Predicting the bioremediation potential of earthworms of different ecotypes through a multi-biomarker approach.

Earthworms are attracting the attention of bioremediation research because of their short-term impact on pollutant fate. However, earthworm-assisted bioremediation largely depends on the earthworm sensitivity to target pollutants and its metabolic capacity to break down contaminants. The most studied species in soil bioremediation has been Eisenia fetida, which inhabits the soil surface feeding on decomposing organic residues. Therefore, its bioremediation potential may be limited to organic matter-rich topsoil. We compared the detoxification potential against organophosphate (OP) pesticides of three earthworm species representative of the main ecotypes: epigeic, anecic, and endogeic. Selected biomarkers of pesticide detoxification (esterases, cytochrome P450-dependent monooxygenase, and glutathione S-transferase) and oxidative homeostasis (total antioxidant capacity, glutathione levels, and glutathione reductase [GR] and catalase activities) were measured in the muscle wall and gastrointestinal tract of E. fetida (epigeic), Lumbricus terrestris (anecic) and Aporrectodea caliginosa (endogeic). Our results show that L. terrestris was the most suitable species to bioremediate OP-contaminated soil for the following reasons: 1) Gut carboxylesterase (CbE) activity of L. terrestris was higher than that of E. fetida, whereas muscle CbE activity was more sensitivity to OP inhibition than that of E. fetida, which means a high capacity to inactivate the toxic oxon metabolites of OPs. 2) Muscle and gut phosphotriesterase activities were significantly higher in L. terrestris than in the other species. 3) Enzymatic (catalase and GR) and molecular mechanisms of free radical inactivation (glutathione) were 3- to 4-fold higher in L. terrestris concerning E. fetida and A. caliginosa, which reveals a higher potential to keep the cellular oxidative homeostasis against reactive metabolites formed during OP metabolism. Together with biological and ecological traits, these toxicological traits suggest L. terrestris a better candidate for soil bioremediation than epigeic earthworms.

Synergistic effects of pesticides and environmental variables on earthworm standard metabolic rate.

Endogeic earthworms such as Aporrectodea caliginosa play an essential role in the agroecosystems because of their continuous burrowing and feeding (geophagous) activity, which causes a profound impact on soil texture, organic matter decomposition, soil carbon storage, microbial activity, soil biodiversity, and nutrient cycling. Accordingly, endogeic earthworms are being proposed as suitable candidates for the ecotoxicity assessment of polluted soils. However, terrestrial ecotoxicology has little considered the interactive effects from pollutants and environmental variables (temperature, moisture). We acclimatized A. caliginosa for 90 days to two contrasting temperatures (10 °C and 20 °C) and moistures (25 % and 35 %, w/v) in soils contaminated with 20 mg kg-1 of chlorpyrifos to examine how these two climate change drivers may modulate the pesticide toxicity. We measured the inhibition of cholinesterase (ChE) activities as indicators of organophosphorus exposure, the standard metabolic rate as an integrative physiological biomarker, and the lipid peroxidation (TBARS) and the total antioxidant capacity (TAC) both as indicators of oxidative stress. The main results were: i) chlorpyrifos strongly inhibited ChE activity (>75 % of controls), demonstrating earthworm bioavailability and acute toxicity at the test concentration; 2) a 50 % mortality and loss of body weight (49 %) were found in the earthworms exposed to the most severe environmental conditions (20 °C, 25 %, and pesticide); 3) this latter experimental group displayed a high SMR, which was concomitant with an increase of the oxidative balance index (TBARS/TAC). We postulated that earthworms acclimatized to stressing environmental conditions experienced a higher pesticide-induced metabolic cost and physiological challenges imposed by adverse environmental conditions.

Biochemical and osmoregulatory responses of the African clawed frog experimentally exposed to salt and pesticide.

