Influence of nano and bulk copper on agile frog development.

Nanotechnology, as one of the fastest-growing industries, offers many benefits in various fields. However, properties that contribute to its positive effects, in other context, can cause adverse effects to various organisms, such as amphibians. Identifying possible negative effects on its survival is crucial since amphibians are the most threatened group of vertebrates. In that context, we investigated the influence of both nano and bulk copper on embryonic development of agile frog, Rana dalmatina. The embryos were exposed to various concentrations (0.01 mg/L, 0.075 mg/L, 0.15 mg/L or 0.3 mg/L) of either nano (CuO, declared size 40-80 nm) or bulk form (CuSO4·5H2O) for 16 days. Upon the experiment, tadpoles were measured and weighted, then homogenized and their protein, lipid, and carbohydrates content determined, as well as the activity of LDH. Our results suggest stronger negative influence of nano copper to size and weight of tadpoles, and bulk copper on lipid content, while both had strong negative effect on carbohydrates content, and LDH activity. In addition, our results suggest agile frog to be more susceptible to negative influence of both, nano and bulk copper, than commonly used Xenopus laevis.

Comprehensive study of the effects of strobilurin-based fungicide formulations on Enchytraeus albidus.

Excessive application of fungicides in crop fields can cause adverse effects on soil organisms and consequently affect soil properties. Existing knowledge on the effects of strobilurin fungicides has been primarily based on toxicity tests with active ingredients, while the effects of fungicide formulations remain unclear. Therefore, this work aims to provide new data on the effects of three commercial formulations of strobilurin fungicides on the soil organism Enchytraeus albidus. The tested fungicide formulations were Retengo® (pyraclostrobin-PYR), Zato WG 50® (trifloxystrobin-TRI) and Stroby WG® (kresoxim-methyl-KM). In laboratory experiments, multiple endpoints were considered at different time points. The results showed that PYR had the greatest impact on survival and reproduction (LC50 = 7.57 mga.i.kgsoil-1, EC50 = 0.98 mga.i.kgsoil-1), followed by TRI (LC50 = 72.98 mga.i.kgsoil-1, EC50 = 16.93 mga.i.kgsoil-1) and KM (LC50 = 73.12 mga.i.kgsoil-1, EC50 ≥ 30 mga.i.kgsoil-1). After 7 days of exposure, MXR activity was inhibited at the highest concentration of all fungicides tested (6 mgPYRkgsoil-1, 15 mgTRIkgsoil-1 and 30 mgKMkgsoil-1). Furthermore, oxidative stress (induction of SOD, CAT and GST) and lipid peroxidation (increase in MDA) were also observed. In addition, there was a decrease in total available energy after exposure to PYR and KM. Exposure to fungicides resulted in a shift in the proportions of carbohydrates, lipids, and proteins affecting the amount of available energy. In addition to the initial findings on the effects of strobilurin formulations on enchytraeids, the observed results suggest that multiple and long-term exposure to strobilurin formulations in the field could have negative consequences on enchytraeid populations.

Toxicity of fungicide azoxystrobin to Enchytraeus albidus: Differences between the active ingredient and formulated product.

Due to the often-excessive usage of fungicides, increasing attention is being paid to their impact on soil and non-target organisms. Risk assessments are usually based on the pure active ingredient and not on the formulated products applied in the environment. The aim of this study was therefore to investigate how azoxystrobin, the best-selling strobilurin fungicide, affects non-target soil organisms Enchytraeus albidus. To investigate the effects of the different types of azoxystrobin, E. albidus was exposed to the pure active ingredient, AZO_AI, and the formulated product, AZO_FP. Survival, reproduction, and molecular biomarkers of E. albidus were determined for different exposure durations (seven and 21 days). AZO_FP (LC50 = 15.3 mga.i./kg) showed a slightly stronger effect on survival than AZO_AI (LC50 = 16.8 mga.i./kg), yet the impact on reproduction was much stronger. Namely, while the tested concentrations of AZO_AI (EC50≥ 8 mga.i./kg) had almost no effect on reproduction, AZO_FP (EC50 = 2.9 mga.i./kg) significantly inhibited reproduction in a dose-dependent manner. Changes in enzyme activities (superoxide dismutase, catalase, glutathione-s-transferase) and malondialdehyde levels in both treatments indicated oxidative stress. Although AZO_FP had a stronger negative effect, the impact depended on the exposure time and the tested concentration. The higher toxicity of AZO_FP was a consequence of increased bioavailability and activity of the active ingredient due to the presence of adjuvants. Overall stronger adverse effects of AZO_FP suggest that the toxicity of azoxystrobin in the agricultural environment on the enchytraeid population may be underestimated. Furthermore, the results of this study highlighted the importance of comparing the toxicity of the active ingredient and the formulated product.

