Susceptibility of epigeic earthworm Eisenia fetida to agricultural application of six insecticides

Ecotoxicological risks of agricultural application of six insecticides to soil organisms were evaluated by acute toxicity tests under laboratory condition following OECD guidelines using the epigeic earthworm Eisenia fetida as the test organism. The organochlorine insecticide endosulfan (LC₅₀ - 0.002 mg kg⁻¹) and the carbamate insecticides aldicarb (LC₅₀ - 9.42 mg kg⁻¹) and carbaryl (LC₅₀ - 14.81 mg kg⁻¹) were found ecologically most dangerous because LC₅₀ values of these insecticides were lower than the respective recommended agricultural dose (RAD). Although E. fetida was found highly susceptible to the pyrethroid insecticide cypermethrin (LC₅₀ - 0.054 mg kg⁻¹), the value was higher than its RAD. The organophosphate insecticides chlorpyrifos (LC₅₀ - 28.58 mg kg⁻¹), and monocrotophos (LC₅₀ - 39.75 mg kg⁻¹) were found less toxic and ecologically safe because the LC₅₀ values were much higher than their respective RAD.     

Using organic-certified rather than synthetic pesticides may not be safer for biological control agents: selectivity and side effects of 14 pesticides on the predator Orius laevigatus

The generalist predator Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) is a key natural enemy of various arthropods in agricultural and natural ecosystems. Releases of this predator are frequently carried out, and it is included in the Integrated Pest Management (IPM) programs of several crops. The accurate assessment of the compatibility of various pesticides with predator activity is key for the success of this strategy. We assessed acute and sublethal toxicity of 14 pesticides on O. laevigatus adults under laboratory conditions. Pesticides commonly used in either conventional or organic farming were selected for the study, including six biopesticides, three synthetic insecticides, two sulfur compounds and three adjuvants. To assess the pesticides’ residual persistence, the predator was exposed for 3 d to pesticide residues on tomato sprouts that had been treated 1 h, 7 d or 14 d prior to the assay. The percentage of mortality and the sublethal effects on predator reproductive capacity were summarized in a reduction coefficient (E_x) and the pesticides were classified according to the IOBC (International Organization for Biological Control) toxicity categories. The results showed that the pesticides greatly differed in their toxicity, both in terms of lethal and sub lethal effects, as well as in their persistence. In particular, abamectin was the most noxious and persistent, and was classified as harmful up to 14 d after the treatment, causing almost 100% mortality. Spinosad, emamectin, metaflumizone were moderately harmful until 7 d after the treatment, while the other pesticides were slightly harmful or harmless. The results, based on the combination of assessment of acute mortality, predator reproductive capacity pesticides residual and pesticides residual persistence, stress the need of using complementary bioassays (e.g. assessment of lethal and sublethal effects) to carefully select the pesticides to be used in IPM programs and appropriately time the pesticides application (as function of natural enemies present in crops) and potential releases of natural enemies like O. laevigatus.     

<Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis>, a relevant earthworm species for a posteriori pesticide risk assessment: current knowledge and recommendations for culture and experimental design

Ecotoxicological tests with earthworms are widely used and are mandatory for the risk assessment of pesticides prior to registration and commercial use. The current model species for standardized tests is Eisenia fetida or Eisenia andrei. However, these species are absent from agricultural soils and often less sensitive to pesticides than other earthworm species found in mineral soils. To move towards a better assessment of pesticide effects on non-target organisms, there is a need to perform a posteriori tests using relevant species. The endogeic species Aporrectodea caliginosa (Savigny, 1826) is representative of cultivated fields in temperate regions and is suggested as a relevant model test species. After providing information on its taxonomy, biology, and ecology, we reviewed current knowledge concerning its sensitivity towards pesticides. Moreover, we highlighted research gaps and promising perspectives. Finally, advice and recommendations are given for the establishment of laboratory cultures and experiments using this soil-dwelling earthworm species.     