Salinization and pollution are two main environmental stressors leading deterioration to water quality and degradation of aquatic ecosystems. Amphibians are a highly sensitive group of vertebrates to environmental disturbance of aquatic ecosystems. However, studies on the combined effect of salinization and pollution on the physiology of amphibians are limited. In this study, we measured the standard metabolic rate (SMR) and biochemical parameters of adult males of the invasive frog Xenopus laevis after 45 days of exposure to contrasting salinity environments (400 and 150 mOsm NaCl) with either 1.0 µg/L of the organophosphate pesticide chlorpyrifos (CPF) or pesticide-free medium. Our results revealed a decrease in SMR of animals exposed to the pesticide and in the ability to concentrate the plasma in animals exposed simultaneously to both stressors. The lack of ability to increase plasma concentration in animals exposed to both salt water and CPF, suggests that osmoregulatory response is decreased by pesticide exposure. In addition, we found an increase of liver citrate synthase activity in response to salt stress. Likewise, the liver acetylcholinesterase (AChE) activity decreased by 50% in frogs exposed to salt water and CPF and 40% in those exposed only to CPF, which suggest an additive effect of salinity on inhibition of AChE. Finally, oxidative stress increased as shown by the higher lipid peroxidation and concentration of aqueous peroxides found in the group exposed to salt water and pesticide. Thus, our results revealed that X. laevis physiology is compromised by salinization and pesticide exposure to both environmental stressors join.

The ecology and evolution of the monito del monte, a relict species from the southern South America temperate forests.

The arboreal marsupial monito del monte (genus Dromiciops, with two recognized species) is a paradigmatic mammal. It is the sole living representative of the order Microbiotheria, the ancestor lineage of Australian marsupials. Also, this marsupial is the unique frugivorous mammal in the temperate rainforest, being the main seed disperser of several endemic plants of this ecosystem, thus acting as keystone species. Dromiciops is also one of the few hibernating mammals in South America, spending half of the year in a physiological dormancy where metabolism is reduced to 10% of normal levels. This capacity to reduce energy expenditure in winter contrasts with the enormous energy turnover rate they experience in spring and summer. The unique life history strategies of this living Microbiotheria, characterized by an alternation of life in the slow and fast lanes, putatively represent ancestral traits that permitted these cold-adapted mammals to survive in this environment. Here, we describe the ecological role of this emblematic marsupial, summarizing the ecophysiology of hibernation and sociality, updated phylogeographic relationships, reproductive cycle, trophic relationships, mutualisms, conservation, and threats. This marsupial shows high densities, despite presenting slow reproductive rates, a paradox explained by the unique characteristics of its three-dimensional habitat. We finally suggest immediate actions to protect these species that may be threatened in the near future due to habitat destruction and climate change.

A toxicological perspective of plastic biodegradation by insect larvae.

Larvae of some insect species (Coleoptera and Lepidoptera) can consume and biodegrade synthetic polymers, including polyethylene, polystyrene, polyvinyl chloride, and polypropylene. Multiple chemical (polymer mass loss and shift of the molecular weight, alterations in chemical functionality, formation of biodegraded intermediates, CO2 production), physical (surface hydrophobicity, thermal analysis), and biological approaches (antibiotic treatment, gut dysbiosis, isolation of plastic microbial degraders) have provided evidence for polymer biodegradation in the larva digestive tract. However, the extent and rate of biodegradation largely depend on the physicochemical structure of the polymer as well as the presence of additives. Additionally, toxicology associated with plastic biodegradation has not been investigated. This knowledge gap is critical to understand the gut symbiont-host interaction in the biodegradation process, its viability in the long term, the effects of plastic additives and their metabolites, and the phenotypic traits linked to a plastic-rich diet might be transferred in successive generations. Likewise, plastic-eating larvae represent a unique case study for elucidating the mechanisms of toxic action by micro- and nanoplastics because of the high concentration of plastics these organisms may be intentionally exposed to. This perspective review graphically summarizes the current knowledge on plastic biodegradation by insect larvae and describes the physiological processes (digestive and immune systems) that may be disrupted by micro- and nanoplastics. It also provides an outlook to advance current knowledge on the toxicity assessment of plastic-rich diets and the environmental risks of plastic-containing by-products (e.g., insect manure used as fertilizer).

