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The impacts of molluscicide pellets on spring and autumn populations of wood mice Apodemus sylvaticus
Agriculture Ecosystems & Environment ELSEVIER
Agriculture, Ecosystems and Environment64 (1997) 211-217
The impacts of molluscicide pellets on spring and autumn populations of wood mice Apodemus sylvaticus R.F. Shore a,*, R.E. Feber b L.G. Firbank a, S.K. Fishwick a, D.W. Macdonald b U. NCrum a a N.E.R.C., Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon PE17 2LS, UK b Wildlife Conservation Research Unit, Department of Zoology, Universi O, of Oxford, South Parks Road, Oxford OX1 3PS, UK
Accepted 3 January 1997
Abstract Methiocarb is a carbamate molluscicide widely used in pelleted form. Wood mice Apodemus sylvaticus are commonly found in arable fields and will eat methiocarb pellets. Two previous field studies on the impacts on arable wood mice of broadcasting methiocarb pellets produced contrasting results, the number of mice being temporarily reduced in one but not the other. The season of application is also likely to influence the degree of impact on populations but the previous trials were both in autumn. The aim of the present study was to determine the effects on wood mice of broadcast applications of methiocarb in both autumn and spring and to determine whether depression of brain and serum cholinesterase (ChE) activity, a biomarker of exposure, could be detected in mice alive on treated fields after application. In trials carried out in October 1994 and April 1995, there were decreases in the number of wood mice on arable fields following pesticide application. The October decline (78%) was greater than that in April (33%), even though the activity of the mice appeared to be more heavily centred on the hedgerows, where pellets were not applied, in the autumn. Despite the reductions in numbers, no depression of ChE activity was detected in mice captured alive after application, suggesting that either exposed animals were not captured or that reactivation of ChE activity was rapid. This study confirmed that broadcast application of methiocarb can reduce wood mouse populations on arable fields. It also demonstrated that such effects can occur irrespective of season and that measurement of ChE activity in mice trapped after application may fail to indicate that exposure of the population has occurred. © 1997 Elsevier Science B.V. Keywords: Methiocarb;Wood mouse; Populations;Cholinesterase;Biomarker
1. Introduction The ban on straw burning in the early 1990s in the U K and the subsequent increase in the ploughing-in of stubble is expected to favour slugs and
* Corresponding author. Tel.: 44-1487-773381; fax: 44-1487773467; e-mail: [email protected]
result in a rise in use of molluscicides. Methiocarb is a non-specific carbamate pesticide widely used to control slugs and snails. It is usually applied as pellets with a cereal base and so is potentially attractive to other, non-target, species such as small mammals. The wood mouse A p o d e m u s s y l v a t i c u s is the commonest small mammal species in arable fields (Green, 1979) and is the only one found consistently through the year (Tew, 1994). It has been shown to
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eat methiocarb pellets (Tarrant and Westlake, 1988) and, therefore, is particularly at risk of exposure. The impacts of broadcast application of methiocarb pellets on arable wood mice were investigated in two years of autumn field trials by Tarrant et al. (1990) who found no changes in capture rates of mice before and after application. Similar trials were also carried out as part of the Boxworth project (Greig-Smith et al., 1992). In contrast to the earlier study, there was a significant decrease in the wood mouse population after broadcast application which was followed by a rapid and sustained recovery, apparently resulting from immigration by juveniles (Johnson et al., 1991). There was no long term decline in numbers, even when autumn treatments were carried out over 6 years, probably because there was a large pool of dispersing animals each year which immigrated into the area. However, the timing of application of a pesticide is likely to be critical (Evans, 1990; Shore and Douben, 1994). Application of pellets in the spring or summer, for instance on potato crops, could have a more marked impact on mouse populations than an autumn treatment because numbers are naturally low at this time and there are few dispersing juveniles which may recolonise depleted areas (Johnson et al., 1991). There have been no reported field studies on the impacts of molluscicides on arable mice in seasons other than autumn. In this study, we investigated the effects on wood mice of broadcast applications of methiocarb to cereal fields carried out in both autumn and spring. This involved capture-mark-release (CMR) studies both before and after application to detect changes in population size. A biomarker, inhibition of cholinesterase (ChE) activity, was used to determine whether there was evidence of exposure of individual mice to methiocarb.
2. Methods 2.1. Experimental area and treatment
Experiments were undertaken at the University of Oxford farm, Wytham, Oxfordshire, UK (grid reference: SP4609). In autumn 1994, three adjacent treatment fields of winter wheat (5.2, 4.5 and 10 ha) and one control field (7.3 ha), which was separated from
the others by a lane, were used. Mice captured in any one field were never captured in any of the other fields during the course of the field trial. In the following spring, the control field was excluded because of poor capture success in the autumn; methiocarb was applied again to only two of the three previously-treated fields and the third (10 ha) field became the control. It was assumed that there would be no carry-over effect from the autumn application, as was the case in the study by Johnson et al. (1991). Methiocarb pellets (Draza, 4% w / w active ingredient, Bayer, Germany) were broadcast on the 10th October 1994 (autumn) and 8th April 1995 (spring) at the commercially recommended rate of 5.5 kg ha- J ; both days were dry.
2.2. Determination of impacts of molluscicide on mouse populations
Square, 2.82 ha grids, consisting of 64 trapping points in an 8 X 8 formation, were positioned in the corner of each field so that two adjacent edges of the grid were in the hedgerow boundaries. The trapping points were regularly spaced at 24 m intervals (Tew et al., 1994a) and one Longworth trap was placed at each trapping point. Thus, 16 traps were in the hedges, the rest in the field. Traps were baited with wheat and blowfly pupae and morning and evening trap rounds were carried out. Captured mice were sexed, assessed for breeding condition, marked with a fur clip and weighed before being released; animals weighing less than 15.5 g were classed as juveniles (Flowerdew and Gardener, 1978). In the autumn, trapping took place for five nights immediately before and three nights immediately after application. Two more nights of trapping were carried out approximately one week after application. In the following spring, trapping was carried out on the same grids on the three fields that were used. However, this time, the traps were left on pre-bait for two nights before being set to catch for five nights immediately before pesticide application. Trapping was then continued for the five nights following application. Thus, in both autumn and spring, the trapping effort before and after molluscicide application was the same (320 trap-nights), allowing direct comparison of trap data.
