International audience | Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg−1 chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (≤1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field. Esterase inhibition combined with oxime reactivation methods is a suitable approach for monitoring organophosphate contamination

Despite being effective in controlling mosquito larvae and a few other target organisms, the application of insecticides into aquatic systems may cause unintended alterations to the physiology or behavioral responses of several aquatic non-target organisms, which can ultimately lead to their death. Here, we firstly evaluated whether the susceptibility of the giant water bug, Belostoma anurum (Hemiptera: Belostomatidae), a predator of mosquito larvae, to pyriproxyfen would be similar to that of its potential prey, larvae of Aedes aegypti (Diptera: Culicidae). Secondly, we recorded the nominal concentrations of pyriproxyfen in water and evaluated whether sublethal exposures would lead to physiological or behavioral alterations on the B. anurum nymphs. We characterized the activities of three major families of detoxification enzymes (i.e., cytochrome P450 monooxygenases, glutathione-S-transferase, and general esterases) and further evaluated the abilities of pyriproxyfen sublethally-exposed B. anurum to prey upon A. aegypti larvae at different prey densities. Our findings revealed that nominal pyriproxyfen concentration significantly decreased (approximately 50%) over the first 24 h. Furthermore, when applied at the concentration of 10 μg a.i./L, pyriproxyfen was approximately four times more toxic to A. aegypti larvae (LT₅₀ = 48 h) than to B. anurum nymphs (LT₅₀ = 192 h). Interestingly, the pyriproxyfen sublethally-exposed (2.5 μg a.i./L) B. anurum nymphs exhibited reduced enzyme activities (cytochrome P450 monooxygenases) involved in detoxication processes and preyed significantly less on A. aegypti larvae when compared to unexposed predators. Collectively, our findings demonstrate that mortality-based pyriproxyfen risk assessments are not always protective of aquatic non-target organisms.

Soil algal bioassays have been limited by their inability to evaluate several toxic endpoints because it is difficult to collect pure soil algae growing on and beneath the soil surface. This study describes the extension of a previously developed paper-disc method for analyzing soil toxicity to algae. The method can be used in conjunction with flow cytometric analysis and facilitates the assessment of previously proposed toxicity endpoints, such as the growth zone, biomass, and photosynthetic activity. We assessed the applicability of this paper-disc soil method using the green algae Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata exposed to nickel-contaminated soil; examined cell sizes, cell granularity, enzyme activity, and oxidative stress as new toxicity endpoints using flow cytometry; and identified morphological changes in green algae assayed. The results showed that, used in conjunction with flow cytometry, the extended paper-disc soil method is sufficiently sensitive to detect decreases in cell granularity in C. reinhardtii and esterase activity in P. subcapitata. The method also revealed decreases in growth zone, biomass, and electron transfer from the reaction center to the quinone pool. Collectively, the results of this study indicate that soil algal bioassays using nonspecific algae can be used to assess soil quality, to derive several toxicity endpoints for individual cells, and to evaluate previously established flow cytometric toxicity endpoints.

Chronic effects of the ionic liquid [C4mim][Cl] (mp 73 °C) towards the microalga, Scenedesmus quadricauda were studied by flow cytometry, monitoring multiple endpoints of cell density, esterase activity, membrane integrity, reactive oxygen species and chlorophyll fluorescence. Toxicity was clearly in evidence, and although increased esterase activity indicated hormesis during initial exposure to [C4mim][Cl], inhibition of both esterase activity and chlorophyll fluorescence became apparent after 3 days. Cell density was also decreased by culturing with [C4mim][Cl], but this effect was clearly concentration-dependent and only became significant during the second half of the experiment. In contrast, [C4mim][Cl] had only a modest effect on reactive oxygen species (ROS) and caused little damage to cell membranes.