Ecotoxicological impact of the antihypertensive valsartan on earthworms, extracellular enzymes and soil bacterial communities.

The use of reclaimed water in agriculture represents a promising alternative to relieve pressure on freshwater supplies, especially in arid or semiarid regions facing water scarcity. However, this implies introducing micropollutants such as pharmaceutical residues into the environment. The fate and the ecotoxicological impact of valsartan, an antihypertensive drug frequently detected in wastewater effluents, were evaluated in soil-earthworm microcosms. Valsartan dissipation in the soil was concomitant with valsartan acid formation. Although both valsartan and valsartan acid accumulated in earthworms, no effect was observed on biomarkers of exposure (acetylcholinesterase, glutathione S-transferase and carboxylesterase activities). The geometric mean index of soil enzyme activity increased in the soils containing earthworms, regardless of the presence of valsartan. Therefore, earthworms increased soil carboxylesterase, dehydrogenase, alkaline phosphatase, ß-glucosidase, urease and protease activities. Although bacterial richness significantly decreased following valsartan exposure, this trend was enhanced in the presence of earthworms with a significant impact on both alpha and beta microbial diversity. The operational taxonomic units involved in these changes were related to four (Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) of the eight most abundant phyla. Their relative abundances significantly increased in the valsartan-treated soils containing earthworms, suggesting the presence of potential valsartan degraders. The ecotoxicological effect of valsartan on microbes was strongly altered in the earthworm-added soils, hence the importance of considering synergistic effects of different soil organisms in the environmental risk assessment of pharmaceutical active compounds.

Elucidating pesticide sensitivity of two endogeic earthworm species through the interplay between esterases and glutathione S-transferases.

Earthworms are common organisms in soil toxicity-testing framework, and endogeic species are currently recommended due to their ecological role in agroecosystem. However, little is known on their pesticide metabolic capacities. We firstly compared the baseline activity of B-esterases and glutathione-S-transferase in Allolobophora chlorotica and Aporrectodea caliginosa. Secondly, vulnerability of these species to pesticide exposure was assessed by in vitro trials using the organophosphate (OP) chlorpyrifos-ethyl-oxon (CPOx) and ethyl-paraoxon (POx), and by short-term (7 days) in vivo metabolic responses in soil contaminated with pesticides. Among B-esterases, acetylcholinesterase (AChE) activity was abundant in the microsomal fraction (80% and 70% of total activity for A. caliginosa and A. chlorotica, respectively). Carboxylesterase (CbE) activities were measured using three substrates to examine species differences in isoenzyme and sensitivity to both in vitro and in vivo exposure. CbEs were mainly found in the cytosolic fraction (80% and 60% for A. caliginosa and A. chlorotica respectively). GST was exclusively found in the soluble fraction for both species. Both OPs inhibited B-esterases in a concentration-dependent manner. In vitro trials revealed a pesticide-specific response, being A. chlorotica AChE more sensitive to CPOx compared to POx. CbE activity was inhibited at the same extent in both species. The 7-d exposure showed A. chlorotica less sensitive to both OPs, which contrasted with outcomes from in vitro experiments. This non-related functional between both approaches for assessing pesticide toxicity suggests that other mechanisms linked with in vivo OP bioactivation and excretion could have a significant role in the OP toxicity in endogeic earthworms.

Earthworms increase the potential for enzymatic bio-activation of biochars made from co-pyrolyzing animal manures and plastic wastes.

