Abstract
As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. Ecotoxicology 21:439-455, 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present.
Highlights
The nematode Caenorahbditis elegans has been utilised widely for toxicity testing of field samples (Mutwakil et al 1997; Traunspurger et al 1997; Power and de Pomerai, 2001), as well as single and mixed toxicants (Leung et al 2008), including pesticides ranging from the fungicide captan (Candido and Jones, 1996) to a series of organophosphates (Rajini et al 2008)
Lethality experiments on C. elegans have not been published for most pesticides tested in our study, but feeding inhibition studies suggested that the concentrations tested were almost all sublethal
Perhaps the most unusual pattern of response was that seen for endosulfan (Figure 1D), where a single gene showed 8-10 fold induction even at the lowest concentration tested (0.2 mg l-1), and this was already evident by 4 hours (Supplementary Material set 1); by contrast, none of the other 23 genes showed any sign of altered expression even at the highest test concentration of 200 mg l-1
Summary
The nematode Caenorahbditis elegans has been utilised widely for toxicity testing of field samples (Mutwakil et al 1997; Traunspurger et al 1997; Power and de Pomerai , 2001), as well as single and mixed toxicants (Leung et al 2008), including pesticides ranging from the fungicide captan (Candido and Jones, 1996) to a series of organophosphates (Rajini et al 2008). 1 mm as adults), transparency, short life-cycle (3.5 days at 25°C), hermaphroditic self-fertilisation (allowing easy maintenance of mutant or transgenic stocks), fully characterised somatic cell lineage (Sulston et al 1983), complete genome sequence (C. elegans Sequencing Consortium 1998), and the possibility of genome-wide RNA interference by feeding (Kamath et al 2003). These powerful methods provide the investigator with an armoury of genetic tools for probing the mechanisms underlying toxicity. This may be due in part to the opportunistic lifestyle of C. elegans in soil and compost, where it faces a variety of heavy metals, organic compounds and bacterial toxins
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