The presence of Bacillus thuringiensis (Bt) protein in earthworms Eisenia fetida has no deleterious effects on their growth and reproduction

Earthworms Eisenia fetida, bred in substances with stover of two genetically-engineered Bacillus thuringiensis (Bt) corns (5422Bt1 (Event Bt11) and 5422CBCL (MON810)) expressing Cry1Ab and their near-isogenic non-Bt corn (5422), were used to investigate the non-target effects of Bt corn on soil-dwelling organisms. Cry1Ab concentrations in substances, casts and guts of E. fetida were also investigated by Enzyme-linked immunosorbent assay (ELISA). More than 90% individuals of E. fetida survived over a period of 30 d, irrespective of whether they received Bt corn or non-Bt corn. Compared to 5422 treatments, significantly higher relative growth rate and more number of new offspring and cocoons of E. fetida were found in 5422Bt1 and 5422CBCL treatments. These results were unlikely to be directly caused by Cry1Ab released from Bt corns but rather by differences in other factors of plants such as plant components (soluble sugar, total organic carbon, total protein and available phosphorus of Bt corns were more than 5422). ELISA results indicated immunoreactive Cry1Ab was detectable in substances, and the casts, guts of E. fetida from Bt corns treatments, of which the highest levels were detected in substances under the corresponding experimental conditions. With the increase of treated time, a strong decline was observed in Cry1Ab from substances and casts of E. fetida, whereas Cry1Ab in guts of E. fetida from 5422Bt1 treatments gradually increased and that from 5422CBCL treatments increased between 14 and 30 d. Therefore, the presence of Cry1Ab in E. fetida had no deleterious effects on their growth and reproduction.     

Ecotoxicological responses of the earthworm Eisenia fetida exposed to soil contaminated with HHCB

Although polycyclic musks have been shown to cause lethal and sub-lethal effects on organisms, their biochemical toxicity to earthworms is not well understood. In the current study, we investigated the responses of antioxidant systems and lipid peroxidation after exposing Eisenia fetida to soil contaminated with 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-γ-2-benzopyran (HHCB). Significant increase in lipid peroxidation level was observed on day 14 at two high concentrations, 50 and 100 mg kg⁻¹. Among antioxidant enzymes, the primary response to chronic HHCB exposure can be attributed to superoxide dismutase (SOD) and catalase (CAT). Of the two enzymes, SOD exhibited more sensitive response to HHCB stress. In addition, these two enzymes could have a combined effect on fighting damage by reactive oxygen species, evidenced by a marked relationship between lipid peroxidation and enzyme activity. On the other hand, dose-dependent inhibition of peroxidase (POD) activity has been observed throughout the test. The results suggest that the variations in investigated parameters of E. fetida could be used as responsive biomarkers for oxidative stress caused by HHCB in a soil environment.     

Life cycle toxicity assessment of pesticides used in integrated and organic production of oranges in the Comunidad Valenciana, Spain

The relative impacts of 25 pesticides including acaricides, fungicides, herbicides, insecticides, and post-harvest fungicides, used in the production of oranges in Spain were assessed with current life cycle impact assessment (LCIA) tools. Chemical specific concentrations were combined with pesticide emission data and information on chemical toxicity to assess human toxicity and freshwater ecotoxicity impacts. As a case study, the relative impacts of two orange production systems in the region of Valencia, integrated pest management (IP) and organic production (OP), were assessed. The evaluation of active ingredients showed that on average acaricides have the highest human toxicity impact scores, while for freshwater ecotoxicity insecticides show the highest impact. In both impact categories the lowest impact scores were calculated for herbicides. In the production of 1 kg of orange fruits, where several kinds of pesticides are combined, results show that post-harvest fungicides can contribute more than 95% to the aggregate human toxicity impacts. More than 85% of aquatic ecotoxicity is generated by fungicides applied before harvest. The potential to reduce impacts on freshwater ecosystems is seven orders of magnitude, while impacts on human health can be reduced by two orders of magnitude. Hence, this stresses the importance of a careful pre-selection of active ingredients. In both impact categories, organic production represents the least toxic pest-control method.     