We assessed the enzymatic activation of four different biochars produced from pyrolyzing swine manure and poultry litter, and by co-pyrolyzing these livestock residues with agricultural spent mulch plastic film wastes (plastichars). Enzymatic activation consisted of incubating biochars in soil inoculated with earthworms (Lumbricus terrestris), which acted as biological vectors to facilitate retention of extracellular enzymes onto biochar surface. The activity of carboxylesterase ‒a pesticide-detoxifying enzyme‒ was measured in non-bioturbed soils (reference), linings of the burrows created by earthworms, casts (feces) and biochar particles recovered from the soil. Our results revealed that: 1) biochar increased soil carboxylesterase activity respect to biochar-free (control) soils, which was more prominent in the presence of earthworms. 2) The maximum enzyme activity was found in soils amended with plastichars. 3) The plastichars showed higher enzyme binding capacities than that of the biochars produced from animal manure alone, corroborating the pattern of enzyme distribution found in soil. 4) The presence of earthworms in soil significantly increased the potential of the plastichars for enzymatic activation. These findings suggest that the plastichars are suitable for increasing and stabilizing soil enzyme activities with no toxicity on earthworms.

Effect of salinity acclimation on osmoregulation, oxidative stress, and metabolic enzymes in the invasive Xenopus laevis.

Aquatic animals often display physiological adjustments to improve their biological performance and hydrosaline balance in saline environments. In addition to energetic costs associated with osmoregulation, oxidative stress, and the activation of the antioxidant system are common cellular responses to salt stress in many species, but the knowledge of osmoregulation-linked oxidative homeostasis in amphibians is scarce. Here we studied the biochemical responses and oxidative responses of Xenopus laevis females exposed for 40 days to two contrasting salinities: hypo-osmotic (150 mOsm·kg-1 ·H2 O NaCl, HYPO group) and hyper-osmotic environments (340 mOsm·kg-1 ·H2 O NaCl, HYPER group). We found an increase of plasma osmolality and plasma urea concentration in the animals incubated in the HYPER treatment. Increases in electrolyte concentration were paralleled with an increase of both citrate synthase and cytochrome c oxidase activities in liver and heart. Interestingly, HYPO group had higher catabolic activity of the skin and liver total antioxidant capacity (TAC), compared with animals from the HYPER group. Moreover, there was an inverse relationship between liver TAC and plasma osmolality; and with the metabolic enzymes from liver. These findings suggest that salinity induces changes in urea metabolism and specific activity of metabolic enzymes, which appears to be tissue-dependent in X. laevis. Contrary to our expectations, we also found a moderate change in the oxidative status as revealed by the increase in TAC activity in the animals acclimated to low salinity medium, but constancy in the lipid peroxidation of membranes.

Beyond survival experiments: using biomarkers of oxidative stress and neurotoxicity to assess vulnerability of subterranean fauna to climate change.

Accurate assessments of species vulnerability to climate change need to consider the physiological capacity of organisms to deal with temperature changes and identify early signs of thermally induced stress. Oxidative stress biomarkers and acetylcholinesterase activity are useful proxies of stress at the cellular and nervous system level. Such responses are especially relevant for poor dispersal organisms with limited capacity for behavioural thermoregulation, like deep subterranean species. We combined experimental measurements of upper lethal thermal limits, acclimation capacity and biomarkers of oxidative stress and neurotoxicity to assess the impact of heat stress (20°C) at different exposure times (2 and 7 days) on the Iberian endemic subterranean beetle Parvospeonomus canyellesi. Survival response (7 days of exposure) was similar to that reported for other subterranean specialist beetles (high survival up to 20°C but no above 23°C). However, a low physiological plasticity (i.e. incapacity to increase heat tolerance via acclimation) and signs of impairment at the cellular and nervous system level were observed after 7 days of exposure at 20°C. Such sublethal effects were identified by significant differences in total antioxidant capacity, glutathione S-transferase activity, the ratio of reduced to oxidized forms of glutathione and acetylcholinesterase activity between the control (cave temperature) and 20°C treatment. At 2 days of exposure, most biomarker values indicated some degree of oxidative stress in both the control and high-temperature treatment, likely reflecting an initial altered physiological status associated to factors other than temperature. Considering these integrated responses and the predicted increase in temperature in its unique locality, P. canyellesi would have a narrower thermal safety margin to face climate change than that obtained considering only survival experiments. Our results highlight the importance of exploring thermally sensitive processes at different levels of biological organization to obtain more accurate estimates of the species capacity to face climate change.

