Evaluation of Earthworms Present on Natural and Agricultural-Livestock Soils of the Center Northern Litoral Santafesino, República Argentina

MARÍA INÉS MAITRE; ALBA RUT RODRIGUEZ; CAROLINA ELISABET MASIN; TAMARA RICARDO

Pesticides Book 1

INTECH - Open Access Publisher

Año: 2012; p. 25 - 50

ABSTRACT INTRODUCTION Importance of edaphic fauna is well known, specially earthworms that are used as bioindicators of soil health (Pérès et al., 2011; Masin et al., 2011; Simonsen et al., 2010; Capowiez et al., 2010; Zerbino et al., 2006; Momo et al., 1993). Earthworms spent their whole life cycle on soil horizons and because of their feeding and burrowing behaviour they directly or indirectly help to improve every physical, chemical and biological process from the soil. Earthworms participate in the mixing of organic and inorganic fractions of the soil, formation of stable clusters, dynamic and recycling of nutrients from the decomposition of organic matter, their burrows help to the aireation, infiltration and drainaige of the soil (Ouellet et al., 2008). Earthworms represent over the 80% of the biomass invertebrate from the soil, therefore they are an useful group to evaluate the effect of pesticides either on field as on laboratory tests. In Argentina, since the 1990’s (20thcentury) the agricultural frontier passed the 11.000.000ha, requiring the application of technologies that include genetically modified seeds and pesticides application (Pengue, 2005). The main crop is transgenic soybean and the most used insecticides are endosulpham, lambda-cyhalothrin, cypermethrin, chlorpyriphos and methamidophos (CASAFE, 2009). Between 2000-2009 the insecticide usage triplicated to control lepidoptera and hemiptera soybean’s parasite (Perrotti et al., 2010). Glyphosate, atrazine and 2,4D are the most widely used herbicides in the control of weed at soybean crops. The ecotoxicological impact of this production system is not well know and needs deeper studies using bioindicators to evaluate the risks from anthropogenic alterations (Candela et al., 2010; Evans et al., 2010; Ricardo et al., 2010; Zhao et al. 2009; Albers et al., 2009; Krzysko-Lupicka & Sudol, 2008; Wei et al., 2009; Lupwayi et al., 2009; Nakamura et al., 2008; Jänsch et al., 2005; Förster et al., 2006; Devotto et al., 2007; Wanner et. al., 2004; European Commission, 2001). Because of that, we performed field studies to determine the biomass, richness and density of earthworms and ecotoxicological assays at laboratory. MATERIALS AND METHODS A) Bioassay design All the laboratory tests were validated according to ISO 11268-1(E) guideline. Acute and chronic bioassays were performed according to the OECD protocol (1999) using OECD artificial soil and 10 clitellated adults Eisenia fetida (Oligochaeta, Lumbricidae) or native reference soil and 6 clitellated adults Aporrectodea trapezoides (Oligochaeta, Lumbricidae) both obtained from our laboratory stock cultures. Prior to the realization of the bioassay organisms were isolated for 24 hours in chambers with filter paper to empty their guts. Experimental units consisted of 500g of dry substrate placed in transparent poliphropilen boxes of 20x10x15 cm with perforated plastic lids; moisture was kept at 25 to 35%, according to each species requirements, using distilled water for the control group and different concentration of pesticide solutions for the assay groups. For the E. fetida experiences organisms were fed with dry triturated cow dung while A. trapezoides were fed with a mixture (1:3) of cow dung and domestic organic residues. Organisms were fed with a frequency of 7-15 days according to the species (E. fetida and A. trapezoides respectively). Survival (ww), number of cocoons and number of juveniles were weekly recorded for E. fetida and monthly for A. trapezoides assays. Statistichal aproximation of LC50 was set by Probit analysis, ANOVA test associated post hoc with Dunnett Multiple Comparison test (p≤ 0.05) was performed for biomass and reproduction parameters. Our goals were to determine endosulpham, glyphosate and lambda-cyhalothrin’s effects on the survival, growth and reproduction of Eisenia fetida and Aporrectodea trapezoides (standard and native species respectively) and the bioaccumulation and potential adsorption of some pesticides tested. a.