Survival and behavior of the insecticide-exposed predators Podisus nigrispinus and Supputius cincticeps (Heteroptera: Pentatomidae)

Pentatomid stinkbugs are important predators of defoliating caterpillars in agricultural and forestry systems, and knowledge of the impact of insecticides on natural enemies is important information for integrated pest management (IPM) programs. Thus, we assessed the toxicity and behavioral sublethal response of the predators Podisus nigrispinus and Supputius cincticeps exposed to deltamethrin, methamidophos, spinosad and chlorantraniliprole, insecticides commonly used to control the velvetbean caterpillar (Anticarsia gemmatalis) in soybean crops. With the exception of deltamethrin for S. cincticeps, all insecticides showed higher acute toxicity to the prey than to these natural enemies providing effective control of A. gemmatalis. The recommended field concentration of deltamethrin, methamidophos and spinosad for controlling A. gemmatalis caused 100% mortality of P. nigrispinus and S. cincticeps nymphs. Chlorantraniliprole was the less toxic and the most selective insecticide to these predators resulting in mortalities of less than 10% when exposed to 10× the recommended field concentration for a period of 72 h. Behavioral pattern changes in predators were found for all insecticides, especially methamidophos and spinosad, which exhibited irritability (i.e., avoidance after contact) to both predator species. However, insecticide repellence (i.e., avoidance without contact) was not observed in any of the insects tested. The lethal and sublethal effects of pesticides on natural enemies is of great importance for IPM, and our results indicate that substitution of pyrethroid and organophosphate insecticides at their field rates by chlorantraniliprole may be a key factor for the success of IPM programs of A. gemmatalis in soybeans.     

Eisenia fetida avoidance behavior as a tool for assessing the efficiency of remediation of Pb, Zn and Cd polluted soil

Remediation by means of soil leaching with ethylenediaminetetraacetic acid (EDTA) is capable of extracting the most labile soil fractions, leaving the residual metals in biologically non-available forms. We evaluated the feasibility of the standardized earthworm (Eisenia fetida) avoidance test for assessing the efficiency of soil remediation of Pb, Zn and Cd polluted soil. Chemical extraction tests (six-step sequential extraction, toxicity characteristic leaching procedure, physiologically based extraction test, diethylenediaminepentaacetic acid extraction) indicated that the mobility, oral bioaccessibility and phytoavailability of Pb, Zn and Cd were consistently reduced. However, the avoidance test showed no significant avoidance of polluted soil in favor of that which had been remediated. Pb, Zn and Cd accumulation in E. fetida mirrored the decreasing pattern of metal potential bioavailability gained by leaching the soil with increasing EDTA concentrations. The calculated bioaccumulation factors indicated the possibility of underestimating the metal bioavailability in soil using chemical extraction tests.     

Influence of earthworm Eisenia fetida on removal efficiency of N and P in vertical flow constructed wetland

This study investigates biomass, density, photosynthetic activity, and accumulation of nitrogen (N) and phosphorus (P) in three wetland plants (Canna indica, Typha augustifolia, and Phragmites austrail) in response to the introduction of the earthworm Eisenia fetida into a constructed wetland. The removal efficiency of N and P in constructed wetlands were also investigated. Results showed that the photosynthetic rate (P _n), transpiration rate (T _r), and stomatal conductance (S _cond) of C. indica and P. austrail were (p?<?0.05) significantly higher when earthworms were present. The addition of E. fetida increased the N uptake value by above-ground of C. indica, T. augustifolia, and P. australis by 185, 216, and 108 %, respectively; and its P uptake value increased by 300, 355, and 211 %, respectively. Earthworms could enhance photosynthetic activity, density, and biomass of wetland plants in constructed wetland, resulting in the higher N and P uptake. The addition of E. fetida into constructed wetland increased the removal efficiency of TN and TP by 10 and 7 %, respectively. The addition of earthworms into vertical flow constructed wetland increased the removal efficiency of TN and TP, which was related to higher photosynthetic activity and N and P uptake. The addition of earthworms into vertical flow constructed wetland and plant harvests could be the significantly sustainable N and P removal strategy.     

