Responses Of Earthworms Research Articles

Biochemical response, histopathological change and DNA damage in earthworm (Eisenia fetida) exposed to sulfentrazone herbicide

• The effects of sulfentrazone on earthworms were investigated using multiple biomarkers. • Sulfentrazone could cause oxidative stress, tissue and DNA damage in the short term. • GST plays a key role in earthworm responses to the sulfentrazone stress. • IBRv2 indicated that the toxicity of sulfentrazone on earthworms was reduced with time. Earthworms are highly important for soil health and functioning. However, the soil-applied agrochemicals may impact their survival and functioning. Sulfentrazone, a triazolinones herbicide, is widely used in modern agriculture while its toxicity to some aquatic organisms and amphibians has been reported. However, we know little about the impact of sulfentrazone on ecologically important soil earthworms such as Eisenia fetida . Thus, we investigated the toxicity of sulfentrazone (0.2, 1.0 and 5.0 mg/kg in soil) on E. fetida at day 7, 14, 21 and 28. We investigated the effects of sulfentrazone on oxidative stress, histopathological change, and DNA damage in earthworms. Our results showed an enhanced production of reactive oxygen species in earthworms corresponding to the sulfentrazone concentrations. The activities of superoxide dismutase, catalase, guaiacol peroxidase and glutathione S-transferase (GST) also exhibited similar trends during sulfentrazone exposure at various time intervals. The malondialdehyde contents increased at day 7, 14 and 21, and returned to the control level at day 28. Sulfentrazone at 1.0 and 5.0 mg/kg concentrations damaged epidermal and intestinal tissues while we observed low DNA damage at day 21. Integrated Biological Response index (IBR) indicated that GST played a key role to minimize the oxidative stress caused by sulfentrazone. Moreover, the IBRv2 values also showed that the effect of sulfentrazone was reduced after 14 days, thus indicating that sulfentrazone (≤5.0 mg/kg) caused reversible toxicological effects to E. fetida . Our findings provide insight into the toxic effects of sulfentrazone on earthworm and may be useful for risk assessment of sulfentrazone on soil ecosystems.

Benzotriazole alleviates copper mediated lysosomal membrane damage and antioxidant defense system responses in earthworms (Eisenia fetida).

Benzotriazole (BTR) is a common corrosion inhibitor used to protect copper (Cu) and Cu alloys. To reveal the combined subacute toxicity of BTR and Cu at environmental levels on terrestrial animals, the activity of antioxidative enzymes and the glutathione levels in earthworms (Eisenia fetida) of the single or co-exposure treatments were determined. The activity of both antioxidant enzymes and non-enzymatic antioxidants was affected by BTR in earthworms. Moreover, the analyses of lysosomal neutral red retention time and total antioxidant capacity indicated a detoxification effect of BTR on Cu-induced impairments of the antioxidant defense capacity in earthworms. The apoptotic rate of coelomocytes in earthworms of the co-exposure treatment was lower than that in earthworms treated with Cu only, indicating that BTR alleviates Cu mediated lysosomal membrane damage and antioxidant defense system responses in earthworms.

Differential histological, cellular and organism-wide response of earthworms exposed to multi-layer graphenes with different morphologies and hydrophobicity

The growing use of graphene-based nanomaterials (GBNs) for various applications increases the probability of their environmental releases and calls for a systematic assessment of their potential impacts on soil invertebrates that serve as an important link along terrestrial food chains. Here, we investigated the response of earthworms (Eisenia fetida) to three types of multi-layer graphenes (MLGs) (G1, G2 and G3 with 12–15 layers) with variable morphology (lateral sizes: 7.4 ± 0.3, 6.4 ± 0.1 and 2.8 ± 0.1 μm; thicknesses: 5.0 ± 0.1, 4.2 ± 0.1 and 4.0 ± 0.2 nm, respectively) and hydrophobicity ((O + N)/C ratios: 0.029, 0.044 and 0.075; contact angles: 122.8, 118.8 and 115.1°, respectively). Exposure to these materials was conducted for 28 days (except for 48-h avoidance test) separately in potting or farm soil at 0.2% and 1% by weight. Earthworms avoided both soils when amended with 1% of the smaller and more hydrophilic MLGs (G2 and G3), leading to a decreased trend in worm cocoon formation. The smallest and most hydrophilic MLG (G3), which was easier to assimilate, also significantly inhibited the viability (20.2–56.0%) and mitochondrial membrane potential (32.0–48.5%) of worm coelomocytes in both soils. In contrast, oxidative damage (indicated by lipid peroxides) was more pronounced upon exposure to more hydrophobic and larger graphenic materials (G1 and G2), which were attributed to facilitated adhesion to and disruption of worm membranes. These findings highlight the importance of MLG morphology and hydrophobicity in their potential toxicity and mode of action, as well as ecological risks associated with incidental and accidental releases.