R. F. Shore et al. / Agriculture, Ecosystems and Environment 64 (1997) 211-217
2.3. Determination of exposure to methiocarb Methiocarb is an anti-ChE pesticide and measuring inhibition of ChE activity is a widely used technique to indicate exposure to such compounds (Thompson, 1991). Brain acetylcholinesterase (ACHE) and serum ChE activities were measured in a randomly selected sample of wood mice trapped on treated fields in the autumn and the spring. To reduce the number of animals that had to be killed, control animals from non-treated fields were not analysed. Instead, data for animals from treated fields were compared with published data on ChE activity in non-exposed wood mice which were measured in a large number of animals using identical analytical techniques (Fishwick et al., 1996). Wood mice which were captured after application of pesticide to fields at the University farm were transported immediately to the laboratory. They were sacrificed by cervical dislocation followed by decapitation and collection of trunk blood. Blood was allowed to clot, separated by centrifugation (2000 rev min -I at 4°C for 10 min) and the serum kept on ice until analysed. The brain was excised, weighed, and homogenised in 25 mM Tris-HC1 buffer (containing 0.1% Triton X-1000) to produce an homogenate of 0.06 g m l - 1; aliquots of this were kept on ice until analysed. The ChE activities in blood and serum were analysed on the day of collection using the colorimetric method of Ellman et al. (1961) as adapted and fully described by Fishwick et al. (1996). Acetylthiocholine iodide was used as the substrate in the assay and between three and five replicate determinations were made on each sample. A commercial reference material (Precinorm U, Boehringer Mannheim, Lewes) was used to confirm that no significant instrument drift occurred and that assay performance was consistent between days and with that used to measure ChE activity in non-exposed animals in the study by Fishwick et al. (1996).
2.4. Statistical analysis Population data were analysed by calendar of captures to calculate the number of individual mice known to be alive before and after application of methiocarb; it was assumed that natural mortality was negligible in the five days immediately before
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application both in the autumn and spring. Comparisons between the observed and expected numbers of mice trapped in the hedge and field were made using Chi-squared tests with Yates' correction. Fisher's Exact test was used to compare both the adult:juvenile and male:female ratios of mice caught before and those captured for the first time after molluscicide application. Data on ChE activities are presented as means and standard deviations of replicate determinations for each mouse.
3. Results
3.1. Population responses In the October trial, no mice were caught in the control field before or after molluscicide application and so population data were only available for the treated fields. The numbers of mice on the three treatment fields were also low, and so data for each field were combined. Analysis of the number of total captures of wood mice in the hedges and fields suggested that the activity of mice focused predominantly on the hedges, there being significantly more captures in hedges than expected from the distribution of the traps (Table 1; X¢]) = 80.3, P < 0.001). Despite this, there was evidence of a marked decline in numbers following application of pellets to the fields (Table 2) with only three of the 41 mice alive before molluscicide application being captured afterwards. There was no evidence of a shift in the sex
Table 1 Distribution of traps and total captures (including recaptures) of wood mice Apodemus sylvaticus in hedges and fields before methiocarb application. Data for different treatment fields are combined Field
Hedge
Total no. of traps Treated fields in autumn Treated fields in spring Control field (both seasons)
144 96 48
48 32 16
Total wood mouse captures Treated fields in autumn Treated fields in spring Control field in spring
11 46 8
42 15 0
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Table 2 Numbers of wood mice Apodemus sylvaticus known to be alive on treatment fields (data for different fields combined) before and after application of methiocarb and on the treatment-free control field. Trapping effort was always equal before and after application No. of wood mice Treatment fields
Control field
Autumn
Spring
Spring
41 9 78
30 20 33
3 1 67
Captured before and after application
3
12
1
% of those caught before that were captured afterwards
7
40
33
Before pesticide application After pesticide application % decline
ratio or age structure of the wood mouse population following pesticide treatment. Twenty-six of the 41 mice captured before application were female (63%)
and 22 were adults (54%) while, of the six new mice caught afterwards, all but one were females (83%) and three were adults (50%). The differences in sex ratio and adults:juvenile ratio between the pre- and post-application populations were not statistically significant (Fisher's Exact test P > 0.05 in both cases). In the April trial, the number of total captures in the hedge and the field were almost exactly in proportion to the distribution of the traps (Table 1), suggesting that activity was centred less on the hedges than in the previous October. Only three mice were captured on the control field, two adult males and an adult female (Table 2). In total, 38 mice were trapped on the treatment fields and there was a decline in the numbers of individuals captured following application of methiocarb (Table 2). This was significantly less pronounced than in the previous autumn (Table 2) in terms of both the loss of marked individuals (Fisher's Exact test comparing between seasons, P = 0.01) and the decline in the total number of
Table 3 Brain and serum cholinesterase activities (mean _+ SD) in a sample of wood mice captured on treated fields after application of methiocarb. Application dates were 10 October 1994 and 8 April 1995 Date of capture
Sex
Breeding condition a
Body weight (g)
Brain AChE activity b (Ixmol rain-i g - i)
12/10/1994 11/10/1994 12/10/1994 12/10/1994 12/10/1994 13/10/1994 10/4/1995 10/4/1995 10/4/1995 10/4/1995 10/4/1995 11/4/1995 11/4/1995 13/4/1995 10/4/1995 11/4/1995 11/4/1995 13/4/1995
M F F F F F M M M M M M M M F F F F
TM Imperf Parous Parous Parous Parous TS TS TS TS TS TS TS TS Perf Perf Parous Parous
14.2 13.6 25.5 26.4 17.3 20.2 21.1 24.6 22.8 24.8 22.4 26.0 27.8 25.7 21.7 18.9 15.7 23.8