Rapid assays for environmental and biological monitoring

Abstract

Rapid, inexpensive, sensitive, and selective enzyme‐linked immunosorbent assays (ELISAs) now are utilized in environmental science. In this laboratory, many ELISAs have been developed for pesticides and other toxic substances and also for their metabolites. Compounds for which ELISAs have recently been devised include insecticides (organophosphates, carbaryl, pyrethroids, and fenoxycarb), herbicides (s‐triazines, arylureas, triclopyr, and bromacil), fungicides (myclobutanil), TCDD, and metabolites of naphthalene and toluene. New rapid assays have been developed for mercury.     

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.     

Effects of perfluorooctanoic acid and perfluorooctane sulfonate on acute toxicity,superoxide dismutase,and cellulase activity in the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis>

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are the two best-known perfluorinated chemicals and have received much attention due to their ubiquity in the environment. In the present study, we evaluated the effects of PFOS and PFOA on acute toxicity, superoxide dismutase (SOD), and cellulase activities in Eisenia fetida. The results of acute toxicity testing using a filter paper contact test and a natural field soil test showed that PFOA and PFOS exhibited acute toxicity in earthworms, and the toxic effect of PFOS was greater than that of PFOA. The results also showed that avoidance behavior is a more sensitive and easy operation biomarker than acute toxicity and will give us information for early diagnosis of soil pollution, well before the lethal effect becomes apparent. Subchronic exposure to PFOA or PFOS resulted in changes in SOD and cellulase activities in E. fetida, and SOD activity was more sensitive than cellulase activity during early exposure. Based on these findings, we suggest that avoidance behavior and SOD activity in earthworms are suitable biomarkers for evaluating the toxicity of PFOA- and PFOS-contaminated soils. These results indicate that exposure to PFOA and PFOS has a potential impact on soil animals and their environment.     

Comparative sensitivity of Eisenia andrei and Perionyx excavatus in earthworm avoidance tests using two soil types in the tropics

Terrestrial avoidance behavior is proposed as a fast and cost-effective method for assessing effects of pesticides on earthworms. Tropical species however, have rarely been used in avoidance tests. Avoidance tests were performed with Perionyx excavatus, a tropical species, and Eisenia andrei as the standard species, using chlorpyrifos and carbofuran in artificial and natural soil. Earthworms were exposed to concentrations of 1–900 (chlorpyrifos) and 1–32 (carbofuran) mg a.i. kg⁻¹ dry soil in a two-chamber system under tropical conditions (26 ± 2 °C, 48 h). No significant difference was found in the control tests comparing the two soils used, suggesting soil type did not affect the distribution of the worms. The results suggest a higher sensitivity of E. andrei, with EC_50S for the effect on avoidance behavior for both pesticides being a factor of 2–3 lowers than for P. excavatus. Earthworm avoidance tests with local species should therefore be used with caution when applied as a tool for pesticide risk assessment in the tropics. Endpoints generated through avoidance tests in this study are shown to be less sensitive than reproduction and more sensitive than survival. This was further confirmed by literature data available. Earthworm avoidance tests therefore can only replace survival tests as an initial screening tool for risk assessment.     