Integration of transcriptomics and metabolomics reveals the responses of earthworms to the long-term exposure of TiO2 nanoparticles in soil

Titanium dioxide nanoparticles (nTiO2) are widely used and their environmental occurrence has raised concerns about the potential toxicity to biota. However, few studies have investigated the effect of long-term exposure to nTiO2 on soil invertebrates. This study therefore for the first time investigated the long-term (120 days) effect of nTiO2 (0, 5, 50, and 500 mg/kg) on the phenotypes, transcriptomic, and metabolomic profiles of earthworm (Eisenia fetida) in soil. The results showed that the long-term exposure to nTiO2 did not significantly affect the growth, reproduction, and Ti content of earthworms. However, the antioxidant system and the transcriptomic and metabolomic profiles of earthworms were significantly affected. The superoxide dismutase (SOD) activity and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio significantly decreased under the 500 mg/kg nTiO2 treatment. The metabolomics analysis showed that glycine and pyroglutamic acid contents involved in the GSH metabolism were significantly altered under the 500 mg/kg treatment. Moreover, transcriptomics and metabolomics data revealed that the long-term exposure to nTiO2 affected the synthesis of carbohydrates, proteins, and lipids. However, the transcriptomics results indicated that the genes involved in ribosome biogenesis in eukaryotes pathway and TGF-beta signaling pathway were upregulated, which could explain why the growth and reproduction of earthworms were apparently not affected by the nTiO2 exposure. The combination of transcriptomics and metabolomics reveals the global responses that cannot be observed by conventional toxicity endpoints, facilitating the assessment of long-term ecological effect of engineered nanoparticles in the environment.

Use of integrated biomarker response for studying the resistance strategy of the earthworm Metaphire californica in Cd-contaminated field soils in Hunan Province, South China

Research was conducted to study the response and detoxification mechanisms of earthworms collected from Cd-contaminated areas in Hunan Province, South China. Metaphire californica, the dominant earthworm species in fields, referred as earthworm-A and -B that collected from low- (0.81 mg kg−1) and high-Cd soil (13.3 mg kg−1), respectively, for exchanging incubation in laboratory. The results showed that earthworm-A gradually accumulated higher Cd when exposed in the high-Cd soil, whereas Cd concentration of earthworm-B decreased after being transferred to low-Cd soil (albeit BAFCd >20). The integrated biomarker response index was calculated with the biomarkers of antioxidant systems (e.g., superoxide dismutase (SOD), peroxidase (POD), glutathione (GSH), glutathione peroxidase (GPx), glutathione-S transferase (GST), and malondialdehyde (MDA)) and energy index (e.g., protein and glycogen) in M. californica. GSH, GPx, and GST contributed the most to the integrated biomarker response (IBR) in earthworm-A when exposed in high-Cd soil for 14 d. Earthworm-B responded with higher GST and GPx activities and decreased protein content in low-Cd soil. For 28 d, the response of earthworm-A was not evident in either low- or high-Cd soil, and the inductive effect of metal stress on earthworm-B tended to be stable, except for the higher MDA content (p < 0.05) when exposed in low-Cd soil. The IBR index of earthworm-B (2.93 and 3.40) in low- and high-Cd soil, respectively, was higher than that of earthworm-A (0.89 and 1.0). Overall, earthworm-A exhibited a detoxification process to resist high-Cd toxicity from low-to high-Cd soil. Earthworm-B exhibited a physiological resilience once its habitat had changed to a normal or low-Cd soil environment, possibly owing to the cost of its resistance adaptation to the historical highly contaminated soil in fields.