12.9 (+0.21) 12.8 (+0.09) 9.80 (_+0.14) 6.40 (_+0.30) 12.1 (_+0.12) 12.0 ( + 0.05) 11.8 (+0.14) 10.7 ( + 0.05) 11.6 (_+0.14) 10.0 (_+0.05) 11.2 ( _+0.39) 10.8 (_+0.17) 11.7 (-+0.49) 12.0 (-+0.43) 11.7 ( _+0.14) 12.4 ( -+ 0.07) 12.9 (-+0.11) 12.4 ( _+0.86)
Serum ChE activity ~ (Izmol min- i ml- l) 2.37 3.16 2.86 2.45
(_+0.02) (__+0.03) (_+0.18) (_+0.16)
2.35 ( ± 0.03) 2.56 (±0.06) 4.11 ( _+0.20) 3.35 (_+0.05) 1.95 (_+0.10) 3.20 ( ± 0.04) 3.35 (-+0.09) 3.03 (±0.15) 3.63 (±0.05) 2.10 ( -+ 0.03) 2.98 ( -+ 0.08) 3.15 (_+0.07) 2.87 ( _+0.00)
TM, testes medial; TS, testes scrotal; perf, perforate; imperf, imperforate. b Brain AChE activity in non-exposed wood mice is usually in the range 10-12 tzmol min - i g - ~. The defined 99% lower confidence limit for normal activity is 8 ~mol min- ~ g - r (Fishwick et al., 1996). c Mean ( + SE) serum ChE activity in non-exposed wood mice is 2.04 ( ± 0.05) I~mol min- I ml- l (n = 93, M + F combined) and there is no significant difference between the sexes (Fishwick et al., 1996).
R.F. Shore et al. / Agriculture, Ecosystems and Environment 64 (1997) 211-217
animals trapped (Fisher's Exact test, P < 0.05). There was no evidence of a shift in either the sex ratio or the population structure following methiocarb application (Fisher's Exact test, P > 0.05 in both cases); five adult males and three adult females were captured for the first time after application of methiocarb and this was an almost identical male:female and adult:juvenile ratio to that of mice captured before pesticide treatment (19 males: 11 females, 87% adults). No dead mice were found on the trapping grids following pesticide application in either October or April. In the two trials combined, 15 mice captured before application of methiocarb were subsequently captured afterwards (Table 2). There was no significant weight loss or gain in these animals during this period (mean ___SE change: - 0 . 1 0 + 0.32 g).
3.2. Detection of exposure of individuals Six mice were analysed for ChE activity in the autumn and 12 in the spring. The levels of brain AChE activity in these mice were typically between 9.8 and 12.9 p~mol min i g - l , the normal range (Table 3). Serum ChE activity levels were similar in males and females and the mean ( + S E ) activity (sexes combined) was 2.91 (+__0.14) txmol min -1 ml-1 (n = 17), significantly greater (Student's t-test, t~m8) = 6.66, P < 0.001) than that measured in laboratory-reared, non-exposed wood mice (Table 3). In the autumn field trial, there was one female mouse with a significantly depressed brain AChE activity of 6.4 ixmol min -1 g - i However, the serum ChE activity in this animal was 2.45 Ixmol min-~ m1-1. This was within the range measured in the other mice captured on the treatment fields which had normal brain AChE activities (Table 3).
4. Discussion The reduction in the numbers of wood mice captured following methiocarb treatment in the autumn is consistent with that observed by Johnson et al. (1991) at Boxworth. Thus, the present study provides further evidence that broadcast application of methiocarb pellets at this time of year can cause a marked decline in arable wood mouse populations.
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Unlike the Boxworth study, there was no statistically significant shift towards a greater proportion of juveniles in the population following pesticide application. This was probably because few new mice were captured on the fields after treatment and, although these were all juveniles, the pre-treatment population already contained a high proportion (54%) of young mice. The present study is the first, as far as we are aware, to investigate the impacts on arable mice of broadcasting methiocarb pellets in spring. Treatment of fields in spring had a similar effect to that in autumn in that there was a decline in the numbers of mice captured, but it was less severe. A less marked impact in April than October was unexpected for two reasons. First, wood mouse densities are normally low in spring (Flowerdew, 1985) and there are few dispersing mice to recolonise depleted areas. However, there was no apparent shortage of immigrants in April in the present study, the number of new individuals captured after application being similar to that in the previous October. Second, the pattern of total captures before pesticide application suggested that mouse activity was more heavily concentrated in the hedges, where pellets were not applied, in the autumn than in the spring. Other studies have also suggested that mice are predominantly found in the hedgerows in arable habitats in autumn (Tew, 1994; Tew et al., 1994b). Hence, it might be expected that mice would be less likely to encounter pellets in autumn than in spring. It is possible that the difference in degree of impact that methiocarb had on mouse populations in October and April may have been due to seasonal variation in factors such as availability of food other than pellets, behavioural response (such as neophobia) to the pellets and the ease with which pellets were found. However, the results of this study need to be replicated to confirm that there truly are seasonal differences in the degree of impact that methiocarb treatment has on wood mouse populations. Inhibition of brain and blood ChE activity is a well established biomarker of exposure to anti-ChE pesticides, including methiocarb, and has been used in previous studies on wood mice (e.g. Westlake et al., 1980; Tarrant et al., 1990; Hardy et al., 1993; Dell'Omo and Shore, 1996; Dell'Omo et al., 1996). Furthermore, serum ChE activity has been shown to
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R.F. Shore et al. /Agriculture, Ecosystems and Environment 64 (1997) 211-217
Table 4 Estimated density of pellets per square metre on treated fields and the number of pellets equivalent to an oral LD50 dose for a wood mouse weighing 14 g (upper figures) and 28 g (lower figures). Values for mouse weights are based on the range in Table 3 Est. LDs0 for wood mouse a (mg)
Mean + SE weight of pellets b (n = 24) (mg)
Weight of active ingredient per pellet c (mg)
No. of pellets per wood mouse LDs0 dose
No. of pellets m -2 of field d
0.18-1.89 0.36-3.78
18.4 + 3.6
0.74
0.25-2.57 0.49-5.14
30
a LDs0 values are based on the range (13-135 mg kg -I ) for oral exposure in the rat (Hayes and Laws, 1991). b R.F. Shore, 1996, unpublished data. c Pellets contain 4% w / w active ingredient. Calculated from value for mean weight of pellets in table and assuming recommended rate of application of 5.5 kg h a - t.