1H NMR-based metabolomics of time-dependent responses of Eisenia fetida to sub-lethal phenanthrene exposure

¹H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida after exposure to sub-lethal concentrations of phenanthrene over time. Earthworms were exposed to 0.025 mg/cm² of phenanthrene (1/64th of the LC₅₀) via contact tests over four days. Earthworm tissues were extracted using a mixture of chloroform, methanol and water, resulting in polar and non-polar fractions that were analyzed by ¹H NMR after one, two, three and four days. NMR-based metabolomic analyses revealed heightened E. fetida responses with longer phenanthrene exposure times. Amino acids alanine and glutamate, the sugar maltose, the lipids cholesterol and phosphatidylcholine emerged as potential indicators of phenanthrene exposure. The conversion of succinate to fumarate in the Krebs cycle was also interrupted by phenanthrene. Therefore, this study shows that NMR-based metabolomics is a powerful tool for elucidating time-dependent relationships in addition to the mode of toxicity of phenanthrene in earthworm exposure studies.     

Effects of some pesticides on Rhizobiomjaponicum and on the seed germination and pathogens of soybean

The relative toxicity of 23 pesticides, including 21 fungicides and two pyrethroid insecticides, on $\text{Rhizobium}$$\text{japonicum}$, four soybean pathogens, $\text{Fusarium}$$\text{oxysporum}$, $\text{Phytophthora}$$\text{megasperma}$, $\text{Phytophthora}$$\text{parasitica}$ and $\text{Pythium}$$\text{ultimum}$, and germination of soybean seeds was compared with a highly toxic broad spectrum germicide, HgCl₂ under laboratory conditions.     

Earthworm ecotoxicological assessments of pesticides used to treat seeds under tropical conditions

Ecotoxicological laboratory tests (lower-tier tests) are fundamental tools for assessing the toxicity of pesticides to soil organisms. In this study, using these tests under tropical conditions, we quantified the impact of the insecticides imidacloprid, fipronil, and thiametoxam, and the fungicides captan and carboxin + thiram, all of which are used in the chemical treatment of crop seeds, on the survival, reproduction, and behavior of Eisenia andrei (Oligochaeta). With the exception of imidacloprid, none of the pesticides tested caused mortality in E. andrei in artificial soils. The LC₅₀ of imidacloprid was estimated as 25.53 mg active ingredient kg^?1 of dry soil. Earthworm reproduction rates were reduced by imidacloprid (EC₅₀ = 4.07 mg kg^?1), fipronil (EC₂₀ = 23.16 mg kg^?1), carboxin + thiram (EC₅₀ = 56.38 mg kg^?1), captan (EC₅₀ = 334.84 mg kg^?1), and thiametoxam (EC₅₀ = 791.99 mg kg^?1). Avoidance behavior was observed in the presence of imidacloprid (AC₅₀ = 0.11 mg kg^?1), captan (AC₅₀ = 33.54 mg kg^?1), carboxin + thiram (AC₅₀ = 60.32 mg kg^?1), and thiametoxam (AC₅₀ = >20 mg kg^?1). Earthworms showed a preference for soils with the insecticide fipronil. Imidacloprid was the most toxic of the substances tested for E. andrei. The avoidance test was the most sensitive test for most pesticides studied, but results varied between pesticides. These results offer new insights on the toxicity of pesticides used to treat seeds in tropical regions. However, they should be complemented with higher-tier tests in order to reduce the uncertainties in risk assessment.     

Effect of fungicides on plant growth promoting activities of phosphate solubilizing Pseudomonasputida isolated from mustard (Brassica compestris) rhizosphere