The accumulation of phthalate acid esters (PAEs) in the environment has aroused a global concern. Microbial degradation is the most promising method for removing PAEs from polluted environment. Di-n-butyl phthalate (DBP) is one of the most widely used PAEs. In this study, a highly efficient DBP-degrading strain, Enterobacter sp. DNB-S2 was isolated from Mollisol in northeast China, and the degradation rate of 500 mg L⁻¹ DBP reached 44.10% at 5 °C and 91.08% at 50 °C within 7 days. A new intermediate, n-butyl benzoate BP, was detected, implying a new degradation pathway. The complete genome of the strain DNB-S2 was successfully sequenced to comprehensively understand of the entire DBP catabolic process. Key genes were proposed to be involved in DBP degradation, such as esterases, 3,4-dihydroxybenzoate decarboxylase and catechol 2,3-dioxygenase genes. Intermediate-utilization tests and real-time quantitative polymerase chain reaction (RT-qPCR) validated the proposed DBP catabolic pathway. The aboriginal bacterium DNB-S2 is a promising germplasm for restoring PAE-contaminated Mollisol regions at low temperature. This study provides novel insight into the catabolic mechanisms and abundant gene resources of PAE biodegradation.

Due to the growing concern about the presence of microplastics (MP) in the environment, the number of studies evaluating the toxicity of these small persistent particles on different marine species has increased in recent years. Few studies have addressed their impact on marine phytoplankton, a subject of great concern since they are primary producers of the aquatic food web. The aim of this study is to unravel the cytotoxicity of 2.5 μg mL⁻¹ unlabelled amino-modified polystyrene beads of different sizes (0.5 and 2 μm) on the marine diatom Chaetoceros neogracile. In addition to traditional growth and photosynthesis endpoints, several physiological and biochemical parameters were monitored every 24 h in C. neogracile cells by flow cytometry during their exponential growth (72 h). Dynamic Light Scattering measurements revealed the strong aggregation and the negative charge of the beads assayed in the culture medium, which seemed to minimize particle interaction with cells and potentially associated impacts. Indeed, MP were not attached to the microalgal cell wall, as evidenced by scanning electron micrographs. Cell growth, morphology, photosynthesis, reactive oxygen species levels and membrane potential remained unaltered. However, exposure to MP significantly decreased the cellular esterase activity and the neutral lipid content. Microalgal oil bodies could serve as an energy source for maintaining a healthy cellular status. Thus, MP-exposed cells modulate their energy metabolism to properly acclimate to the stress conditions.

The study was prompted to characterize the B-type esterase activities in the terrestrial snail Xeropicta derbentina and to evaluate its sensitivity to organophosphorus and carbamate pesticides. Specific cholinesterase and carboxylesterase activities were mainly obtained with acetylthiocholine (Km = 77.2 mM; Vmax = 38.2 mU/mg protein) and 1-naphthyl acetate (Km = 222 mM, Vmax = 1095 mU/mg protein) substrates, respectively. Acetylcholinesterase activity was concentration-dependently inhibited by chlorpyrifos-oxon, dichlorvos, carbaryl and carbofuran (IC50 = 1.35 x 10-5-3.80 x 10-8 M). The organophosphate-inhibited acetylcholinesterase activity was reactivated in the presence of pyridine-2-aldoxime methochloride. Carboxylesterase activity was inhibited by organophosphorus insecticides (IC50 = 1.20 x 10-5-2.98 x 10-8 M) but not by carbamates. B-esterase-specific differences in the inhibition by organophosphates and carbamates are discussed with respect to the buffering capacity of the carboxylesterase to reduce pesticide toxicity. These results suggest that B-type esterases in X. derbentina are suitable biomarkers of pesticide exposure and that this snail could be used as sentinel species in field monitoring of Mediterranean climate regions. Characterization of the B-type esterases in the terrestrial snail Xeropicta derbentina in order to evaluate pesticide exposure.

The house fly, Musca domestica L., is a cosmopolitan insect pest of public and animal health importance that serves as a mechanical vector of pathogens. Aimed at prospective resistance management to reduce environmental pollution, we characterized the inheritance pattern, realized heritability, fitness cost, cross resistance, stability and mechanism of clothianidin resistance in M. domestica that were collected from the poultry farm. By continuous selection with clothianidin for 11 generations, the clothianidin selected M. domestica strain (Clotha-SEL) developed a 3827-fold resistance compared to a susceptible strain. However, resistance to clothianidin was proved to be unstable when selection with clothianidin was removed for five generations (G₇ to G₁₂). Inheritance pattern analysis at G₈ of Clotha-SEL (RR = 897) revealed that resistance to clothianidin was polygenic, autosomal and incompletely dominant. Realized heritability (h²) for resistance value was 0.38 (at G₁₁) in the tested strain. Synergist bioassays showed that microsomal oxidases and esterases might not contribute significantly in resistance evolution. Fitness costs of clothianidin resistance were present, for example, reduction in growth potential of the Clotha-SEL strain in comparison to the untreated counterpart strain (UNSEL) was observed. No cross resistance to bifenthrin and fipronil and a very low cross-resistance to spinosad were observed. These insecticides could be alternated with clothianidin as an insecticide resistance management tool to sustain its efficacy for a longer time period. These results shall be utilized to devise a proactive resistance management strategy for use of clothianidin against M. domestica that will be helpful to alleviate the allied threats to environmental and human health.

