The potential genotoxicity (nuclear anomalies, damage to single-strand DNA) and pinocytic adherence activity of two (glyphosate-based and paraquat-based) commercial herbicides to earthworm coelomocytes (immune cells in the coelomic cavity) were assessed. Coelomocytes were extracted from earthworms (Pheretima peguana) exposed to concentrations

To determine genotoxicity to coelomocytes, Pheretima peguana earthworms were exposed in filter paper studies to cadmium (Cd) and lead (Pb) for 48 h, at concentrations less than the LC 10 -Cd: 0.09, 0.19, 0.38, 0.75, and 1.50 µg cm −2 ; Pb: 1.65, 3.29, 6.58, 13.16, and 26.32 µg cm −2 . For Cd at 0.75 µg cm −2 , in the micronucleus test (detects chromosomal aberrations), significant increases (P < .05) in micronuclei and binucleate cells were observed, and in the comet assay (detects DNA single-strand breaks), tail DNA% was significantly increased. Lead was less toxic with minimal effects on DNA, but the binucleates were significantly increased by Pb at 3.29 µg cm −2 . This study shows that Cd is more acutely toxic and sublethally genotoxic than Pb to P. peguana. Cadmium caused chromosomal aberrations and DNA single-strand breaks at 45% of the LC 10 concentration. Lead, in contrast, did not induce DNA damage but caused cytokinesis defects.

A chronic toxicity study was conducted in earthworms (Pheretima peguana) exposed to soil spiked with chlorpyrifos at concentrations of 0, 0.1, 1, 10, and 100 mg/kg soil dry matter for 7, 14, and 28 days. The integrity of the coelomocyte lysosomal membrane, nervous system, and male reproductive tissue was monitored using, respectively, the neutral-red retention assay, acetylcholinesterase (AChE) enzyme assay, and histomorphology of spermatogenic cells in the seminal vesicles and cocoon production (at 28 days after 28 days' exposure). Chlorpyrifos decreased the coelomocyte neutral-red retention time (NRRT) significantly (p < 0.05) at concentrations > 0.1 mg/kg soil as early as day 7 of exposure and was dose- and time-dependent. Chlorpyrifos inhibition of AChE activity was greater at day 7 than at day14 (p < 0.05) indicating possibly nerve recovery. Chlorpyrifos induced concentration-dependent damage to spermatogenic cells and cytophores in premature stages. The number and size of premature, maturing, and fully mature spermatogenic stages were increased at low concentrations (<1 mg/kg) but a number of these maturation stages declined at higher concentrations (10 and100 mg/kg) on day 28. The most severe effects were observed in the maturing and fully mature stages at the highest chlorpyrifos concentration, and this had an adverse impact on cocoon production and cocoon viability. Collectively, the results suggest induction of widespread effects on multiple organ systems in P. peguana exposed to chlorpyrifos. Although NRRT and AChE activity were the most sensitive of the biomarkers, cocoon production and cocoon viability could still be considered as diagnostic tools for monitoring effects from low-dose long-term chlorpyrifos toxicity and for evaluating population effects. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1450-1459, 2016.

Cypermethrin is a synthetic pyrethroid insecticide used worldwide in agriculture, home pest control, disease vector control, and food safety. It accumulates in soil. Therefore, traces of cypermethrin may frequently appear in vegetables grown in contaminated soil. There is a push now to develop biomarkers as early warning indicators of environmental pollution. In this study, DNA damage (tail DNA%, tail length, and olive tail moment), the micronucleus, neutral red retention (NRR) time, and pinocytic adherence ability of coelomocytes were investigated in Pheretima peguana earthworms exposed to cypermethrin in filter paper tests. The NRR time of earthworm coelomocytes decreased significantly at a concentration of 3.5 × 10(-3) µg · cm(-2) (1/100 LC50 ) after 48 h exposure, with a highly negative correlation with cypermethrin concentration. Pinocytic adherence ability of coelomocytes also declined significantly at a cypermethrin concentration of 3.5 × 10(-2) µg · cm(-2) (1/10 LC50 ). The DNA damage to earthworm coelomocytes (tail DNA%, tail length, and olive tail moment) increased considerably at the highest concentration (3.5 × 10(-1) µg · cm(-2) ) although the correlation between tail DNA% and cypermethrin concentration was low. Thus, physiological biomarkers were more sensitive than the genotoxic effects in earthworms exposed to commercial cypermethrin. Although a suite of earthworm biomarkers could be used to evaluate cypermethrin terrestrial pollution, the NRR test is easier to conduct and a more sensitive indicator.

The LC 50 of commercial neem extract (Sadao Thai III containing azadirachtin; NEEM) on filter paper in the earthworm Pheretima peguana at 48 h and 72 h was 3.79 and 3.33 µg cm −2 , respectively. In earthworms exposed to five NEEM concentrations from 0.39 (∼10% of 48-h LC 50 ) to 3.13 (∼80% of 48-h LC 50 ) µg cm −2 , the radial thickness of the epidermis and body wall significantly (P < .05) decreased, and thickness of intestinal epithelium increased but only at high doses, approximately 25-fold above the concentration permitted for use as an insecticide in field applications (0.09 µg cm −2 ). NEEM significantly (P < .05) increased the number of binucleated coelomocytes in the micronucleus test (detects chromosomal aberrations) at 3.13 µg cm −2 , approximately 35-fold higher than the recommended dose, but it did not cause coelomocyte DNA single-strand breaks in the comet assay. Thus, NEEM is cytotoxic (increase in binucleates through the inhibition of cytokinesis) but not genotoxic to earthworm coelomocytes. This study demonstrates that the recommended dosage of commercial neem extract as an insecticide in agricultural practices is safe for earthworms.