This study was navigated to examine the effects of fungicide-stress on the activities of plant-growth-promoting rhizobacterium Pseudomonasputida with inherent phosphate solubilizing activity. The fungicide-tolerant and phosphate solubilizing P.putida strain PS9 was isolated from the mustard rhizosphere and tentatively identified following standard morphological, physiological and biochemical tests. To further consolidate the identity of the strain PS9, the 16S rDNA sequence analysis was performed. Following the BLAST program, the strain PS9 was identified as P.putida. In the presence of the varying concentrations (0-3200 μg mL⁻¹; at a two fold dilution interval) of four fungicides of different chemical families (tebuconazole, hexaconazole, metalaxyl and kitazin) amended in minimal salt agar medium, the P.putida strain PS9 showed a variable tolerance levels (1400-3200 μg mL⁻¹) against the tested fungicides. The strain PS9 produced plant-growth-promoting (PGP) substances in significant amount in the absence of fungicides. In general, fungicides applied at the recommended, two and three times of the recommended rates, decreased the PGP attributes of P.putida the strain PS9 and affected the PGP activities in concentration-dependent manner. Fungicides at the recommended dose had minor reducing effect while the doses higher than the recommended dose significantly reduced the PGP activities (phosphate solubilization, salicylic acid, 2,3-dihydroxy benzoic acid, and indole-3-acetic acid production except exo-polysaccharides, hydrogen cyanate and ammonia production). Of the four fungicides, tebuconazole generally, showed maximum toxicity to the PGP activities of the strain PS9. This study inferred that fungicides must be examined in vitro for their possible adverse effects on soil micro flora before their application in agricultural fields. Moreover, the results also suggested the prerequisite of application of fungicide-tolerant PGPR strains as bioinoculants so that their PGP activities may not be suppressed under fungicide stress.     

The dissipation of three fungicides in a biobed organic substrate and their impact on the structure and activity of the microbial community

Biopurification systems (BPS) have been introduced to minimise the risk for point source contamination of natural water resources by pesticides. Their depuration efficiency relies mostly on the high biodegradation of their packing substrate (biomixture). Despite that, little is known regarding the interactions between biomixture microflora and pesticides, especially fungicides which are expected to have a higher impact on the microbial community. This study reports the dissipation of the fungicides azoxystrobin (AZX), fludioxonil (FL) and penconazole (PC), commonly used in vineyards, in a biomixture composed of pruning residues and straw used in vineyard BPS. The impact of fungicides on the microbial community was also studied via microbial biomass carbon, basal respiration and phospholipid fatty acid analysis. AZX dissipated faster (t _1/2?=?30.1 days) than PC (t _1/2?=?99.0 days) and FL (t _1/2?=?115.5 days). Fungicides differently affected the microbial community. PC showed the highest adverse effect on both the size and the activity of the biomixture microflora. A significant change in the structure of the microbial community was noted for PC and FL, and it was attributed to a rapid inhibition of the fungal fraction while bacteria showed a delayed response which was attributed to indirect effects by the late proliferation of fungi. All effects observed were transitory and a full recovery of microbial indices was observed 60 days post-application. Overall, no clear link between pesticide persistence and microbial responses was observed stressing the complex nature of interactions between pesticides in microflora in BPS.     

How to assess the feeding activity in ecotoxicological laboratory tests using enchytraeids?

The risk assessment of pesticides on soil fauna is an issue to protect agroecosystem sustainability. Enchytraeids are recognized as relevant soil bioindicators of chemical stress in agroecosystems. In laboratory, the reproduction test was found to be sensitive to reveal chemical impacts on enchytraeids. However, it does not allow to assess the impacts on ecological functions in which enchytraeids are involved. The objectives of this study were (i) to explore the feasibility of the bait-lamina test with enchytraeids under laboratory conditions and (ii) to compare its sensitivity with the Enchytraeid Reproduction Test. We exposed individuals of Enchytraeus albidus to two commercial formulations of fungicides widely used in Europe. The EC50 reproduction for the Swing^® Gold (50 g L^?1 epoxiconazole and 133 g L^?1 dimoxystrobin) and the Cuprafor micro^® (50% copper oxychloride) were respectively estimated at 1.66?±?0.93 times the recommended dose and >?496 mg kg^?1 of copper. However, no impact was found on the feeding activity of enchytraeids. The bait-lamina test thus appeared less sensitive than the Enchytraeid Reproduction Test to the tested fungicides. Despite that, this test which is achievable under laboratory conditions and allows assessing indirect effects of pesticides is quick, cheap, and easy to perform. It would deserve to be used to explore longer-exposure effects through the repeated addition of bait-lamina sticks.