Tetrabromobisphenol A inhibits carboxylesterase activity of marine organisms from different trophic levels.

Tetrabromobisphenol A (TBBPA), a brominated flame retardant used in synthetic polymers and electronics, is present in the aquatic environment and recent evidence suggests it can be potentially biomagnified in the marine ecosystem. However, the toxicity of TBBPA in the marine biota has not been investigated in detail. In this study we aimed to understand the role of carboxylesterases (CEs) in xenobiotic metabolism under the exposure of marine organisms to a chemical of environmental concern, TBBPA. Specifically, we tested for in vitro inhibition of CE activity in a range of marine organisms covering different ecological niches, from species from low (mussels and copepods), medium (sardines and anchovies) and high trophic levels (tuna). The results revealed that the highest inhibition of CE activity to 100 µM TBBPA was recorded in mussels (66.5% inhibition) and tunids (36.3-76.4%), whereas copepods and small pelagic fish showed comparatively lower effects (respectively, 30% and 36.5-55.6%). Our results suggest that CE-mediated detoxification and physiological processes could be compromised in TBBPA-exposed organisms and could ultimately affect humans as many of them are market species.

Exploring the potential enzymatic bioremediation of vermicompost through pesticide-detoxifying carboxylesterases.

Vermicompost is a known biofertilizer of potential use in soil bioremediation. This study was undertaken to explore the capacity of grape marc-derived vermicompost to inactivate methyl carbamate (MC) and organophosphorus (OP) pesticides via exploring the carboxylesterase (CE) activity level and its response to pesticide exposure. We first optimized the method for enzyme activity assay comparing the CE activity in two contrasting homogenization procedures (30-min mixing and mortar grinding). Thereafter, we assessed the sensitivity of the enzyme by both in vitro and vermicompost incubation trials with selected pesticides. The main findings can be summarized as follows: i) grinding the vermicompost in water (2% w/v) yielded maximum enzyme activity; ii) at concentrations around 10-4 M, highly toxic oxygen-analog metabolites of OPs strongly inhibited the CE activity (76-93% inhibition), but MC did not inhibit the enzyme activity; iii) liquid vermicompost was able to degrade chlorpyrifos and inactivate its highly toxic metabolite chlorpyrifos-oxon. Our results suggest that liquid vermicompost is the most appropriate preparation to increase the enzymatic potential of vermicompost in pesticide-contaminated soils.

Biochar and earthworms working in tandem: Research opportunities for soil bioremediation.

Intensive use of agrochemicals is considered one of the major threats for soil quality. In an attempt to mitigate their side-effects on non-target organisms and soil functioning, many engineering and biological remediation methodologies are currently available. Among them, the use of biochar, a carbonaceous material produced from pyrolysing biomass, represents an attractive option enhancing both remediation and soil carbon storage potentials. Currently, activation of biochar with chemical or physical agents seeks for improving its remediation potential, but most of them have some undesirable drawbacks such as high costs and generation of chemical wastes. Alternatively, the use of biological procedures to activate biochar with extracellular enzymes is gaining acceptance mainly due to its eco-friendly nature and cost-effectiveness. In these strategies, microorganisms play a key role as a source of extracellular enzymes, which are retained on the biochar surface. Recently, several studies point out that soil macrofauna (earthworms) may act as a biological vector facilitating the adsorption of enzymes on biochar. This paper briefly introduces current biochar bioactivation methodologies and the mechanisms underlying the coating of biochar with enzymes. We then propose a new conceptual model using earthworms to activate biochar with extracellular enzymes. This new earthworm-biochar model can be used as a theoretical framework to produce a new product "vermichar", vermicompost produced from blended feedstock, earthworms, and biochar that can be used to improve soil quality and remove soil contaminants. This model can also be used to develop innovative in-situ "vermiremediation" technologies utilizing the beneficial effects of both earthworms and biochar. Since biochar may contain toxic chemicals generated during its production stages or later concentrated when applied to polluted soils, this paper also highlights the need for an ecotoxicological knowledge around earthworm-biochar interaction, promoting further discussion on suitable procedures for assessing the environmental risk of this conceptual model application in soil bioremediation.

