Validation of biomarkers for neonicotinoid exposure in Folsomia candida under mutual exposure to diethyl maleate.

Neonicotinoid insecticides are harmful to non-target soil invertebrates, which are crucial for sustainable agriculture. Gene expression biomarkers could provide economic and high-throughput metrics of neonicotinoid exposure and toxicity to non-target invertebrates. Thereby, biomarkers can help guide remediation efforts or policy enforcement. Gene expression of Glutathione S-Transferase 3 (GST3) has previously been proposed as a biomarker for the neonicotinoid imidacloprid in the soil ecotoxicological model species Folsomia candida (Collembola). However, it remains unclear how reliably gene expression of neonicotinoid biomarkers, such as GST3, can indicate the exposure to the broader neonicotinoid family under putative GST enzymatic inhibition. In this work, we exposed springtails to two neonicotinoids, thiacloprid and imidacloprid, alongside diethyl maleate (DEM), a known GST metabolic inhibitor that imposes oxidative stress. First, we determined the influence of DEM on neonicotinoid toxicity to springtail fecundity. Second, we surveyed the gene expression of four biomarkers, including GST3, under mutual exposure to neonicotinoids and DEM. We observed no effect of DEM on springtail fecundity. Moreover, the expression of GST3 was only influenced by DEM under mutual exposure with thiacloprid but not with imidacloprid. The results indicate that GST3 is not a robust indicator of neonicotinoid exposure and that probable GST enzymatic inhibition mediates the toxicity of imidacloprid and thiacloprid differentially. Future research should investigate biomarker reliability under shifting metabolic conditions such as provided by DEM exposure.

Effects of Life Stage on the Sensitivity of Folsomia candida to Four Pesticides.

The registration of pesticides in the European Union requires the assessment of the toxicity of active substances to soil invertebrates. The most commonly tested soil microarthropod species is Folsomia candida (Collembola), for which toxicity tests usually start with juveniles and determine survival and reproduction after 28 days of exposure, following Organisation for Economic Co-Operation and Development test guideline 232. Test duration may be shortened to 21 days by starting exposures with adult animals. The toxicity of chemicals can, however, vary significantly between different life stages (e.g., juveniles or adults) of the same species. In the present study, we assessed the toxicity of four active substances (cyproconazole, teflubenzuron, imidacloprid, and thiacloprid) to F. candida aged approximately 10 days (juveniles) and 20 days (adults) at the beginning of the tests. Tests were performed in LUFA 2.2 standard soil at 20 ±  2 °C, and effect concentration (ECx) values compared using likelihood ratio tests. The tests lasted 21 days for older springtails and 28 days for the younger ones. Life stage did affect the sensitivity of the springtails, with the survival and reproduction of younger animals being a factor of 2-6.5 more sensitive to the insecticides but not to the fungicide. For teflubenzuron and imidacloprid, the EC50 for younger springtails were 0.025 and 0.111 mg a.s. kg-1 soildw , respectively, and for adults 0.048 and 0.264 mg a.s. kg-1 soildw , respectively. For the younger animals the median lethal concentration values for teflubenzuron, imidacloprid, and thiacloprid were 0.353, 0.224, and 1.02 mg a.s. kg-1 soildw , respectively, and 0.571, 0.446, and 6.91 mg a.s. kg-1 soildw , respectively, for older animals. We discuss the implication of these differences for the risk assessment of pesticides to soil arthropods. Environ Toxicol Chem 2023;42:1782-1790. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Combining time-resolved transcriptomics and proteomics data for Adverse Outcome Pathway refinement in ecotoxicology.

