-free plot locations).
fenbendazole treatment or position were detectable in 2000. 3.3. Species composition In both grazing seasons, the earthworm community consisted of 11 species belonging to the following ecological groups (Bouché, 1977). Anecic worms: Lumbricus terrestris (mean adult wet weight 1.81 g) and Aporrectodea longa (1.32 g); endogeic species: Allolobophora chlorotica (0.19 g), Aporrectodea caliginosa (0.33 g), A. rosea (0.15 g), Octolasion cyaneum (1.09 g) and O. tyrtaeum (0.52 g); epigeic worms: L. castaneus (0.12 g), L. rubellus (0.46 g), Eiseniella tetraedra (0.06 g) and Dendrodrilus rubidus (0.06 g). The juvenile worms were separated into two groups: juvenile Lumbricus spp. (mean wet weight 0.10 g) and ‘other juveniles’ (0.13 g). Figs. 4 and 5 show the relative contribution of each group to
the total biomass under dung-free plot locations and in/under dung pats. Sub-adult specimens of L. terrestris (mean wet weight 0.80 g) and A. longa (0.66 g) are included in the anecic group. There were no negative effects of ivermectin or fenbendazole on the biomass of the five most abundant species L. terrestris, A. longa, A. chlorotica, A. caliginosa and L. castaneus when tested separately (data not shown). Generally, the biomass of L. terrestris, A. longa and L. castaneus was significantly higher in the presence of dung pats, whereas the two endogeic species, A. chlorotica and A. caliginosa, were less attracted to dung pats. The remaining species were not tested separately. The abundance of juveniles and adults depended significantly on time of the year. In and under dung pats, the biomass of juvenile Lumbricus spp. was significantly (P < 0.001) higher than under dung-free locations, whereas this was seen for ‘other juveniles’ in 1999 only (P < 0.005). There
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Fig. 4. The relative contribution of ecological groups and juveniles to the total biomass of earthworms extracted in 1999 from plots receiving C: control, I: ivermectin or F: fenbendazole dung.
were no effects of drug treatments on the biomass of juvenile worms.
4. Discussion 4.1. Methodology The concentration of ivermectin or fenbendazole in the dung pats were not determined by chemical analysis. However, the experimental pats (as well as the additional pats) consisted of a mixture of dung from at least seven treated animals. This ensured that these pats did contain residues of parasiticide, even if a bo-
lus in one of the animals had failed to work properly. In both years we observed (visual judgement) a much smaller number of dung-beetle larvae (Aphodius spp.) in the ivermectin pats compared with controls, and their development was clearly stunted. In contrast, we found that the number and development of dung beetle-larvae in pats containing fenbendazole and in control pats were similar. This is in accordance with the findings of Strong et al. (1996) and confirms that ivermectin residues were indeed present in the ‘ivermectin pats’. Any differences in dung pat disappearance due to variations in size and surface area were reduced by the use of standardised, artificially formed pats in contrast
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Fig. 5. Relative contribution of ecological groups and juveniles to the total biomass of earthworms extracted in 2000 from plots receiving C: control, I: ivermectin or F: fenbendazole dung.
to naturally voided pats studied by Barth et al. (1993, 1995) and Wratten et al. (1993). The dung pats were deposited in a cattle-free enclosure and covered by netting to prevent any differences due to trampling by cattle or disturbance by birds. Finally, because the dung pats were placed on pieces of net, the sampling was standardised as the material above the net was defined as remaining dung, whereas everything below the net was regarded as soil. Contamination with soil, probably due to earthworm activity, may strongly increase the dry weight of dung pats and hence invalidate studies where the degradation of dung pats has been measured as simple dry weight loss (Wall and Strong, 1987; Wratten et al., 1993). To overcome this
difficulty, at least in part, we quantified dung disappearance in terms of decline of the amount of organic matter in the pats as recommended by Holter (1979) and Herd et al. (1993). The initial moisture content of dung pats has been seen as an important factor affecting the rate of dung disappearance. Barth et al. (1993, 1995) have pointed out the lack of this information in several papers dealing with the effect of ivermectin on decomposition, as dung from untreated cattle might have higher moisture content as a result of infection with parasitic nematodes. According to Barth et al. (1993, 1995), even small differences in moisture content of 1–2% may have an effect on dung disappearance, i.e. the
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higher water content leads to an increase in dung disappearance. However, we found no correlation (r = 0.07–0.15) between initial moisture content of the dung (which did not differ much between paddocks, cf. Table 1) and the subsequent pat degradation at any time after deposition. Furthermore, judged from our observations on C/N ratios (Table 1), the dung from the three paddocks did not differ substantially as regards other basic characteristics. Hence, it seems unlikely that the present results have been seriously affected by the necessary use of three, apparently similar, paddocks that were managed in the same way. 4.2. Dung pat disappearance and abundance of earthworms The present study confirms that dung disappearance is a result of several factors of which drug residues is only one. In this comparison of ivermectin, fenbendazole and control pats, differences in the disappearance could not be ascribed to drug treatment alone. The effect of season was highly significant, and so was the location of