Surface coating and particle size are main factors explaining the transcriptome-wide responses of the earthwormLumbricus rubellusto silver nanoparticles

We present transcriptome responses of earthworms exposed to differently sized and coated silver nanoparticles (AgNPs), which are used in important industrial and biomedical applications.

Assessment of the immobilization effectiveness of several amendments on a cadmium-contaminated soil using Eisenia fetida.

Proper protocols for assessing the remediation effectiveness of contaminated soils are an important part of remediation projects. In the present study, the residual immobilization effectiveness of hydrated lime (L), hydroxyapatite (H), biochar (B) and organic fertilizer (F) alone and in combination was assessed by Eisenia fetida. The results showed that the application of amendments had no significant effect on the death rate and average fresh weight loss of earthworms. The earthworm Cd concentration increased with prolonged exposure time, however, the significant immobilization efficacy of amendments observed on the 7th day nearly disappeared after 28 days of exposure. The immobilization efficiencies, estimated by the earthworms internal Cd concentration, of L, H and B on the 7th day were 38.6%, 37.8% and 20.7%, respectively. These values decreased to 4.9%, 19.8% and 15.1%, respectively, on the 28th day. The detoxification effect of amendments was confirmed by the Cd subcellular fractionation in earthworms with lower proportions of Cd distributed in the metal-sensitive fractions in L, H and B treatments. The level of oxidative stress response of earthworms increased with exposure duration and amendments alleviated the oxidative damage induced by Cd to the earthworms. In addition, the pH and CaCl2-Cd in soils were both increased due to earthworm life activities and gut-related ingestion. In summary, the assessment of immobilization effectiveness of heavy metal-contaminated soils using Eisenia fetida was time-dependent. The immobilization efficacy of L and H performed better than B and F on the 7th day, while H and B performed better than L and F on the 28th day. Accordingly, the short-term earthworm exposure experiment (7 days) was recommended to be an alternative approach to time-consuming plant bioassays in assessment of reduced phytoavailability in chemical immobilization remediation. But the impact of earthworms on the immobilization effect of amendments needs to be considered in practical remediation.

Individual and cellular responses of earthworms (Eisenia fetida) to endosulfan at environmentally related concentrations

The presence of endosulfan at high levels in soils poses a potential risk for terrestrial ecosystems and human health via the food chain. Therefore, the effects of endosulfan at environmentally related doses on the terrestrial biota are of great concern. The present study measured the mortality, growth inhibition and ultrastructure of the stomach and skin of earthworms exposed to endosulfan at environmentally related concentrations to identify the individual and cellular effects of endosulfan on terrestrial biota. The results demonstrated that the growth inhibition of earthworms was significantly and positively correlated with the endosulfan dose and little mortality was found. The nuclei, microvilli and cuticles in the stomachs and skin cells of earthworms exhibited marked abnormalities. Endosulfan injured the ultrastructure of the nucleus even at low doses (0.5 mg·kg-1). Endosulfan seriously affected stomach microvilli and the cuticle structure of the skin, and this damage increased with increased exposure time and dose. Notably, cuticle damage was worse than the microvilli damage. These experiments demonstrated that the morphological changes in the tissue ultrastructure of the earthworm were more sensitive than growth inhibition, and these changes may be used as an early warning indicator of endosulfan pollution. The degree of damage to microvilli and cuticle is a promising bio-indicator to evaluate pesticide risk. The results of this study provide evidence of endosulfan toxicity and the importance of risk assessment on the terrestrial ecosystem.