be more sensitive and to recover more slowly than brain AChE activity in wood mice following exposure to an OP pesticide (Dell'Omo et al., 1996). Given the reduction in the numbers of wood mice following methiocarb application in the present study, it was surprising that reduced ChE activities were not detected in at least some of the surviving animals. The one mouse which did have an abnormally low brain AChE activity did not have inhibited serum ChE activity, which suggested that the reduced brain activity may not have been a result of exposure to methiocarb. The overall lack of detectable ChE depression in the surviving mice suggests that either mice which were exposed did not enter traps, only non-exposed animals being captured, or that spontaneous recovery of ChE activity in sub-lethally exposed mice was rapid. Failure to catch exposed animals may have been because there was an 'all or nothing' effect in that some mice found pellets and subsequently consumed a lethal dose, after which they quickly died, while others failed to locate or ingest pellets at all. It can be calculated that wood mice probably need to eat only between a quarter and five pellets to ingest an oral LDs0 dose of methiocarb and the density of pellets on fields is high (Table 4), facilitating their discovery by foraging mice. However, it is also possible that mice captured on the fields had been exposed to methiocarb and that their ChE activity had spontaneously reactivated (Martin et al., 1981). The elevated serum ChE levels in these animals might thus be explained by 'overcompensation' where, after exposure, serum ChE activities can rebound to levels above normal (Thompson, 1991). Whatever the reason for the lack of detectable ChE
inhibition in mice captured after pesticide treatment, the results of this study do suggest that monitoring for depression in ChE activity after application may fail to indicate that exposure of the population to molluscicide pellets has occurred. In conclusion, the results of this study confirm that broadcast application of methiocarb can reduce populations of wood mice and such effects can occur irrespective of the season when application is carried out. One of the most important potential consequences of this is that predators could be adversely affected either due to a reduction in prey availability or by secondary poisoning. The effects of reduced mouse numbers on predators are likely to depend upon the time taken for the population to recover and this, in turn, is likely to depend upon the proximity of source areas which contain dispersing mice. Effects on predators may, therefore, be site-specific. However, the potential for secondary poisoning of avian and mammalian predators appears to be high. Tawny owl Strix aluco, kestrel Falco tinunculus and weasel Mustela nivalis all prey on wood mice in arable areas and have body weights in the region of 500 g, 200 g and 60-120 g, respectively (Mikkola, 1983; Village, 1990; King, 1991). Using an oral LDs0 value of 10 mg kg -j for the raptors (the median of the values reported by Schafer (1972) for 17 wild bird species) and the rat oral LDs0 values for the weasel, it can be calculated that the amount of methiocarb equivalent to an LD50 dose in tawny owls, kestrels and weasels is 5 mg, 2 mg and 0.816.2 mg, respectively. These doses are similar or within the range of the amounts of methiocarb estimated to be taken in by a single mouse which has ingested the equivalent of an LDso dose (Table 4).
R.F. Shore et al. / Agriculture, Ecosystems and Environment 64 (1997) 211-217
Given these estimates, the potential impacts of molluscicide application on predator populations merit further investigation.
Acknowledgements This work was funded by the Natural Environment Research Council as part of the Organic Farming Study, which is also funded by the Biotechnology and Biological Sciences Research Council and the Economic and Social Research Council. We are grateful to G. Berry and E. Brown for assistance in the field and to G. Dell'Omo for comments on an earlier version of the manuscript.