- Endosulpham Endosulpham is a widely used insecticide even if their use was limitated since this year. Prior to the bioassay, the percent of active ingredient commercial endosulpham (Atanor® 35%) was determined by gas chromatography (GC) using a VARIAN 3700 with electronic capture detector. Due to the high lipophilicity of endosulpham, was mixed with pesticide quality hexane to obtain the desired concentrations. Each replicate was put under hood to evaporate the solvent and then distilled water was added to obtain the required moisture percent (35%). Lastly 10 clitellated adults (300mg ea) were placed. The concentrations for LC50 were 10, 18, 32, 58, 105 mg.kg–1 dw according to the ISO 11268-1:2008 protocol. Sublethal concentrations were 2, 3, 4, 7, 10 mg.kg–1 dw with a 56 day period to asses survival and biomass, and a 84 day period for reproduction. For soil degradation analyses, 10g samples were taken weekly from the 2, 4 and 10 mg.kg–1 dw groups and were sent to the GC laboratory for further analyses. Samples were analyzed by triplicate according to Miller & Miller (1993). Detection limits were 0.003, 0.003 and 0.005 mg.kg–1 for endosulpham I, II and endosulpham sulphate respectively. b.- glyphosate Transgenic soybean crops required the utilization of glyphosate. Their persistence on the soil matrix could vary from days to months and depends on multiple edaphic and climatic factors. Bioassays were performed at concentrations 7, 11, 18, 30 and 50 mg.kg–1 dw of commercial glyphosate (Round up Monsanto® 48%). For each concentration and the control group four replicates were used, each one consisting of 500g of reference soil from San Vicente (Dpto. Castellanos, Pcia de Santa Fe) and 10 clitellated adults with a weight of 450mg ea. Solutions were mixed on distilled water and commercial glyphosate to obtain a moist level of 50%. The same moist level was used on control groups. Exposure time was 28 days and prior to the bioassay the a.i. percent was determined by HPLC with a Waters 600 chromatographic system with fluorescence detector, post column derivatisation and Millenium32 data adquisition system. Detection limits were 0.05 µg.g-1 for glyphosate and their metabolyte AMPA. c.- lambda cyhalothrin Bioassays with this 4th generation pyrethroid were performed using commercial lambda cyhalothrin (Cilambda Ciagro® 5%). Because of their high liphophilicity the pesticide was mixed with pesticide quality hexane as explained in section (a). Moist level was 25% and 10 clitellated adults with a weight of 300mg ea were used. Concentrations were 1.25, 7.5, 16.25, 32.5 and 65 mg.kg–1 dw for Avoidance Behaviour Test according to ISO 17512-1:2008 protocol and 1, 2, 4, 8 mg.kg–1 dw for the chronic bioassay being exposure times 48h and 56 days respectively. After the end of chronic test, surviving organisms from 2, 4 and 8 mg.kg–1 dw were separated to determine bioaccumulation factor using the BAF=Cb/Cs according ASTM (1998) protocol. For determination of lambda cyhalotrin degradation in soil, samples of 10g from the 2, 4 and 8 mg.kg–1 dw were obtained at days 0, 56 and 86 of the chronic test and sent to the GC laboratory as described in section (a.