Biochar increases pesticide-detoxifying carboxylesterases along earthworm burrows.

Herein, we examined whether synergistic effects of earthworms (Lumbricus terrestris) and pine needle-derived biochar result in biochar-coated burrows with enhanced carboxylesterase (CE) activity (a pesticide-detoxifying enzyme). Biochar was placed at the top of soil columns at two doses (2.5 and 5% w/w dry mass), with an additional biochar-free treatment as control. Carboxylesterase and dehydrogenase activities were measured in the burrow walls sampled at three depths (0-4, 8-12, and 18-22 cm). Biochar was recovered from these samples to confirm its vertical transport and enzymatic activation. We tested whether biochar protected CE activity against desiccation stress of burrow wall samples. Likewise, the role of earthworm mucus in stabilizing CE onto biochar surface was also investigated by measuring the enzyme activity in fresh biochar particles previously incubated in the presence of earthworm mucus and purified esterase. Finally, we checked for the sensitivity of biochar-bound CE activity against selected organophosphorus pesticides. The main results were: i) co-application of earthworms and biochar caused a significant increase of CE activity in the first 12 cm of the soil column, ii) recovered biochar particles displayed CE activity which was significantly higher in the particles collected from the bottom of columns, iii) soil desiccation decreased the enzymatic activity, although such effect depended on biochar treatment and column depth (22-77% inhibition). Nevertheless, CE activity still was higher in the samples from the 5%-biochar treatment than activity in control and 2.5%-biochar treatments, iv) earthworm mucus favored the retention of CE onto the biochar surface, and v) the activity of biochar-bound CE was sensitive to inhibition by chlorpyrifos-oxon. These results suggest that the joint application of L. terrestris and biochar may be a suitable vermiremediation strategy to inactivate OP pesticides.

Assessing biochar impact on earthworms: Implications for soil quality promotion.

Potential harmful effects of spent coffee grounds (SCGs)-derived biochar on earthworms (Lumbricus terrestris) were investigated through two complementary experiments, which assessed the avoidance response of earthworms to biochar-amended soils (experiment 1), and the response of oxidative stress biomarkers and digestive enzymes (experiment 2). The main results were: 1) the highest dose of biochar (5% w/w) caused a significant avoidance response of earthworms (75% individuals avoided these treated soils after 48 h); 2) signs of oxidative stress were early detected in earthworms exposed to biochar (1 and 5% w/w) as indicated by the integrated biological response index; 3) earthworms exposed to biochar-amended soils for 30 d experienced a significant increase of digestive enzyme activities measured in both the gastrointestinal tissue and the luminal content; 4) interaction between earthworms and biochar led to a higher soil extracellular enzyme activities in the 1% biochar treatment than that of control and 5% biochar treatments. These findings suggest that the joint application of SCG-biochar and L. terrestris is a workable approach for improving soil quality in terms of soil biochemical promotion, although earthworms may develop some physiological mechanisms of biochar tolerance (antioxidant defenses).

Terrestrial birds in coastal environments: metabolic rate and oxidative status varies with the use of marine resources.