Conventional Environmental Risk Assessment (ERA) of pesticide pollution is based on soil concentrations and apical endpoints, such as the reproduction of test organisms, but has traditionally disregarded information along the organismal response cascade leading to an adverse outcome. The Adverse Outcome Pathway (AOP) framework includes response information at any level of biological organization, providing opportunities to use intermediate responses as a predictive read-out for adverse outcomes instead. Transcriptomic and proteomic data can provide thousands of data points on the response to toxic exposure. Combining multiple omics data types is necessary for a comprehensive overview of the response cascade and, therefore, AOP development. However, it is unclear if transcript and protein responses are synchronized in time or time lagged. To understand if analysis of multi-omics data obtained at the same timepoint reveal one synchronized response cascade, we studied time-resolved shifts in gene transcript and protein abundance in the springtail Folsomia candida, a soil ecotoxicological model, after exposure to the neonicotinoid insecticide imidacloprid. We analyzed transcriptome and proteome data every 12 h up to 72 h after onset of exposure. The most pronounced shift in both transcript and protein abundances was observed after 48 h exposure. Moreover, cross-correlation analyses indicate that most genes displayed the highest correlation between transcript and protein abundances without a time-lag. This demonstrates that a combined analysis of transcriptomic and proteomic data from the same time-point can be used for AOP improvement. This data will promote the development of biomarkers for the presence of neonicotinoid insecticides or chemicals with a similar mechanism of action in soils.

Heat stress delays detoxification of phenanthrene in the springtail Folsomia candida.

Climate change has intensified the occurrence of heat waves, resulting in organisms being exposed to thermal and chemical stress at the same time. The effects of mild heat shock combined with sublethal concentrations of phenanthrene (PHE) on defense mechanisms in springtails Folsomia candida were investigated. The transcription of Heat Shock Protein 70 (HSP70) was significantly upregulated by heat shock but tended to reach the control levels after 42 h of recovery. The transcription of cytochrome P450 3A13 (CYP3A13) was upregulated 3-13 fold by PHE but suppressed by heat shock. The suppression by heat shock might contribute to the reduced detoxification of PHE during high-temperature exposure. In line with this, we found that the internal PHE concentration was approximately 70% higher in heat-shocked springtails than in animals kept at control temperature. In general, the transcription of genes encoding enzymes of detoxification phase Ⅱ (glutathione S-transferase 3) and phase Ⅲ (ABC transporter 1) and the activity of antioxidant defense enzymes (superoxide dismutase and catalase) were less influenced than genes encoding phase I detoxification mechanisms (CYP3A13). These results indicate that heat shock delays the detoxification of PHE in springtails.

Biomarker development for neonicotinoid exposure in soil under interaction with the synergist piperonyl butoxide in Folsomia candida.

Pesticide toxicity is typically assessed by exposing model organisms to individual compounds and measuring effects on survival and reproduction. These tests are time-consuming, labor-intensive, and do not accurately capture the effect of pesticide mixtures. Moreover, it is unfeasible to screen the nearly infinite combinations of mixtures for synergistic effects on model organisms. Therefore, reliable molecular indicators of pesticide exposure have to be identified, i.e., biomarkers. These biomarkers can form the basis of rapid and economical screening procedures to assess the toxicity of pesticides even under synergistic interaction with other pollutants. In this study, we screened the expression patterns of eight genes for suitability as a biomarker for neonicotinoid exposure in the soil ecotoxicological model Folsomia candida (springtails). Springtails were exposed to the neonicotinoids imidacloprid and thiacloprid either alone or with various levels of piperonyl butoxide (PBO), which inhibits cytochrome P450 enzymes (CYPs): a common point of synergistic interaction between neonicotinoid and other pesticides. First, we confirmed PBO as a potency enhancer for neonicotinoid toxicity to springtail fecundity, and then used it as a tool to confirm biomarker robustness. We identified two genes that are reliably indicative for neonicotinoid exposure even under metabolic inhibition of CYPs by PBO, nicotinic acetylcholine receptor-subunit alpha 1 (nAchR) and sodium-coupled monocarboxylate transporter (SMCT). These results can form the basis for developing high-throughput screening procedures for neonicotinoid exposure in varying mixture compositions.