Defense responses in earthworms (Eisenia fetida) exposed to low-density polyethylene microplastics in soils

The potential threats of microplastics to global health are a new problem. However, little is known about the influence of microplastics on soil organisms. Here, we investigated the effects of low-density polyethylene (LDPE, < 400 μm) on earthworms (Eisenia fetida) under different concentrations (0.1, 0.25, 0.5, 1.0, 1.5 g/kg dry) with three replicates in artificial soil. Results showed that surface damage of earthworms was observed at the concentration of 1.5 g/kg LDPE after exposure 28 days. The microplastics were ingested in a dose-response manner. Smaller sizes of LDPE microplastics were found in the casts of E. fetida, and approximately 30% of the microplastics egested (size < 100 μm) were increased compared with initial microplastics in the soil. The catalase activity and malondialdehyde content increased significantly at the concentration of 1.0 g/kg LDPE after exposure 28 days, and acetylcholine esterase was significantly stimulated at concentrations of 1.5 and 1.0 g/kg LDPE on days 21 and 28, respectively. The results of this study demonstrate the potential risk of LDPE microplastics to E. fetida and may provide a reference for the impact of microplastics on terrestrial creatures.

Green autofluorescence eleocytes from earthworm as a tool for detecting environmental iron pollution

• Earthworm coelomocytes are composed of amoebocytes and autofluorescent eleocytes. • The mean fluorescence intensity (MFI) of the eleocytes were significantly decreased in FeCl 3 exposed. • The eleocytes of earthworm can be used as a fluorescence indicator for iron pollution in soil and environmental. The riboflavin content of eleocytes is involved in immune responses in earthworms and serves as a sensitive bioindicator of soil condition, such as metal ions pollution. Previous studies have shown that the transition metals Ni and Cu robustly decrease riboflavin content of eleocytes when earthworms were exposed to transition metals-polluted soil. However, there is no previous study of earthworm responses to environmental stressors associated with the transition metal Fe. Therefore, this study aimed to uncover the effect of Fe exposure on the riboflavin content of eleocytes from Eisenia fetida ( E. fetida ) exposed to 1 mM FeCl 3 for 4 days using agar gels as exposure media, and riboflavin exposure as a positive control. In this study, we found that two cell types exist in the coelomic fluid of E. fetida . One cell type is the amoebocytes (about 70%), and the other is riboflavin-storing eleocytes (about 20%). Interestingly, eleocytes can spontaneously emit green autofluorescence, and the mean fluorescence intensity (MFI) and percentages of this cell population were decreased when exposed to FeCl 3 in vitro. These findings were also confirmed by in vivo experiments. However, the riboflavin content of celomic fluid supernatants and total coelomocyte numbers were unaffected. These results indicate that Fe 3+ can potentially quench the fluorescence of riboflavin in eleocytes. Therefore, by detecting the number and MFI of eleocytes, we provide a novel link between Fe 3+ and the green autofluorescent cells of earthworms, which can be used as a novel tool for detecting the iron pollution in soil and the environment.

Growth, DNA damage and biochemical toxicity of cyantraniliprole in earthworms (Eisenia fetida)

Cyantraniliprole is a second-generation diamide insecticide that exhibited excellent biological efficacy against a variety of pests. To assess the toxic impact of cyantraniliprole on earthworms, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), as well as DNA damage were measured after exposed to five cyantraniliprole concentrations ranging from 0 to 10.00 mg/kg for 7, 14, 21 and 28 days. In most treatment groups, the ROS levels increased significantly before exposure time of 14 days and then returned to normal levels. However, the SOD and CAT activities showed different response with activities were first significantly decreased and subsequently increased. The peroxidase (POD) activity showed no significant differences between treatment and control groups at first and then significantly increased. However, the opposite pattern characterized the GST activity. Also, maybe being dose-dependent before 14 days. The MDA concentration was used as a measure of lipid peroxidation (LPO). During experiment period, the MDA concentrations significantly increased when treated by this pesticide. The olive tail moment (OTM) was used as a measure of DNA damage. At higher concentrations of cyantraniliprole and longer exposure times, the OTM gradually increased, and DNA damage in the earthworms gradually increased. The weight of the high-dose (i.e., 5 mg/kg, 10 mg/kg) earthworms showed a significant trend of decrease phenomenon. Overall, the results suggest that sub-chronic exposure to cyantraniliprole causes DNA damage and LPO, weight loss and growth inhibition, leading to antioxidant defence responses in earthworms.

