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Short communication

Non-avoidance of organophosphate insecticides by the earthworm

Aporrectodea caliginosa

(lumbricidae)

S. Hodge, K.M. Webster, L. Booth, V. Hepplethwaite, K. O'Halloran*

Centre for Environmental Toxicology (CENTOX), P.O. Box 84, Lincoln University, Canterbury, New Zealand

Received 6 April 1999; received in revised form 20 July 1999; accepted 16 August 1999

Earthworms sometimes avoid surface layers of soil contaminated by agrochemicals (Keogh and White-head, 1975; Cook et al., 1980) and, in the laboratory, they have been repelled by a range of soil contami-nants (Gunn and Sadd, 1994; Slimak, 1997).

This study examined the potentially repellent eects of two organophosphate insecticides on the earthworm

Aporrectodea caliginosa(Savigny). The pesticides tested were chlorpyrifos (Lorsban 40EC; a.i. 400 g/L, DowE-lanco, NZ) and diazinon (Basudin 600EW; a.i. 600 g/ L, Ciba-Geigy, NZ). The manufacturers' recommended ®eld application rates for pasture are 4 L for diazinon and 2 L for chlorpyrifos, both in 200 L of water/ha. The aims of the investigation were to evaluate avoid-ance behaviour as a potential bioindicator of soil con-tamination (see Yeardley et al., 1996) and also to estimate how any population declines observed after exposure to these chemicals could be explained by dis-persal behaviour rather than mortality. A hierarchy of experimental systems was employed to assess the range of conditions under which avoidance of these chemi-cals occurred; these were Petri dish experiments, micro-cosm experiments and ®eld studies.

Three experiments assessing the avoidance of pesti-cide residues in glass Petri dishes (14 cm diameter) were performed, each using a dierent substrate; bare glass, a 1-cm layer of reconstituted loam soil (25% water by mass) or a 0.5-cm layer of 1% agar. Half of each dish was sprayed with pesticide using a computer controlled spraying apparatus (Burkard, UK), set to spray the dishes with the same amount of liquid as rec-ommended for ®eld applications 12mL=cm2). To

ensure non-mixing and a sharp boundary between the

solutions, the pesticide was allowed to dry before the other half of the dish was sprayed with water (to act as a control). For the ®rst two substrates, the rec-ommended ®eld concentration of both pesticides was used. In the third experiment, the dishes were treated with chlorpyrifos only, at concentrations ranging from half to 16 times the recommended rate. When the dishes were dry, one juvenile (2.5±4 months) labora-tory-reared A. caliginosa was placed in each dish and the lids replaced. Juvenile A. caliginosa were used as they have previously been found to be more sensitive to pesticide contamination than adults. The dishes were placed in a dark room at 20218C and the pos-ition of the earthworms recorded after 0.5, 1, 2 and 24 h.

Microcosms consisted of two 2020 cm glass plates separated by a 0.5-cm thick band of insulating foam, so that the plates stood vertically and the bottom and side edges were sealed by the foam. The microcosms were ®lled with a commercially-produced compost (Patio and Tub Mix, Yates, NZ) with a water content of 25%. Four juvenile A. caliginosa were placed in each microcosm and the open (upper) ends sealed with adhesive tape. The microcosms were maintained in darkness, at a temperature ¯uctuating between 17 and 218C. Every second day, 5 ml of water was added to each microcosm, and they were then resealed using fresh tape. The mean depth of the earthworms in each microcosm was recorded once each day for 5 days. After 5 days, 1 ml of pesticide solution was added evenly to the upper surface of each microcosm using a plastic pipette (water was used as a control). Although only 1 ml of solution was used, this represented 25 times the recommended amount of active ingredient per unit area for pasture. The depths of the earth-worms were recorded 1, 2, 6 and 24 h after applying the pesticides and then daily for a further 4 days.

Soil Biology & Biochemistry 32 (2000) 425±428

www.elsevier.com/locate/soilbio

* Corresponding author. Tel.: +64-3-325-6701.

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Field experiments were carried out during July, August and September in 1997 and 1998 on an estab-lished Lolium perenne pasture on Templeton silt loam soil (Plant Science Research Unit, Lincoln University, New Zealand). There was no history of agrochemical use on the plot for at least two years and, before ex-periments were initiated, the pasture was grazed by sheep which produced a sward height of approximately 5 cm.

In the ®rst ®eld trial, both chemicals and a water control were applied at the recommended rate using a conventional spray boom at a pressure of 300 kPa. Each treatment was replicated six times, in 1010 m square plots. Two 202020 cm soil samples were taken from each plot 2, 7 and 28 days after spraying. The samples were divided into the top and bottom 10

cm, the layers sorted by hand and the number of ju-venileA. caliginosain each soil fraction recorded.

In the second ®eld experiment, chlorpyrifos was applied at the recommended rate and at seven times that rate. Each treatment was replicated three times. One soil sample was taken from each plot 2, 7, 14 and 28 days after spraying. The samples were divided hori-zontally into 5-cm sections, sorted by hand and the number of juvenile A. caliginosa in each section was counted.