Life in saline environments represents a major physiological challenge for birds, particularly for passerines that lack nasal salt glands and hence are forced to live in environments that do not contain salty resources. Increased energy costs associated with increased salt intake, which in turn increases the production of reactive oxygen species, is likely a major selection pressure for why passerines are largely absent from brackish and marine environments. Here we measured basal metabolic rates (BMR) and oxidative status of free-ranging individuals of three species of Cinclodes, a group of passerine birds that inhabit marine and freshwater habitats in Chile. We used a combination of carbon, nitrogen, and hydrogen isotope data from metabolically active (blood) and inert (feathers) tissues to estimate seasonal changes in marine resource use and infer altitudinal migration. Contrary to our expectations, the consumption of marine resources did not result in higher BMR values and higher oxidative stress. Specifically, the marine specialist C. nigrofumosus had lower BMR than the other two species (C. fuscus and C. oustaleti), which seasonally switch between terrestrial and marine resources. C. fuscus had significantly higher total antioxidant capacity than the other two species (C. nigrofumosus and C. oustaleti) that consumed a relatively high proportion of marine resources. Nearly all studies examining the effects of salt consumption have focused on intraspecific acclimation via controlled experiments in the laboratory. The mixed results obtained from field- and lab-based studies reflect our poor understanding of the mechanistic link among hydric-salt balance, BMR, and oxidative stress in birds.

Interspecific differences in biochemical and behavioral biomarkers in endogeic earthworms exposed to ethyl-parathion.

Earthworms are common organisms in the soil toxicity-testing framework, and the epigeic Eisenia andrei and E. fetida are the recommended species. However, Eisenia species are rarely found in agricultural soils and recent studies have pointed out endogeic species are more sensitive to pesticides than Eisenia. Allolobophora chlorotica and Aporrectodea caliginosa are two endogeic soil-dwelling species that are abundant in the agroecosystem. However, knowledge on pesticide impact on this ecological group of earthworms is still incipient. Herein, we compared the biochemical (acetylcholinesterase [AChE] and carboxylesterase [CbE] activities) and behavioral (burrowing, casting and feeding) biomarker responses of these two endogeic earthworm species exposed for 7 days to soils contaminated with 0.1, 1 and 10 mg kg-1 ethyl-parathion. The results showed marked species-specific differences in both groups of biomarkers, suggesting A. caliginosa the most sensitive species to this organophosphorus pesticide under the exposure conditions in this study. Moreover, an in vitro inhibition trial with ethyl-paraoxon evidenced a higher sensitivity of A. caliginosa AChE activity compared with that of A. chlorotica. This finding suggested that this molecular target endpoint could contribute to the interspecific differences of behavioral responses rather than CbE activity; this latter considered a potent mechanism of OP removal. Our results suggest the inclusion of more than one endogeic earthworm species to assess toxicity from organophosphorus insecticides. However, the use of A. caliginosa in the environmental risk assessment framework of organophosphorus contamination is highly recommended because of its higher sensibility to this class of pesticides, in addition to its abundance in the agroecosystem.

Response of digestive enzymes and esterases of ecotoxicological concern in earthworms exposed to chlorpyrifos-treated soils.

Assessment of organophosphorus (OP) pesticide exposure in non-target organisms rarely involves non-neural molecular targets. Here we performed a 30-d microcosm experiment with Lumbricus terrestris to determine whether the activity of digestive enzymes (phosphatase, ß-glucosidase, carboxylesterase and lipase) was sensitive to chlorpyrifos (5 mg kg-1 wet soil). Likewise, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the wall muscle and gastrointestinal tissues as indicators of OP exposure. Chlorpyrifos inhibited the acid phosphatase (34% of controls), carboxylesterase (25.6%) and lipase activities (31%) in the gastrointestinal content. However, in the gastrointestinal tissue, only the carboxylesterase and lipase activities were significantly depressed (42-67% carboxylesterase inhibition in the foregut and crop/gizzard, and 15% lipase inhibition in the foregut). Chlorpyrifos inhibited the activity of both cholinesterases in the gastrointestinal tissues, whereas the AChE activity was affected in the wall muscle. These results suggested chlorpyrifos was widely distributed throughout the earthworm body after 30 d of incubation. Interestingly, we found muscle carboxylesterase activity strongly inhibited (92% of control) compared with that detected in the gastrointestinal tissues of the same OP-exposed individuals. This finding was explained by the occurrence of pesticide-resistant esterases in the gastrointestinal tissues, which were evidenced by zymography. Our results suggest that digestive processes of L. terrestris may be altered by chlorpyrifos, as a consequence of the inhibitory action of the insecticide on some digestive enzymes.