dung pats within the experimental area. On the other hand, there were no strong toxic effects of faecally excreted ivermectin or fenbendazole on pastureland earthworm populations following bolus implantation in grazing cattle. Likewise, there was no indication of important negative, indirect effects such as lower attractiveness of dung from treated animals. These results are supported by laboratory experiments (Svendsen et al., 2002; Svendsen et al., unpublished) in which long-term exposure of ivermectin or fenbendazole in dung had no lethal or adverse sub-lethal effects on L. terrestris. The effect of season on the disappearance of dung is probably a result of the influence of weather on earthworm activity (Holter, 1979). In both years, the highest disappearance rate of dung occurred in autumn, with high soil humidity and low temperatures (Fig. 1) and hence a high biomass of (active) earthworms (Figs. 2 and 3). Moreover, weather may also have affected the activity of insects, including their impact on dung disappearance. Thus, the faster disappearance of fenbendazole pats compared with those containing ivermectin in the summers of 1999 and 2000, i.e. during periods of high soil temperatures (Fig. 1) and low earthworm activity, might be attributed to the fact that ivermectin is toxic to insects
whereas fenbendazole is not. Reduced insect activity in the ivermectin dung could explain part of the delay in pat disappearance compared with the controls, as there were no differences in earthworm abundance between treatments. Furthermore, compared with 1999, dung pats in all treatments disappeared faster in the autumn of 2000 which experienced heavy rainfall soon after the placement of experimental pats in September (Fig. 1). The associated weathering of these newly exposed pats may have facilitated the dung dispersal by earthworms and thus made up for any missing insect activity (due to the ivermectin treatment) that might otherwise have delayed the disappearance of pats. Earlier studies in Denmark showed delayed disappearance of dung from cattle given ivermectin either as a subcutaneous injection of 200 g kg−1 or as a 0.5 mg kg−1 pour-on treatment (Madsen et al., 1990; Sommer et al., 1992). These studies followed the disappearance of pats in the field from August and throughout the autumn in a single year. The same result was seen in the present study in late summer and autumn in 1999 but not in 2000, which emphasises the importance of season and weather for dung disappearance, and the value of longer-term studies. Herd et al. (1996) measured steady-state ivermectin concentrations of 2.9–4.0 g g−1 dry weight and Alvinerie et al. (1998) measured steady-state concentrations of about 9 g g−1 dry weight in dung from cattle administered with the same sized bolus as that used in the current study. The concentrations of ivermectin in the dung used by Madsen et al. (1990) and Sommer et al. (1992) were up to 9.0 g g−1 dry weight (at 1–2 days after pour-on treatment, maximum concentration after injection was 3.9 g g−1 dry weight). However, Madsen et al. (1990) and Sommer et al. (1992) observed a reduced disappearance rate of dung produced up to 20 days after ivermectin administration (pour-on or injection), when the concentration in the dung was probably below 0.3 g g−1 dry weight (Sommer et al., 1992; Sommer and Steffansen, 1993; Herd et al., 1996). Hence, the lack of consistent effects of ivermectin on dung disappearance in the current study is unlikely to be a result of low residual concentrations. In our study, all dung pats, including the untreated controls, disappeared at a higher rate than the control pats in the earlier Danish field studies, indicating a more abundant earthworm fauna in our study and thus a less pronounced effect on dung disappearance due to missing
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insect activity. The high activity of earthworms may be a result of the regular addition of dung pats in each plot (i.e. the addition of dung pats every second week besides the experimental pats), which is an improvement in comparison with the earlier studies, and essential to investigate long-term effects on the earthworm population. 4.3. Concluding remarks The results reported here suggest that under temperate field conditions with a high level of earthworm activity, disappearance of dung pats from cattle administered sustained-release boluses of ivermectin and fenbendazole is not significantly affected by the faecally-excreted drug residues. Earthworms were equally attracted to dung pats irrespective of drug treatment and no long-term effects on the studied population were observed. Faecally-excreted ivermectin has detrimental effects on the activity of insect larvae in dung pats, but the impact on dung disappearance may be overridden by the effect of earthworms, when these are favoured by suitable weather. This partly explains the different findings in earlier field trials under varying, albeit temperate climatic conditions.
Acknowledgements We are grateful to Lise and Jørgen Due for access to cattle and pastureland. Malene Michel, Jan Tækker Christiansen, Hanne Lynge Christiansen, Hanne Rawat, Annette Spangenberg, Leif Stausholm Jensen and Torben Frost are thanked for their excellent technical assistance. Thanks to Torben Martinussen and Ib Skovgaard for statistical advice and to Keith Wardhaugh for improving the manuscript. Valuable comments from John Finn and an anonymous reviewer are also gratefully acknowledged. The Danish Agricultural and Veterinary Research Council financially supported this study. References Alvinerie, M., Sutra, J.F., Galtier, P., Lifschitz, A., Virkel, G., Sallovitz, J., Lanusse, C., 1998. Persistence of ivermectin in plasma and faeces following administration of a sustainedrelease bolus to cattle. Res. Vet. Sci. 66, 57–61.
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