Multigenerational and transgenerational effects of azoxystrobin on Folsomia candida.

Soil organisms are exposed to various pollutants during several generations. However standard toxicity tests are often based on exposure in only one generation. Research of multigenerational (MG) and transgenerational (TG) effects are still quite scarce, however evidence accumulates that effects observed in one generation can be significantly different in some of the following generations, with different effects observed. Some studies suggest adaptation to pollutants, while others report severe effects in following generations. Azoxystrobin is commonly used in the prevention and treatment of fungal diseases in a wide range of economically important crops. The main aim of this study was to assess the toxic effects of azoxystrobin (AZO) on F. candida over 3 generations through the application of biochemical and population level biomarkers. Results of reproduction tests showed a significant decrease in estimated EC50 values, with EC50 for F0 being estimated at 104.44 mga.i./kgD.W.soil and only 15.4 mga.i./kgD.W.soil for F1. In F1 a significant reduction in the number of juveniles was observed, and at AZO concentration of 50 mga.i./kgD.W and higher, F1 did not reproduce. Significant oxidative stress was observed in all generations, with increased SOD and lipid damage that slowly decreased in subsequent generations. Transgenerational effects were also observed, with a significantly reduced number of juveniles in F1 and significant oxidative stress and lipid damage in all generations. IBRv2 showed that F1 was most affected, followed by F0, and least affected was F3. When considering the whole body energy budget, F1 to F3 had significantly higher WBEB compared to F0, and a shift in proportion of energy reserves occurred in F1, where the proportion of lipids increased while protein decreased. Results of this research show that considering standard toxicity tests, risks for populations of soil organisms are possibly severely underestimated. Therefore, standard toxicity guidelines should be supplemented by multigenerational tests, when possible.

Mitigation of OMW toxicity toward Enchytraeus albidus with application of additives.

The olive oil industry generates considerable amounts of olive mill wastewater (OMW) which is treated and used in agriculture, energy production, or discharged into evaporating ponds where OMW contaminated soil (OMWS) is formed. Due to the extremely high phenol content, untreated OMWS is not suitable for plants and soil organisms. This study aimed to determine the adverse effects of OMWS on Enchytraeus albidus and whether the addition of clay and diatomaceous earth can reduce these effects. Without additives, reproduction was reduced up to 35%, with high number of unhatched cocoons, indicated hatching impairment. Furthermore, acetylcholinesterase (AChE) activity decreased significantly at the highest OMWS ratio (40%), as did glutathione-S-transferase (GST) activity at two ratios (40%), indicating neurotoxic effects and oxidative stress. The application of additives (clay and diatomaceous earth) decreased phenol concentration up to 35 and 45%, respectively. Consequently, the number of juveniles increased significantly compared to the control and AChE and GST activities were not affected. However, an increased number of unhatched cocoons and lipid peroxidation were observed after diatomaceous earth treatment, indicating the occurrence of stress. Although additives can bind phenols, neutralize harmful effects and allow the use of OMWS as a fertilizer in agriculture, they should be carefully selected taking into account soil organisms as well. The use of additives to reduce the adverse effects of OMWS can be a turning point in the remediation of olive contaminated soil.

Effects of olive mill waste (OMW) contaminated soil on biochemical biomarkers and reproduction of Dendrobaena veneta.

Olive oil industry is economically important in Mediterranean countries. Disposal of olive mill waste (OMW) presents an environmental concern in those countries due to its high salinity and its high level of polyphenols. In order to reuse OMW, those properties have to change either through the filtration process and addition of adsorbents or by composting. One of the most important organisms in composting of organic wastes is earthworms. However, data on the effects of OMW on earthworms are scarce. The main aim of our study was to investigate whether OMW contaminated soil (OMW CS) causes adverse effects on molecular and organism level in epigeic earthworm Dendrobaena veneta and on microbiological activity. Changes of measured biochemical biomarkers (AChE, CAT, GST, lipids, MDA) varied depending on the quantity of added OMW CS and the exposure duration. Oxidative stress occurred after 7 days of exposure, while in most cases enzyme activity recovered after 28 days. At the highest ratio of contaminated soil (50%), reproduction was completely inhibited. The second aim was to investigate the impact of earthworms on phenol degradation and microbial activity, indicating an important role in the bioremediation of contaminated soils. Our results show that above a certain quantity an OMW CS has an adverse effect on earthworms, while the impact of earthworms on soil microbial activity was positive but transient. Yet, as the results also imply that earthworms have an impact on phenol degradation, they can be used to help remediation of OMW CS and its subsequent usage in agriculture. However, the quantity of OMW CS that can be safely added should be determined first.

