Assessment of short and long-term effects of imidacloprid on the burrowing behaviour of two earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) by using 2D and 3D post-exposure techniques.

Adverse effects of agrochemicals on earthworms' burrowing behaviour can have crucial impacts on the entire ecosystem. In the present study, we have therefore assessed short- and long-term effects on burrowing behaviour in the earthworm species Aporrectodea caliginosa and Lumbricus terrestris after exposure to a range of imidacloprid concentrations (0.2-4 mg kg(-1) dry weight (DW)) for different exposure times (1, 7, 14 d). 2D-terraria were used for the examination of post-exposure short-term effects (24-96 h), while post-exposure long-term effects were assessed by means of X-ray burrow reconstruction in three dimensional soil cores (6 weeks). For the latter each core was incubated with two specimens of L. terrestris and four of A. calignosa. Short-term effects on the burrowing behaviour (2D) of A. caliginosa were already detected at the lowest test concentration (0.2 mg kg(-1) DW), whereas such effects in L. terrestris were not observed until exposure to concentrations 10 times higher (2 mg kg(-1) DW). For both species tested in the 2D-terraria, "total burrow length after 24 h" and "maximal burrow depth after 24 h" were the most sensitive endpoints. 3D reconstructions of the burrow systems made by both earthworm species in the repacked soil cores revealed a significant linear decrease in burrow volume with increasing imidacloprid concentration. Since many of the observed effects occurred at imidacloprid concentrations relevant to natural conditions and since reduced activities of earthworms in soils can have crucial impacts on the ecosystem level, our results are of environmental concern.

Physiological and behavioural effects of imidacloprid on two ecologically relevant earthworm species (Lumbricus terrestris and Aporrectodea caliginosa).

Earthworms play key roles in soils and sub-lethal effects of environmental toxicants on these organisms should be taken seriously, since they might have detrimental effects on higher ecological levels. In laboratory experiments we have assessed sub-lethal effects (body mass change and cast production) of imidacloprid on two earthworm species commonly found in different agricultural soils (Lumbricus terrestris and Aporrectodea caliginosa). After 7 days of exposure in contaminated soil, a significant loss of body mass was found in both species exposed to imidacloprid concentrations as low as 0.66 mg kg(-1) dry soil. These losses ranged from 18.3 to 39% for A. caliginosa and from 7.4 to 32.4% for L. terrestris, respectively. Changes in cast production, a new biomarker previously validated using L. terrestris, was assessed by soil sieving using the recommended mesh size (5.6 mm) for L. terrestris and three different mesh sizes for A. caliginosa (5.6, 4 and 3.15 mm). The 4 mm mesh size proved to be the most suitable sieve size for A. caliginosa. Cast production increased by 26.2% in A. caliginosa and by 28.1% in L. terrestris at the lowest imidacloprid concentration tested (0.2 mg kg(-1) dry soil), but significantly decreased at higher concentrations (equal to and above 0.66 mg kg(-1) dry soil) in both earthworm species after the 7 days exposure experiment. These decreases in cast production ranged from 44.5 to 96.9% in A. caliginosa and from 42.4 to 95.7% in L. terrestris. The EC(50) for cast production were 0.84 (L. terrestris) and 0.76 mg kg(-1) dry soil (A. caliginosa), respectively. The detected sub-lethal effects were found close to the predicted environmental concentration (PEC) of imidacloprid, which is in the range of 0.33-0.66 mg kg(-1) dry soil. The biomarkers used in the present study, body mass change and changes in cast production, may be of ecological relevance and have shown high sensitivity for imidacloprid exposure of earthworms. The measurement of changes in cast production should be considered for inclusion in current standard tests.

Earthworm cast production as a new behavioural biomarker for toxicity testing.

There is currently a lack of ecotoxicity tests adapted to earthworm species of higher ecological relevance and whose endpoints could be directly related to their ecological role in the soil. We propose a new and relatively simple ecotoxicity test based on the estimation of cast production (CP) by Lumbricus terrestris under laboratory conditions. CP was found to be linearly correlated to earthworm biomass and to be greatly influenced by soil water content. Azinphos-methyl had no effect on CP at all the concentrations tested. Significant decreases were observed at the normal application rate for other pesticides with (imidacloprid, carbaryl, methomyl) or without (ethyl-parathion and chlorpyrifos-ethyl) a clear concentration-effect response. For the highest concentration tested, reduction in CP varied between 35 and 67%. CP is straightforward and rapidly measured and ecologically meaningful. We thus believe it to be of great use as an endpoint in ecotoxicity testing.

Snail phenotypic variation and stress proteins: do different heat response strategies contribute to Waddington's widget in field populations?

On the basis of studies with laboratory strains of Drosophila and Arabidopsis, it has been hypothesized that potential buffers to the expression of phenotypic morphological variation, such as Hsp90 and possibly Hsp70, represent important components of Waddington's widget, which may confer capacitive evolution. As studies on field populations of living organisms to test this hypothesis are lacking, we tested whether a heat response strategy involving high stress protein levels is associated with low morphological variation and vice versa, using four natural populations of Mediterranean pulmonate snails. In response to 8 hr of elevated temperatures, a population of Xeropicta derbentina with uniform shell pigmentation pattern showed remarkably high Hsp70 but low Hsp90 levels. In contrast, a highly variable population of Cernuella virgata kept both Hsp90 and Hsp70 levels low when held at diverse though environmentally relevant temperatures. Two other populations (Theba pisana and another X. derbentina population) with intermediate variation in shell pigmentation pattern were also intermediate in inducing Hsp70, though Hsp90 was maintained at a low level. The observed correlation of stress protein levels and coloration pattern variation provide the first indirect evidence for an association of stress proteins with Waddington's widget under natural conditions.