Mortality, growth and metabolic responses by 1H-NMR-based metabolomics of earthworms to sodium selenite exposure in soils

The rapid development of selenium-enriched agriculture leads to the accumulation of selenium in the soil, which has an adverse impact on terrestrial ecosystems. In the present study, the mortality, growth inhibition rate and metabolism of earthworms were examined to investigate the toxicological effects of sodium selenite (Na2SeO3) on earthworms (Eisenia fetida) after exposuring for 14 days (d). We used 1H-NMR-based metabolomics to identify sensitive biomarkers and explored the metabolic responses of earthworms exposed to Na2SeO3. The mortality and growth inhibition rate of earthworms exposed to 70 and 90 mg/kg Na2SeO3 were significantly higher than the rate of control group. The LC50 (the median lethal concentration) of Na2SeO3 was 57.4 mg/kg in this artificial soil test of E. fetida exposed to Na2SeO3 for 14 d. However, there was no significant differences when earthworms were exposed to different concentrations of Na2SeO3. The selected metabolic markers were ATP, lactic acid, leucine, alanine, valine, glycine, glutamic acid, lysine, α-glucose and betaine. Na2SeO3 affected the metabolic level of earthworms, as the percentage of metabolic markers in the earthworm changes when exposed to different concentrations of Na2SeO3. The metabolic disturbances were greater with increasing concentrations of Na2SeO3. The differential metabolic markers were significantly changed when exposed to Na2SeO3 comparing to those in the control group, affecting the tricarboxylic acid cycle process and breaking the metabolic balance. This study showed that Na2SeO3 had toxic effect on the growth and development of earthworms. In addition, this study provided a biochemical insights for the development of selenium-enriched agriculture.

Comparison of metabolomic responses of earthworms to sub-lethal imidacloprid exposure in contact and soil tests.

Eisenia fetida earthworms were exposed to sub-lethal levels of imidacloprid for 48h via contact filter paper tests and soil tests. After the exposure, 1H nuclear magnetic resonance (NMR) metabolomics was used to measure earthworm sub-lethal responses by analyzing the changes in the polar metabolite profile. Maltose, glucose, malate, lactate/threonine, myo-inositol, glutamate, arginine, lysine, tyrosine, leucine, and phenylalanine relative concentrations were altered with imidacloprid exposure in soil. In addition to these metabolites (excluding leucine and phenylalanine), fumarate, ATP, inosine, betaine, scyllo-inositol, glutamine, valine, tryptophan, alanine, tyrosine, and isoleucine relative concentrations shifted with imidacloprid exposure during contact tests. Metabolite changes in E. fetida earthworms exposed to imidacloprid showed a non-linear concentration response and an upregulation in gluconeogenesis. Overall, imidacloprid exposure in soil induces a less pronounced response in metabolites glucose, maltose, fumarate, adenosine-5'-triphosphate (ATP), inosine, scyllo-inositol, lactate/threonine, and tyrosine in comparison to the response observed via contact tests. Thus, our study highlights that tests in soil can result in a different metabolic response in E. fetida and demonstrates the importance of different modes of exposure and the extent of metabolic perturbation in earthworms. Our study also emphasizes the underlying metabolic disruption of earthworms after acute sub-lethal exposure to imidacloprid. These observations should be further examined in different soil types to assess the sub-lethal toxicity of imidacloprid to soil-dwelling earthworms.

Temperature and body mass drive earthworm (Eisenia fetida) sensitivity to a popular glyphosate-based herbicide

Researchers have analyzed the effects of glyphosate and glyphosate-based formulations on earthworm health. A full literature review shows negative, positive, and non-significant impacts on a suite of outcomes. Pilot studies from our lab have produced similar inconsistencies. This study aimed to determine which factors drive earthworm sensitivity to Roundup-Ready-To-Use III®, a popular herbicide. Taking cues from literature and our pilot work, we examined the effects of soil temperature, initial earthworm body mass, and the interaction between the two on a) final earthworm mass after a month of exposure to contamination and b) survival time during a stress test. We found that both earthworm responses varied with intrinsic worm characteristics (e.g. initial body mass) and environmental characteristics (e.g. soil temperature). Specifically, only initially heavy worms grown in heated soil responded to contamination, and they responded by growing significantly heavier than their uncontaminated counterparts. Worms of lighter mass and worms grown in a cooler temperature did not respond to contamination by altering final body mass. Additionally, earthworms living in unheated soil survived significantly fewer minutes during the stress test, with herbicide-exposed worms surviving the shortest number of minutes overall. Initial earthworm body mass did not affect the outcome of this test. This study shows that by varying initial body condition and soil temperature, earthworms can respond positively, negatively or not at all to exposure to a glyphosate-based formulation. Earthworm sensitivity varies with specific environmental and somatic conditions, and this may explain variation in published literature, where neither initial earthworm body mass nor soil temperature is generally considered.