Assessment of adverse effects of olive mill waste water and olive mill waste contaminated soil on springtail Folsomia candida.

In recent years significant attention has been given to the problem of olive mill waste towards the environment. Still, there is a considerable gap in the knowledge of the impact of the olive mill wastewater (OMWW) and the olive mill waste contaminated soil (OMW CS) on non-target soil organisms. Springtails, as an important group of non-target soil organisms, are frequently used in ecotoxicological research. However, information on olive mill waste impact on the model species Folsomia candida is scarce. Therefore, in this study, we determined the effects of OMWW and OMW CS on survival, reproduction, neurotoxicity, oxidative stress, and available energy in springtail F. candida. The exposure to different ratios of OMWW and OMW CS showed higher toxicity of OMW CS in terms of survival (LC50 = 32.34% of OMWW; LC50 = 45.36% of OMW CS) and reproduction (EC50 = 10.10% of OMWW; EC50 = 19.44% of OMW CS). Furthermore, neurotoxicity (AChE induction), oxidative stress (SOD, GST, and MDA induction), and changes in available energy (decrease in lipid and carbohydrate content) have been observed. Those negative effects are likely consequences of the high phenol content specific to OMWW and OMW CS. Obtained results indicate that for the ecotoxicological assessment of various wastes it is essential to consider different tier level biomarkers to have a clear insight into the mechanism of action.

Effects of single and combined exposure to nano and bulk zinc-oxide and propiconazole on Enchytraeus albidus.

Organisms in soil are often exposed to different mixtures of contaminants. These contaminants may interact with each other and, consequently, may have a different effect on organisms than each of them alone. We wanted to investigate possible effects of ZnO mixtures in bulk and nano form and fungicide propiconazole (PCZ) on biochemical parameters and reproduction in Enchytreus albidus. These compounds were applied separately and in binary mixture. In the single exposure experiment the EC50 values for the number of juveniles were calculated: nano ZnO (641.21 ±â€¯88.82 mg kg-1), bulk ZnO (445.78 ±â€¯148.4 mg kg-1) and PCZ (3.63 ±â€¯1.68 mg kg-1), respectively. These concentrations were subsequently used in the binary exposure experiment. Calculated combination indices (CI), that allow quantitative determination of chemical interactions at different concentration and effect level, indicated additive or antagonistic interactions (CI ≥ 1) of applied mixture ratios. The only ratio that showed synergistic interaction (CI < 1) was 75% EC50 nZnO/25% EC50 PCZ. Both ZnO forms caused acetylcholinesterase (AChE) activity increase of up to 40% of control level, as well as increased catalase (CAT) and glutathione S-transferase (GST) activities and malondialdehyde (MDA) level. PCZ did not affect AChE and CAT activities, yet it increased GST activity and MDA level. Induced levels of measured biomarkers indicate an oxidative stress after binary exposure, as well. These effects were not enhanced after binary exposure but reflected the effects on biomarkers that corresponding concentrations of these compounds generated in a single exposure experiment.

Acute and subchronic effects of three herbicides on biomarkers and reproduction in earthworm Dendrobaena veneta.

Earthworms are exposed to herbicides both through their skin and digestive system. Herbicides can influence earthworms' survival, physiology and reproduction. However, there is a lack of data on herbicide effects on earthworms as they are often regarded as low or non-toxic. The aim of our study was to investigate whether widely used commercial formulations of glyphosate (GLF), tembotrione (TBT) and nicosulfuron (NCS) each applied at three environmentally relevant concentrations have adverse effects on various biomarkers and reproduction in epigeic earthworm Dendrobaena veneta. The activities of measured biomarkers varied depending on the herbicide used and the exposure duration and suggest that oxidative stress plays an important role in the toxicity of tested herbicides. Namely, GLF caused an acetylcholinesterase (AChE) activity induction after seven days, and NCS after 28 days, while TBT caused an inhibition up to 47% (6.6 µg kgdw soil-1) after seven days. Only TBT caused a significant change (H2 = 13.96, p = 0.002) to catalase (CAT) after seven days of exposure. Malondialdehyde concentrations (MDA) were increased all the time after NCS exposure, but only after seven days in GLF and 28 days in TBT treatments, respectively. The tested herbicides did not have a significant effect on reproduction success, expect of NCS which increased the number of juveniles (p < 0.05).