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Smith, P.W., Fletcher, M.R., Tarrant, K.A., Stanley, P.I., 1993. An intensive field trial to assess hazards to birds and mammals from the use of methiocarb as a bird repellent on ripening cherries. Ecotoxicology 2, 1-31. Hayes, W.J., Laws, E.R. (Eds.), 1991. Handbook of Pesticide Toxicology. Academic Press, San Diego, 1576 pp. Johnson, I.P., Flowerdew, J.R., Hare, R., 1991. Effects of broadcasting and of drilling methiocarb molluscicide pellets on field populations of wood mice, Apodemus sylvaticus. Bull. Environ. Contam. Toxicol. 46, 84-91. King, C.M., 1991. Weasel. In: Corbet, G.B., Harris, S. (Eds.), The Handbook of British Mammals. Blackwell Scientific Publications, Oxford, pp. 387-395. Martin, A.D., Norman, G., Stanley, P.I., Westlake, G.E., 1981. Use of reactivation techniques for differential diagnosis of organophosphorus and carbamate pesticide poisoning in birds. Bull. Environ. Contam. Toxicol. 26, 775-780. Mikkola, H., 1983. Owls of Europe. Poyser, Calton, 397 pp. Schafer, E.W., 1972. The acute oral toxicity of 369 pesticidal, pharmaceutical and other chemicals to wild birds. Toxicol. Appl. Pharmacol. 21,315-330. Shore, R.F., Douben, P.E.T., 1994. Predicting ecotoxicological impacts of environmental contaminants on terrestrial small mammals. Revs. Environ. Contam. Toxicol. 134, 49-89. Tarrant, K.A., Westlake, G.E., 1988. Laboratory evaluation of the hazard to wood mice, Apodemus sylvaticus, from the agricultural use of methiocarb molluscicide pellets. Bull. Environ. Contam. Toxicol. 40, 147-152. Tarrant, K.A., Johnson, I.P., Flowerdew, J.R., Greig-Smith, P.W., 1990. Effects of pesticide applications on small mammals in arable fields, and the recovery of their populations. In: British Crop Protection Conference--Pests and Diseases. The British Crop Protection Council, Farnham, pp. 173-182. Tew, T., 1994. Farmland hedgerows: habitat, corridors or irrelevant? A small mammals perspective. In: Watt, T.A., Buckley, G.P. (Eds.), Hedgerow Management and Nature Conservation. Wye College Press, Wye, pp. 80-94. Tew, T.E., Todd, I.A., Macdonald, D.W., 1994a. The effects of trap spacing on population estimates of small mammals. J. Zool. 223, 340-344. Tew, T.E., Todd. I.A., Macdonald, D.W., 1994. Field margins and small mammals. In: Boatman, N. (Ed.), Field Margins: Integrating Agriculture and Conservation. B.C.P.C. Monograph No. 58, Surrey, pp. 85-94. Thompson, H.M., 1991. Serum 'B' esterases as indicators of exposure to pesticides. In: Mineau, P. (Ed.), Cholinesterase-inhibiting Insecticides, their Impact on Wildlife and the Environment. Elsevier, Amsterdam, pp. 109-125. Village, A., 1990. The Kestrel. Poyser, London, 352 pp. Westlake, G.E., Blunden, C.A., Brown, P.M., Bunyan, P.J., Martin, A.D., Sayers, P.E., Stanley, P.I., Tarrant, K.A., 1980. Residues and effects in mice after drilling wheat treated with chlorfenvinphos and an organomercurial fungicide. Ecotoxicol. Environ. Safety 4, 1-16.
Agriculture, Ecosystems and Environment64 (1997) 211-217
The impacts of molluscicide pellets on spring and autumn populations of wood mice Apodemus sylvaticus R.F. Shore a,*, R.E. Feber b L.G. Firbank a, S.K. Fishwick a, D.W. Macdonald b U. NCrum a a N.E.R.C., Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon PE17 2LS, UK b Wildlife Conservation Research Unit, Department of Zoology, Universi O, of Oxford, South Parks Road, Oxford OX1 3PS, UK
Accepted 3 January 1997
Abstract Methiocarb is a carbamate molluscicide widely used in pelleted form. Wood mice Apodemus sylvaticus are commonly found in arable fields and will eat methiocarb pellets. Two previous field studies on the impacts on arable wood mice of broadcasting methiocarb pellets produced contrasting results, the number of mice being temporarily reduced in one but not the other. The season of application is also likely to influence the degree of impact on populations but the previous trials were both in autumn. The aim of the present study was to determine the effects on wood mice of broadcast applications of methiocarb in both autumn and spring and to determine whether depression of brain and serum cholinesterase (ChE) activity, a biomarker of exposure, could be detected in mice alive on treated fields after application. In trials carried out in October 1994 and April 1995, there were decreases in the number of wood mice on arable fields following pesticide application. The October decline (78%) was greater than that in April (33%), even though the activity of the mice appeared to be more heavily centred on the hedgerows, where pellets were not applied, in the autumn. Despite the reductions in numbers, no depression of ChE activity was detected in mice captured alive after application, suggesting that either exposed animals were not captured or that reactivation of ChE activity was rapid. This study confirmed that broadcast application of methiocarb can reduce wood mouse populations on arable fields. It also demonstrated that such effects can occur irrespective of season and that measurement of ChE activity in mice trapped after application may fail to indicate that exposure of the population has occurred. © 1997 Elsevier Science B.V. Keywords: Methiocarb;Wood mouse; Populations;Cholinesterase;Biomarker
1. Introduction The ban on straw burning in the early 1990s in the U K and the subsequent increase in the ploughing-in of stubble is expected to favour slugs and
* Corresponding author. Tel.: 44-1487-773381; fax: 44-1487773467; e-mail: [email protected]
result in a rise in use of molluscicides. Methiocarb is a non-specific carbamate pesticide widely used to control slugs and snails. It is usually applied as pellets with a cereal base and so is potentially attractive to other, non-target, species such as small mammals. The wood mouse A p o d e m u s s y l v a t i c u s is the commonest small mammal species in arable fields (Green, 1979) and is the only one found consistently through the year (Tew, 1994). It has been shown to
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eat methiocarb pellets (Tarrant and Westlake, 1988) and, therefore, is particularly at risk of exposure. The impacts of broadcast application of methiocarb pellets on arable wood mice were investigated in two years of autumn field trials by Tarrant et al. (1990) who found no changes in capture rates of mice before and after application. Similar trials were also carried out as part of the Boxworth project (Greig-Smith et al., 1992). In contrast to the earlier study, there was a significant decrease in the wood mouse population after broadcast application which was followed by a rapid and sustained recovery, apparently resulting from immigration by juveniles (Johnson et al., 1991). There was no long term decline in numbers, even when autumn treatments were carried out over 6 years, probably because there was a large pool of dispersing animals each year which immigrated into the area. However, the timing of application of a pesticide is likely to be critical (Evans, 1990; Shore and Douben, 1994). Application of pellets in the spring or summer, for instance on potato crops, could have a more marked impact on mouse populations than an autumn treatment because numbers are naturally low at this time and there are few dispersing juveniles which may recolonise depleted areas (Johnson et al., 1991). There have been no reported field studies on the impacts of molluscicides on arable mice in seasons other than autumn. In this study, we investigated the effects on wood mice of broadcast applications of methiocarb to cereal fields carried out in both autumn and spring. This involved capture-mark-release (CMR) studies both before and after application to detect changes in population size. A biomarker, inhibition of cholinesterase (ChE) activity, was used to determine whether there was evidence of exposure of individual mice to methiocarb.