-) B) Field Experiences To understand the influence of different production systems on the edaphic fauna, several areas from the northern of Santa Fe, Argentine were sampled. Agricultural, livestock and horticultural systems were selected (sites 1 to 7) to assess presence, density and diversity of earthworms. In each site a zig-zag transects was placed according to the TSBF standard method. At laboratory, adult, cocoons and juveniles were collected. Sexually mature organisms were kept and frozen as described by Moreno and Borges (2004) to further analysis following the Mischis (1991) taxonomy. Species richness index (S), Shannon-Wiener diversity index (H’) and Pielou equitativity index (J’) were determined. After the recolection of organisms, 1kg of soil obtained by quarting (mixing of the 15 replicates of each sample) were reserved to perform physical and chemical analyses according to the IRAM (2005) protocol. Statystichal analyses were performed using ANOVA and Tukey’s Multiple Test (p0.05). The number and size of juveniles was lower in treatment groups than in controls for all the tested pesticides. Bioaccumulation factors for lambda cyhalothrin were 0.0076, 0.0056 and 0.0845 for the 2, 4 and 8 mg.kg–1 dw concentrations respectively. Median life of endosulpham I and II and endosulpham sulphate were 44, 80 and 57 days respectively, degradation of lambda cyhalothrin was 99% after 86 days. B) Field experiences Site 1: over 5 years cropping system of soybean, wheat and alfalfa hay, fertilized with hen manure tertilization and glyphosate (800 cm3/ha), endosulpham (150 cm3/ha) and cypermethrin (150 cm3/ha) applications. Samples (summer and fall, 2008) showed a density of 72 and 52 ind/m2 respectively and a richness of 3 species: Aporrectodea trapezoides, Aporrectodea rosea and Amynthas morrisi. Site 2: over 20 years of cropping system of soybean, sunflower, cotton, corn and oat. Fertilized with calcium superphosphate (65 kg/ha) with cow dung and fumigated with glyphosate (4 l/ha) and chlorimuron (60 kg/ha). Samples were taken at summer, 2011 showing a 87 ind/m2 density and species richness was 2 (A. trapezoides and A. rosea). Site 3: over 10 years of agricultural activity with crops of soybean, wheat and alfalfa hay with minimun tillage, fumigated with glyphosate (5kg/ha). Samples (summer and fall, 2008) showed densities of 44 and 23 ind/m2 respectively, and richness of 2 species: A. trapezoides and A. rosea. Site 4: over 30 years of activity and crops of sunflower, soybean, corn and sorghum with no tillage fumigated with glyphosate (4.5 l/ha) and atrazine (3 l/ha). Samples (summer, 2011) showed a density of 9 ind/m2 and the presence of A. trapezoides. Site 5: ovine cattle with rotationally grazed pasture, no agrochemicals or mechanic activity to control weeds. Samples (summer, 2011) showed a density of 31 ind/m2 and richness was 3 species: A. trapezoides, A. rosea and Octolasion tyrtaeum. Site 6: bovine cattle with rotationally grazed pasture. Samples (spring, 2009 and summer, 2011) showed densities of 49 and 54 ind/m2 and richness was 3 species: A. trapezoides, A. rosea and Microscolex dubius. Site 7: over 30 years of horticultural activities, crops of lettuce, tomato, cabbage, squash and onion with tillage. Application of cypermethrin (150 cm3/ha), endosulpham (150 cm3/ha), glyphosate (2 l/ha), lambda cyhalothrin (1.5 l/ha), chlorpiriphos (120 cm3/ha) and copper sulfate (20 g/ha). Samples taken at summer and fall (2008) showed a density of 46 and 7 ind/m2 respectively and presence of A. trapezoides. Conclusions - A decrease in the movility and burrowing behaviour of earthworms showed a compactation of the soil on the lambda cyhalothrin, glyphosate and endosulpham assays. - Number and size of juveniles was lower on pesticide exposure treatments. - On field studies, the activity performed at each soil determine the taxocenosis of earthworms. Usage of agrochemicals leads to a decrease of diversity and abundance of earthworms. Alternative agricultural practices and livestock activities improve the growth and persistence of soil invertebrates. References • Albers C.H.; G.T. Banta; P.E. Hansen & O.S. Jacobsen. 2009. 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