Chronic exposure to pyrethroid insecticides can result in strong selective pressures on non-target species in aquatic systems and drive the evolution of resistance and population-level changes. Characterizing the underlying mechanisms of resistance is essential to better understanding the potential consequences of contaminant-driven microevolution. The current study found that multiple mechanisms enhance the overall tolerance of Hyalella azteca to the pyrethroid permethrin. In H. azteca containing mutations in the voltage-gated sodium channel (VGSC), both adaptation and acclimation played a role in mitigating the adverse effects of pyrethroid exposures. Pyrethroid resistance is primarily attributed to the heritable mutation at a single locus of the VGSC, resulting in reduced target-site sensitivity. However, additional pyrethroid tolerance was conferred through enhanced enzyme-mediated detoxification. Cytochrome P450 monooxygenases (CYP450) and general esterases (GE) significantly contributed to the detoxification of permethrin in H. azteca. Over time, VGSC mutated H. azteca retained most of their pyrethroid resistance, though there was some increased sensitivity from parent to offspring when reared in the absence of pyrethroid exposure. Permethrin median lethal concentrations (LC50s) declined from 1809 ng/L in parent (P₀) individuals to 1123 ng/L in the first filial (F₁) generation, and this reduction in tolerance was likely related to alterations in acclimation mechanisms, rather than changes to target-site sensitivity. Enzyme bioassays indicated decreased CYP450 and GE activity from P₀ to F₁, whereas the VGSC mutation was retained. The permethrin LC50s in resistant H. azteca were still two orders-of-magnitude higher than non-resistant populations indicating that the largest proportion of resistance was maintained through the inherited VGSC mutation. Thus, the noted variation in tolerance in H. azteca is likely associated with inducible traits controlling enzyme pathways. A better understanding of the mechanistic and genomic basis of acclimation is necessary to more accurately predict the ecological and evolutionary consequences of contaminant-driven change in H. azteca.

The complexation with extracellular polymeric substances (EPS) greatly reduces the toxicity of heavy metals towards organisms in the environment. However, the molecular mechanism of EPS−metal complexation remains unclear owing to the limitation of precise analysis for key fractions and functionalities in EPS that associate with metals. Herein, we explored the EPS−Cd (II) complexation by fluorescence excitation emission matrix coupled with parallel factor (EEM−PARAFAC), two-dimensional Fourier transform infrared correlation spectroscopy (2D-FTIR−COS) and X-ray photoelectron spectroscopy (XPS), attempting to explain the mechanisms of EPS in alleviating Cd (II) toxicity toward a green alga Chlorella vulgaris (C. vulgaris). When the algal EPS were removed, the cell internalizations of Cd (II), growth inhibition rate and chlorophyll autofluorescence increased, but the surface adsorption and esterase activities decreased, indicating that the sorption of Cd (II) by EPS was crucial in alleviating the algal toxicity. Moreover, the complexation with proteins in EPS controlled the sorption of Cd (II) to algal EPS, resulting in the chemical static quenching of the proteins fluorescence by 47.69 ± 2.37%. Additionally, the complexing capability of the main functionalities, COO⁻ and C–OH in proteins with Cd (II) was stronger than that of C–O(H) and C–O–C in polysaccharides or C–OH in the humus-related substances. Oxygen atom in protein carboxyl C–O might be the key site of EPS−Cd (II) complexation, supported by the modified Ryan−Weber complexation model and the obvious shift of oxygen valence-electron signal. These findings provide deep insights into understanding the interaction of EPS with heavy metals in aquatic environment.