2. Methods 2.1. Experimental area and treatment
Experiments were undertaken at the University of Oxford farm, Wytham, Oxfordshire, UK (grid reference: SP4609). In autumn 1994, three adjacent treatment fields of winter wheat (5.2, 4.5 and 10 ha) and one control field (7.3 ha), which was separated from
the others by a lane, were used. Mice captured in any one field were never captured in any of the other fields during the course of the field trial. In the following spring, the control field was excluded because of poor capture success in the autumn; methiocarb was applied again to only two of the three previously-treated fields and the third (10 ha) field became the control. It was assumed that there would be no carry-over effect from the autumn application, as was the case in the study by Johnson et al. (1991). Methiocarb pellets (Draza, 4% w / w active ingredient, Bayer, Germany) were broadcast on the 10th October 1994 (autumn) and 8th April 1995 (spring) at the commercially recommended rate of 5.5 kg ha- J ; both days were dry.
2.2. Determination of impacts of molluscicide on mouse populations
Square, 2.82 ha grids, consisting of 64 trapping points in an 8 X 8 formation, were positioned in the corner of each field so that two adjacent edges of the grid were in the hedgerow boundaries. The trapping points were regularly spaced at 24 m intervals (Tew et al., 1994a) and one Longworth trap was placed at each trapping point. Thus, 16 traps were in the hedges, the rest in the field. Traps were baited with wheat and blowfly pupae and morning and evening trap rounds were carried out. Captured mice were sexed, assessed for breeding condition, marked with a fur clip and weighed before being released; animals weighing less than 15.5 g were classed as juveniles (Flowerdew and Gardener, 1978). In the autumn, trapping took place for five nights immediately before and three nights immediately after application. Two more nights of trapping were carried out approximately one week after application. In the following spring, trapping was carried out on the same grids on the three fields that were used. However, this time, the traps were left on pre-bait for two nights before being set to catch for five nights immediately before pesticide application. Trapping was then continued for the five nights following application. Thus, in both autumn and spring, the trapping effort before and after molluscicide application was the same (320 trap-nights), allowing direct comparison of trap data.
R. F. Shore et al. / Agriculture, Ecosystems and Environment 64 (1997) 211-217
2.3. Determination of exposure to methiocarb Methiocarb is an anti-ChE pesticide and measuring inhibition of ChE activity is a widely used technique to indicate exposure to such compounds (Thompson, 1991). Brain acetylcholinesterase (ACHE) and serum ChE activities were measured in a randomly selected sample of wood mice trapped on treated fields in the autumn and the spring. To reduce the number of animals that had to be killed, control animals from non-treated fields were not analysed. Instead, data for animals from treated fields were compared with published data on ChE activity in non-exposed wood mice which were measured in a large number of animals using identical analytical techniques (Fishwick et al., 1996). Wood mice which were captured after application of pesticide to fields at the University farm were transported immediately to the laboratory. They were sacrificed by cervical dislocation followed by decapitation and collection of trunk blood. Blood was allowed to clot, separated by centrifugation (2000 rev min -I at 4°C for 10 min) and the serum kept on ice until analysed. The brain was excised, weighed, and homogenised in 25 mM Tris-HC1 buffer (containing 0.1% Triton X-1000) to produce an homogenate of 0.06 g m l - 1; aliquots of this were kept on ice until analysed. The ChE activities in blood and serum were analysed on the day of collection using the colorimetric method of Ellman et al. (1961) as adapted and fully described by Fishwick et al. (1996). Acetylthiocholine iodide was used as the substrate in the assay and between three and five replicate determinations were made on each sample. A commercial reference material (Precinorm U, Boehringer Mannheim, Lewes) was used to confirm that no significant instrument drift occurred and that assay performance was consistent between days and with that used to measure ChE activity in non-exposed animals in the study by Fishwick et al. (1996).
2.4. Statistical analysis Population data were analysed by calendar of captures to calculate the number of individual mice known to be alive before and after application of methiocarb; it was assumed that natural mortality was negligible in the five days immediately before
213
application both in the autumn and spring. Comparisons between the observed and expected numbers of mice trapped in the hedge and field were made using Chi-squared tests with Yates' correction. Fisher's Exact test was used to compare both the adult:juvenile and male:female ratios of mice caught before and those captured for the first time after molluscicide application. Data on ChE activities are presented as means and standard deviations of replicate determinations for each mouse.