In the Petri dishes, the earthworms did not signi®-cantly avoid chlorpyrifos or diazinon sprayed directly on to glass plates or on laboratory-prepared soil at any time (Table 1). A statistically signi®cant avoidance of chlorpyrifos occurred only at 8 and 16 times the recommended ®eld concentration, but this was not apparent on all occasions (Table 1).

In the microcosms, although there was a signi®cant dierence in earthworm depth at dierent times (P< 0.001), there was no eect of pesticide treatment on earthworm depth on any occasion (Fig. 1).

In the ®rst ®eld trial, there was no eect of pesticide treatment on the number of juveniles present or the proportion of earthworms in the top 10 cm of soil (Fig. 2a). In the second trial, even at seven times the recommended ®eld application rate, there was no eect of chlorpyrifos on the density of juvenile A. caliginosa

or the proportion in the top 5 cm of soil (Fig. 2b). These ®ndings disagree with other studies where earthworm behaviour has been modi®ed by agrochem-icals. A. caliginosa may be less sensitive to soil con-tamination than are some other species of earthworms (Dean-Ross, 1983; Ma and Bodt, 1993) or the chemi-cals tested here may be intrinsically less toxic to earth-worms than those in previous studies (i.e. the fungicides benomyl and carbendazim; Keogh and Whitehead, 1975). Pesticides can become bound to

or-Table 1

Percentage ofA. caliginosa found on the control side of the Petri disha

Substrate Compound tested Time (h)

0.5 1.0 2.0 24

Bare glassn20) diazinon 50 61 61 61 chlorpyrifos 67 67 72 50 Soiln18) diazinon 55 55 60 45 chlorpyrifos 60 60 40 60 Agar chlorpyrifos at:

Signi®cant avoidance of pesticide indicated by a w2 analysis,

P< 0.05.

Fig. 1. The depth distribution of earthworms in microcosms before and after the application of diazinon and chlorpyrifosN5; S.E. bars have been omitted for clarity).

S. Hodge et al. / Soil Biology & Biochemistry 32 (2000) 425±428

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ganic matter in soils and their bioavailability can become accordingly reduced. However, laboratory tests investigating other sublethal responses in A. cali-ginosa suggest this is not the case with these com-pounds (Booth et al., 1998). Soil residue analysis indicated ®eld concentrations of 112mg=kg for

diazi-non and10:04mg=kgfor chlorpyrifos one month after

application of recommended rates and the results of the laboratory studies conducted here suggest that any avoidance response is likely to occur only at concen-trations far in excess of these.

From these results, it can be inferred that migration of A. caliginosa from soils contaminated with these pesticides is unlikely to occur at concentrations expected in the ®eld. Also, the avoidance response of

A. caliginosa appears to be a poor indicator of soil contamination by these chemicals at the concentrations tested. Rather than acting purely as a deterrent, pesti-cides have been shown to aect more qualitative aspects of earthworm behaviour (Hans and Beg, 1992; Addison and Holmes, 1995). The eect of these orga-nophosphates on other behavioural traits in A. caligi-nosaremains to be investigated.

Acknowledgements

Thanks to Mike Bowie, Charles Eason, Don Heer, Dave Jack, Rhonda Pearce, Eric Scott and Steve Wrat-ten. Financial support was provided by the New Zeal-and FRST (project No. 96-LCR-15-4844).

References

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Booth, L.H., Heppelthwaite, V., Eason, C.T., 1998. Cholinesterase and glutathione S-transferase in the earthwormAporrectodea cali-ginosaas biomarkers of organophosphate exposure. Proceedings of the Fifty-®rst New Zealand Plant Protection Society Conference, 138±142.

Cook, A.G., Critchley, B.R., Critchley, U., Perfect, T.J., Yeadon, R., 1980. Eects of cultivation and DDT on earthworm activity in a forest soil in the sub-humid tropics. Journal of Applied Ecology 17, 21±29.

Fig. 2. The proportion of juvenileA. caliginosain (a) the upper 10 cm of pasture soil after application of ®eld rate of diazinon and chlorpyrifos (b) the upper 5 cm of pasture soil after application of chlorpyrifos at ®eld rate and seven times ®eld rate.

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Dean-Ross, D., 1983. Methods for the assessment of the toxicity of environmental chemicals to earthworms. Regulatory Toxicology and Pharmacology 3, 48±59.

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Keogh, R.G., Whitehead, P.H., 1975. Observations on some eects of pasture spraying with benomyl and carbendazim on earthworm

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Ma, W.-C., Bodt, J., 1993. Dierences in toxicity of the insecticide chlorpyrifos to six species of earthworms (Oligochaeta, Lumbricidae) in standardized soil tests. Bulletin of Environmental Contamination and Toxicology 50, 864±870.

Slimak, K.M., 1997. Avoidance response as a sublethal eect of pes-ticides on Lumbricus terrestris (Oligochaeta). Soil Biology and Biochemistry 29, 713±715.

Yeardley, R.B., Lazorchak, J.M., Gast, L.C., 1996. The potential of an earthworm avoidance test for evaluation of hazardous waste sites. Environmental Toxicology and Chemistry 15, 1532±1537.

S. Hodge et al. / Soil Biology & Biochemistry 32 (2000) 425±428