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).

Biochemical and transcriptomic response of earthworms Eisenia andrei exposed to soils irrigated with treated wastewater.

In order to ensure better use of treated wastewater (TWW), we investigated the effect of three increasing doses of TWW, 10%, 50%, and 100%, on biochemical and transcriptomic statuses of earthworms Eisenia andrei exposed during 7 and 14 days. The effect of TWW on the oxidative status of E. andrei was observed, but this effect was widely dependent on the dilution degree of TWW. Results showed a significant decrease in the catalase (CAT) activity and an increase in the glutathione-S-transferase (GST) activity, and considerable acetylcholinesterase (AChE) inhibition was recorded after 14 days of exposure. Moreover, malondialdehyde (MDA) accumulation was found to be higher in exposed animals compared to control worms. The gene expression level revealed a significant upregulation of target genes (CAT and GST) during experimentation. These data provided new information about the reuse of TWW and its potential toxicity on soil organisms.

Oxidative stress, energy metabolism and molecular responses of earthworms (Eisenia fetida) exposed to low-density polyethylene microplastics.

Soils are both a sink and a pathway of plastic wastes, but there is a great lack of knowledge regarding their impacts on soil biota. To tackle the mechanisms of toxicity of these contaminants to soil invertebrates, earthworms (Eisenia fetida Savigny, 1826) were exposed during 28days to different concentrations of low-density polyethylene microplastics (62, 125, 250, 500 and 1000mg MPs kg-1 soildw) with sizes ranging between 250 and 1000μm, in an artificial soil. The ecotoxicological responses were evaluated by analysing various oxidative stress biomarkers (catalase, glutathione S-transferase and thiobarbituric acid reactive substances), a biomarker of energy metabolism (lactate dehydrogenase) and overall organism molecular changes by Fourier transform infrared spectrometry (FTIR) and nuclear magnetic resonance (NMR) analyses. Significant effects resulting from an unbalanced oxidative stress system, expressed in terms of thiobarbituric acid reactive substances levels were recorded on earthworms exposed at the three highest concentrations tested. Despite that, no significant changes were recorded on the molecular profiles of earthworms by FTIR-ATR. NMR analysis pointed out for differences from the control, only for earthworms exposed to the lowest concentration of MPs. Considering that stress responses are complex, and involve multiple mechanisms, a cluster analysis taking into account all the parameters assessed, clearly identified two groups of earthworms separated by the concentration of 250mg MPs kg-1 soildw, above each meaningful effects were recorded.

Bioavailability of biosolids-borne ciprofloxacin and azithromycin to terrestrial organisms: Microbial toxicity and earthworm responses