3. Results
3.1. Population responses In the October trial, no mice were caught in the control field before or after molluscicide application and so population data were only available for the treated fields. The numbers of mice on the three treatment fields were also low, and so data for each field were combined. Analysis of the number of total captures of wood mice in the hedges and fields suggested that the activity of mice focused predominantly on the hedges, there being significantly more captures in hedges than expected from the distribution of the traps (Table 1; X¢]) = 80.3, P < 0.001). Despite this, there was evidence of a marked decline in numbers following application of pellets to the fields (Table 2) with only three of the 41 mice alive before molluscicide application being captured afterwards. There was no evidence of a shift in the sex
Table 1 Distribution of traps and total captures (including recaptures) of wood mice Apodemus sylvaticus in hedges and fields before methiocarb application. Data for different treatment fields are combined Field
Hedge
Total no. of traps Treated fields in autumn Treated fields in spring Control field (both seasons)
144 96 48
48 32 16
Total wood mouse captures Treated fields in autumn Treated fields in spring Control field in spring
11 46 8
42 15 0
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Table 2 Numbers of wood mice Apodemus sylvaticus known to be alive on treatment fields (data for different fields combined) before and after application of methiocarb and on the treatment-free control field. Trapping effort was always equal before and after application No. of wood mice Treatment fields
Control field
Autumn
Spring
Spring
41 9 78
30 20 33
3 1 67
Captured before and after application
3
12
1
% of those caught before that were captured afterwards
7
40
33
Before pesticide application After pesticide application % decline
ratio or age structure of the wood mouse population following pesticide treatment. Twenty-six of the 41 mice captured before application were female (63%)
and 22 were adults (54%) while, of the six new mice caught afterwards, all but one were females (83%) and three were adults (50%). The differences in sex ratio and adults:juvenile ratio between the pre- and post-application populations were not statistically significant (Fisher's Exact test P > 0.05 in both cases). In the April trial, the number of total captures in the hedge and the field were almost exactly in proportion to the distribution of the traps (Table 1), suggesting that activity was centred less on the hedges than in the previous October. Only three mice were captured on the control field, two adult males and an adult female (Table 2). In total, 38 mice were trapped on the treatment fields and there was a decline in the numbers of individuals captured following application of methiocarb (Table 2). This was significantly less pronounced than in the previous autumn (Table 2) in terms of both the loss of marked individuals (Fisher's Exact test comparing between seasons, P = 0.01) and the decline in the total number of
Table 3 Brain and serum cholinesterase activities (mean _+ SD) in a sample of wood mice captured on treated fields after application of methiocarb. Application dates were 10 October 1994 and 8 April 1995 Date of capture
Sex
Breeding condition a
Body weight (g)
Brain AChE activity b (Ixmol rain-i g - i)
12/10/1994 11/10/1994 12/10/1994 12/10/1994 12/10/1994 13/10/1994 10/4/1995 10/4/1995 10/4/1995 10/4/1995 10/4/1995 11/4/1995 11/4/1995 13/4/1995 10/4/1995 11/4/1995 11/4/1995 13/4/1995
M F F F F F M M M M M M M M F F F F
TM Imperf Parous Parous Parous Parous TS TS TS TS TS TS TS TS Perf Perf Parous Parous
14.2 13.6 25.5 26.4 17.3 20.2 21.1 24.6 22.8 24.8 22.4 26.0 27.8 25.7 21.7 18.9 15.7 23.8
12.9 (+0.21) 12.8 (+0.09) 9.80 (_+0.14) 6.40 (_+0.30) 12.1 (_+0.12) 12.0 ( + 0.05) 11.8 (+0.14) 10.7 ( + 0.05) 11.6 (_+0.14) 10.0 (_+0.05) 11.2 ( _+0.39) 10.8 (_+0.17) 11.7 (-+0.49) 12.0 (-+0.43) 11.7 ( _+0.14) 12.4 ( -+ 0.07) 12.9 (-+0.11) 12.4 ( _+0.86)
Serum ChE activity ~ (Izmol min- i ml- l) 2.37 3.16 2.86 2.45
(_+0.02) (__+0.03) (_+0.18) (_+0.16)
2.35 ( ± 0.03) 2.56 (±0.06) 4.11 ( _+0.20) 3.35 (_+0.05) 1.95 (_+0.10) 3.20 ( ± 0.04) 3.35 (-+0.09) 3.03 (±0.15) 3.63 (±0.05) 2.10 ( -+ 0.03) 2.98 ( -+ 0.08) 3.15 (_+0.07) 2.87 ( _+0.00)
TM, testes medial; TS, testes scrotal; perf, perforate; imperf, imperforate. b Brain AChE activity in non-exposed wood mice is usually in the range 10-12 tzmol min - i g - ~. The defined 99% lower confidence limit for normal activity is 8 ~mol min- ~ g - r (Fishwick et al., 1996). c Mean ( + SE) serum ChE activity in non-exposed wood mice is 2.04 ( ± 0.05) I~mol min- I ml- l (n = 93, M + F combined) and there is no significant difference between the sexes (Fishwick et al., 1996).
R.F. Shore et al. / Agriculture, Ecosystems and Environment 64 (1997) 211-217
animals trapped (Fisher's Exact test, P < 0.05). There was no evidence of a shift in either the sex ratio or the population structure following methiocarb application (Fisher's Exact test, P > 0.05 in both cases); five adult males and three adult females were captured for the first time after application of methiocarb and this was an almost identical male:female and adult:juvenile ratio to that of mice captured before pesticide treatment (19 males: 11 females, 87% adults). No dead mice were found on the trapping grids following pesticide application in either October or April. In the two trials combined, 15 mice captured before application of methiocarb were subsequently captured afterwards (Table 2). There was no significant weight loss or gain in these animals during this period (mean ___SE change: - 0 . 1 0 + 0.32 g).
3.2. Detection of exposure of individuals Six mice were analysed for ChE activity in the autumn and 12 in the spring. The levels of brain AChE activity in these mice were typically between 9.8 and 12.9 p~mol min i g - l , the normal range (Table 3). Serum ChE activity levels were similar in males and females and the mean ( + S E ) activity (sexes combined) was 2.91 (+__0.14) txmol min -1 ml-1 (n = 17), significantly greater (Student's t-test, t~m8) = 6.66, P < 0.001) than that measured in laboratory-reared, non-exposed wood mice (Table 3). In the autumn field trial, there was one female mouse with a significantly depressed brain AChE activity of 6.4 ixmol min -1 g - i However, the serum ChE activity in this animal was 2.45 Ixmol min-~ m1-1. This was within the range measured in the other mice captured on the treatment fields which had normal brain AChE activities (Table 3).
4. Discussion The reduction in the numbers of wood mice captured following methiocarb treatment in the autumn is consistent with that observed by Johnson et al. (1991) at Boxworth. Thus, the present study provides further evidence that broadcast application of methiocarb pellets at this time of year can cause a marked decline in arable wood mouse populations.