Biochar activation with exoenzymes induced by earthworms: A novel functional strategy for soil quality promotion.

The activation of biochar (carbonaceous material generated by pyrolysis of biomass) with extracellular enzymes generated from soil biological processes was studied via a 2-month microcosm using earthworms. The isolation of biochar particles (post-incubated biochar) from earthworm-treated soils allowed to confirm an enrichment of biochar with extracellular enzymes associated to biogeochemical (alkaline phosphatase, ß-glucosidase and arylsulfatase) and bioremediation pathways (carboxylesterase). The hydrolytic activity of this biochar incubated with earthworms was up to 8 times higher compared with that of control biochar (incubated in earthworm-free soils). More interestingly, the enzyme carboxylesterase was stabilized onto biochar surface as demonstrated by its unchanged hydrolytic activity after multiple water rinses of the post-incubated biochar, and the remaining activity following heat stress. Moreover, this biochar-bound carboxylesterase activity was sensitive to inhibition by organophosphorus (23-100% of control activity) and methyl carbamate pesticides (37-57%), demonstrating that bioactivated biochar with carboxylesterases may inactivate these agrochemicals by providing stable ligands of enzymatic nature.

Soil enzyme dynamics in chlorpyrifos-treated soils under the influence of earthworms.

Earthworms contribute, directly and indirectly, to contaminant biodegradation. However, most of bioremediation studies using these annelids focus on pollutant dissipation, thus disregarding the health status of the organism implied in bioremediation as well as the recovery of indicators of soil quality. A microcosm study was performed using Lumbricus terrestris to determine whether earthworm density (2 or 4individuals/kg wet soil) and the time of exposure (1, 2, 6, 12, and 18wk) could affect chlorpyrifos persistence in soil initially treated with 20mg active ingredientkg-1 wet soil. Additionally, selected earthworm biomarkers and soil enzyme activities were measured as indicators of earthworm health and soil quality, respectively. After an 18-wk incubation period, no earthworm was killed by the pesticide, but clear signs of severe intoxication were detected, i.e., 90% inhibition in muscle acetylcholinesterase and carboxylesterase (CbE) activities. Unexpectedly, the earthworm density had no significant impact on chlorpyrifos dissipation rate, for which the measured half-life ranged between 30.3d (control soils) and 44.5d (low earthworm density) or 36.7d (high earthworm density). The dynamic response of several soil enzymes to chlorpyrifos exposure was examined calculating the geometric mean and the treated-soil quality index, which are common enzyme-based indexes of microbial functional diversity. Both indexes showed a significant and linear increase of the global enzyme response after 6wk of chlorpyrifos treatment in the presence of earthworms. Examination of individual enzymes revealed that soil CbE activity could decrease chlorpyrifos-oxon impact upon the rest of enzyme activities. Although L. terrestris was found not to accelerate chlorpyrifos dissipation, a significant increase in the activity of soil enzyme activities was achieved compared with earthworm-free, chlorpyrifos-treated soils. Therefore, the inoculation of organophosphorus-contaminated soils with L. terrestris arises as a complementary bioremediation strategy in terms of recovery of soil biochemical performance and quality.