Information on bioavailability of two antibiotic TOrCs, ciprofloxacin (CIP) and azithromycin (AZ), to terrestrial organisms is severely limited, especially in the biosolids context. Responses of two terrestrial organisms, earthworms and microbes, to a range of environmentally relevant concentrations of biosolids-borne CIP and AZ were assessed in laboratory incubation studies involving 3H-labeled compounds. Earthworm assessments were based on the Earthworm Sub-chronic Toxicity Test (OCSPP 850.3100). Microbial impacts were assessed using respiration and reverse transcriptase-quantitative PCR (mRNA) analyses of nutrient (N and P) cycling genes as toxicity markers. Antibiotic extractability and stability during incubations were assessed using sequential extractions with CaCl2, methanol:water, and accelerated solvent extraction and analyses using thin layer chromatography. Subsample combustion, in addition to sequential extraction, recovered nearly 100% of the added antibiotic. The two compounds persisted (estimated half-lives ≥ 3 y), but extractable fractions (especially of CIP) decreased over time. Neither biosolids-borne antibiotic significantly impacted overall respiration or N and P cycling. Microbial toxicity responses were minimal; complementary DNA (cDNA) concentrations of ammonia oxidizing bacterial genes were affected, but only initially. Similarly, earthworms showed no apparent response related to toxicity to environmentally relevant (and much greater) concentrations of biosolids-borne CIP and AZ. Earthworms, however, accumulated both compounds, and the bioaccumulation factor (BAF) values (dry weight basis) were ~4 (CIP) and ~7 (AZ) in depurated worms and ~20 (CIP and AZ) in un-depurated worms. The microbial and earthworm responses strongly to moderately correlated with “bioaccessible” fractions of the target TOrCs. The results suggest that biosolids-borne CIP and AZ toxicity to terrestrial microbes and earthworms is minimal, but there is a potential for target TOrC entry into ecological food web.

Effect of treated wastewater irrigation in East Central region of Tunisia (Monastir governorate) on the biochemical and transcriptomic response of earthworms Eisenia andrei

Treated wastewater (TWW) reuse for irrigation has become an excellent way to palliate water scarcity in Mediterranean arid regions. However, the toxicological effects of these effluents on the soil's organisms, especially earthworms, have not been well studied as yet. In this paper, earthworms Eisenia andrei were exposed for 7 days and 14 days to five agricultural soils irrigated with TWW for different periods: 1 year, 8 years, and 20 years. In addition, they were also exposed to soil from one reference site sampled from the Ouardenin perimeter in the Monastir Governorate in Tunisia. The effect on earthworms was assessed at the biochemical level by evaluating for catalase (CAT), glutathione-S-transferase (GST), malondialdehyde accumulation (MDA) and acetylcholinesterase inhibition (AChE). On the other hand, genotoxicity and transcriptomic responses were evaluated using micronuclei test (MNT) and gene expression level of CAT and GST. Moreover, metals uptake by earthworms was analyzed. Results showed that CAT and GST activity in the earthworm increased significantly when they were exposed to soils irrigated with TWW for 1, 8 and 20 years. Furthermore, MDA concentration also increased significantly with the increase in exposure period. However, AChE activity decreased and MNi frequency increased in earthworms after 7 and 14 days of exposure to soils irrigated with TWW for more than a year. The gene expression level of CAT and GST showed a significant variability, thus data are discussed in relation to the studied biomarkers (CAT and GST). These data provide new insights into the effect of toxicity of TWW on the soil's macro fauna, which is strongly affected by the trace elements and other organic compounds accumulated in soils after 20 years of TWW irrigation.

Integration of chemical and toxicological tools to assess the bioavailability of copper derived from different copper-based fungicides in soil

Because the extensive use of Cu-based fungicides, the accumulation of Cu in agricultural soil has been widely reported. However, little information is known about the bioavailability of Cu deriving from different fungicides in soil. This paper investigated both the distribution behaviors of Cu from two commonly used fungicides (Bordeaux mixture and copper oxychloride) during the aging process and the toxicological effects of Cu on earthworms. Copper nitrate was selected as a comparison during the aging process. The distribution process of exogenous Cu into different soil fractions involved an initial rapid retention (the first 8 weeks) and a following slow continuous retention. Moreover, Cu mainly moved from exchangeable and carbonate fractions to Fe-Mn oxides-combined fraction during the aging process. The Elovich model fit well with the available Cu aging process, and the transformation rate was in the order of Cu(NO3)2 > Bordeaux mixture > copper oxychloride. On the other hand, the biological responses of earthworms showed that catalase activities and malondialdehyde contents of the copper oxychloride treated earthworms were significantly higher than those of Bordeaux mixture treated earthworms. Also, body Cu loads of earthworms from different Cu compounds spiked soils were in the following order: copper oxychloride > Bordeaux mixture. Thus, the bioavailability of Cu from copper oxychloride in soil was significantly higher than that of Bordeaux mixture, and different Cu compounds should be taken into consideration when studying the bioavailability of Cu-based fungicides in the soil.