215
Unlike the Boxworth study, there was no statistically significant shift towards a greater proportion of juveniles in the population following pesticide application. This was probably because few new mice were captured on the fields after treatment and, although these were all juveniles, the pre-treatment population already contained a high proportion (54%) of young mice. The present study is the first, as far as we are aware, to investigate the impacts on arable mice of broadcasting methiocarb pellets in spring. Treatment of fields in spring had a similar effect to that in autumn in that there was a decline in the numbers of mice captured, but it was less severe. A less marked impact in April than October was unexpected for two reasons. First, wood mouse densities are normally low in spring (Flowerdew, 1985) and there are few dispersing mice to recolonise depleted areas. However, there was no apparent shortage of immigrants in April in the present study, the number of new individuals captured after application being similar to that in the previous October. Second, the pattern of total captures before pesticide application suggested that mouse activity was more heavily concentrated in the hedges, where pellets were not applied, in the autumn than in the spring. Other studies have also suggested that mice are predominantly found in the hedgerows in arable habitats in autumn (Tew, 1994; Tew et al., 1994b). Hence, it might be expected that mice would be less likely to encounter pellets in autumn than in spring. It is possible that the difference in degree of impact that methiocarb had on mouse populations in October and April may have been due to seasonal variation in factors such as availability of food other than pellets, behavioural response (such as neophobia) to the pellets and the ease with which pellets were found. However, the results of this study need to be replicated to confirm that there truly are seasonal differences in the degree of impact that methiocarb treatment has on wood mouse populations. Inhibition of brain and blood ChE activity is a well established biomarker of exposure to anti-ChE pesticides, including methiocarb, and has been used in previous studies on wood mice (e.g. Westlake et al., 1980; Tarrant et al., 1990; Hardy et al., 1993; Dell'Omo and Shore, 1996; Dell'Omo et al., 1996). Furthermore, serum ChE activity has been shown to
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Table 4 Estimated density of pellets per square metre on treated fields and the number of pellets equivalent to an oral LD50 dose for a wood mouse weighing 14 g (upper figures) and 28 g (lower figures). Values for mouse weights are based on the range in Table 3 Est. LDs0 for wood mouse a (mg)
Mean + SE weight of pellets b (n = 24) (mg)
Weight of active ingredient per pellet c (mg)
No. of pellets per wood mouse LDs0 dose
No. of pellets m -2 of field d
0.18-1.89 0.36-3.78
18.4 + 3.6
0.74
0.25-2.57 0.49-5.14
30
a LDs0 values are based on the range (13-135 mg kg -I ) for oral exposure in the rat (Hayes and Laws, 1991). b R.F. Shore, 1996, unpublished data. c Pellets contain 4% w / w active ingredient. Calculated from value for mean weight of pellets in table and assuming recommended rate of application of 5.5 kg h a - t.
be more sensitive and to recover more slowly than brain AChE activity in wood mice following exposure to an OP pesticide (Dell'Omo et al., 1996). Given the reduction in the numbers of wood mice following methiocarb application in the present study, it was surprising that reduced ChE activities were not detected in at least some of the surviving animals. The one mouse which did have an abnormally low brain AChE activity did not have inhibited serum ChE activity, which suggested that the reduced brain activity may not have been a result of exposure to methiocarb. The overall lack of detectable ChE depression in the surviving mice suggests that either mice which were exposed did not enter traps, only non-exposed animals being captured, or that spontaneous recovery of ChE activity in sub-lethally exposed mice was rapid. Failure to catch exposed animals may have been because there was an 'all or nothing' effect in that some mice found pellets and subsequently consumed a lethal dose, after which they quickly died, while others failed to locate or ingest pellets at all. It can be calculated that wood mice probably need to eat only between a quarter and five pellets to ingest an oral LDs0 dose of methiocarb and the density of pellets on fields is high (Table 4), facilitating their discovery by foraging mice. However, it is also possible that mice captured on the fields had been exposed to methiocarb and that their ChE activity had spontaneously reactivated (Martin et al., 1981). The elevated serum ChE levels in these animals might thus be explained by 'overcompensation' where, after exposure, serum ChE activities can rebound to levels above normal (Thompson, 1991). Whatever the reason for the lack of detectable ChE
inhibition in mice captured after pesticide treatment, the results of this study do suggest that monitoring for depression in ChE activity after application may fail to indicate that exposure of the population to molluscicide pellets has occurred. In conclusion, the results of this study confirm that broadcast application of methiocarb can reduce populations of wood mice and such effects can occur irrespective of the season when application is carried out. One of the most important potential consequences of this is that predators could be adversely affected either due to a reduction in prey availability or by secondary poisoning. The effects of reduced mouse numbers on predators are likely to depend upon the time taken for the population to recover and this, in turn, is likely to depend upon the proximity of source areas which contain dispersing mice. Effects on predators may, therefore, be site-specific. However, the potential for secondary poisoning of avian and mammalian predators appears to be high. Tawny owl Strix aluco, kestrel Falco tinunculus and weasel Mustela nivalis all prey on wood mice in arable areas and have body weights in the region of 500 g, 200 g and 60-120 g, respectively (Mikkola, 1983; Village, 1990; King, 1991). Using an oral LDs0 value of 10 mg kg -j for the raptors (the median of the values reported by Schafer (1972) for 17 wild bird species) and the rat oral LDs0 values for the weasel, it can be calculated that the amount of methiocarb equivalent to an LD50 dose in tawny owls, kestrels and weasels is 5 mg, 2 mg and 0.816.2 mg, respectively. These doses are similar or within the range of the amounts of methiocarb estimated to be taken in by a single mouse which has ingested the equivalent of an LDso dose (Table 4).
R.F. Shore et al. / Agriculture, Ecosystems and Environment 64 (1997) 211-217
Given these estimates, the potential impacts of molluscicide application on predator populations merit further investigation.
Acknowledgements This work was funded by the Natural Environment Research Council as part of the Organic Farming Study, which is also funded by the Biotechnology and Biological Sciences Research Council and the Economic and Social Research Council. We are grateful to G. Berry and E. Brown for assistance in the field and to G. Dell'Omo for comments on an earlier version of the manuscript.
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