Innovative statistical approaches: the use of neural networks reduces the sample size in the splenectomy-MCAO mouse model.

AIM: To compare the effectiveness of artificial neural network (ANN) and traditional statistical analysis on identical data sets within the splenectomy-middle carotid artery occlusion (MCAO) mouse model. METHODS: Mice were divided into the splenectomized (SPLX) and sham-operated (SPLX-sham) group. A splenectomy was conducted 14 days before middle carotid artery occlusion (MCAO). Magnetic resonance imaging (MRI), bioluminescent imaging, neurological scoring (NS), and histological analysis, were conducted at two, four, seven, and 28 days after MCAO. Frequentist statistical analyses and ANN analysis employing a multi-layer perceptron architecture were performed to assess the probability of discriminating between SPLX and SPLX-sham mice. RESULTS: Repeated measures ANOVA showed no significant differences in body weight (F (5, 45)=0.696, P=0.629), NS (F (2.024, 18.218)=1.032, P=0.377) and brain infarct size on MRI between the SPLX and SPLX-sham groups post-MCAO (F (2, 24)=0.267, P=0.768). ANN analysis was employed to predict SPLX and SPL-sham classes. The highest accuracy in predicting SPLX class was observed when the model was trained on a data set containing all variables (0.7736±0.0234). For SPL-sham class, the highest accuracy was achieved when it was trained on a data set excluding the variable combination MR contralateral/animal mass/NS (0.9284±0.0366). CONCLUSION: This study validated the neuroprotective impact of splenectomy in an MCAO model using ANN for data analysis with a reduced animal sample size, demonstrating the potential for leveraging advanced statistical methods to minimize sample sizes in experimental biomedical research.

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.

Avoidance Behaviour of Six Collembolan Species Shows Species-Specific Sensitivity-Impact of Ag NM300K.

Although standard testing guidelines use a species as a representative surrogate, species-specific sensitivity is well-known. The aim of this study was to investigate the species-specific difference in avoidance behaviour among Collembola species exposed to silver (Ag) nanomaterials (NM) (Ag NM300K). The avoidance test was performed with Folsomia candida, an international standard species in laboratory tests, and five widely distributed species with different life history traits, commonly used in small multispecies systems (Folsomia fimetaria, Proisotoma minuta, Mesaphorura macrochaeta, Protaphorura fimata and Ceratophysella denticulata). There was higher avoidance in euedaphic species, such as F. candida and F. fimetaria, compared to the epiedaphic species C. denticulata, which showed the least avoidance behaviour. An explanation may be that euedaphic species (living in deeper soil layers) are more directly exposed within the soil pores and have developed a pronounced avoidance behaviour. In contrast, species living on the surface are likely less directly exposed and hence only avoid at higher total concentrations. Additionally, difference in cuticula between the groups, providing different degrees of protection against exposure, can explain the different behaviours. The present results highlight the importance of biodiversity for the ecosystem and raise awareness on species sensitivity.

Investigating the Ability of Growth Models to Predict In Situ Vibrio spp. Abundances.

Vibrio spp. have an important role in biogeochemical cycles; some species are disease agents for aquatic animals and/or humans. Predicting population dynamics of Vibrio spp. in natural environments is crucial to predicting how the future conditions will affect the dynamics of these bacteria. The majority of existing Vibrio spp. population growth models were developed in controlled environments, and their applicability to natural environments is unknown. We collected all available functional models from the literature, and distilled them into 28 variants using unified nomenclature. Next, we assessed their ability to predict Vibrio spp. abundance using two new and five already published longitudinal datasets on Vibrio abundance in four different habitat types. Results demonstrate that, while the models were able to predict Vibrio spp. abundance to an extent, the predictions were not reliable. Models often underperformed, especially in environments under significant anthropogenic influence such as aquaculture and urban coastal habitats. We discuss implications and limitations of our analysis, and suggest research priorities; in particular, we advocate for measuring and modeling organic matter.

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.

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.

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.

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.

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.

Effects of strobilurin fungicides (azoxystrobin, pyraclostrobin, and trifloxystrobin) on survival, reproduction and hatching success of Enchytraeus crypticus.

Large quantities of strobilurin fungicides (SFs) are used worldwide, resulting in adverse effects on non-target organisms. SFs affect the reproduction and embryonic development of aquatic organisms, while the impact on soil organisms has been insufficiently researched. Therefore, we investigated the effects of three SFs (azoxystrobin (AZO), pyraclostrobin (PYR), and trifloxystrobin (TRI)) on the survival, reproduction, and hatching success of the non-target soil oligochaete Enchytraeus crypticus. The standard enchytraeid reproduction test (ERT) showed that, regarding survival, TRI (LC50 = 2.34 mg/kg) was the most toxic, followed by PYR (LC50 = 4.26 mg/kg) and AZO (LC50 ≥150 mg/kg). Reproduction was affected in the same order (TRI EC50 = 0.045 mg/kg, PYR EC50 = 1.85 mg/kg, and AZO EC50 = 93.10 mg/kg). Exposure to AZO and PYR showed a negative impact on hatching success with a significant increase in the number of unhatched cocoons. Prolonged hatching test was consequently carried out. As a result, a hatching delay was observed at lower AZO and PYR concentrations, while at higher concentrations hatching was completely stopped as the cocoons were no longer viable. Hence, hatching test enabled a discrimination between hatching delay and hatching impairment. Besides demonstrating the adverse effects of AZO, PYR, and TRI on the survival, reproduction, and hatching success of E. crypticus, the obtained results indicate the convenience of using several endpoints in reproduction tests. The usage of prolonged hatching tests and monitoring of hatching dynamics could fill the gap between standard reproduction tests and multigeneration tests and allow a better understanding of the adverse effects on reproduction.

Measurement of multixenobiotic resistance activity in enchytraeids as a tool in soil ecotoxicology.

The multixenobiotic resistance (MXR) mechanism is the first defense line against xenobiotics. Enchytraeids, a model organism in soil ecotoxicology, are often exposed to various xenobiotics, some of which may influence MXR activity. Since MXR activity has not been studied in these organisms, the aim of this paper was to establish a methodology for the implementation of the dye assay in enchytraeids. Enchytraeus albidus and Enchytraeus crypticus were exposed to model chemosensitizers: cyclosporine A (CA), dexamethasone (DEX), ivermectin (IVM), rifampicin (RIF), verapamil (VER), and fungicide propiconazole (PCZ). Thereafter, a dye assay with specific fluorescent dyes rhodamine B and rhodamine 123 was performed. Changes in MXR activity caused by variations in dye accumulation were measured fluorometrically. CA, IVM, and VER were found to inhibit the MXR system and increase the fluorescence 2.2-fold, while DEX and RIF induced the MXR system and decreased the fluorescence. CA was the strongest inhibitor in both E. albidus (IC50 5.48 ± 1.25 µM) and E. crypticus (IC50 5.20 ± 3.10 µM). In the validation experiment, PCZ was found to inhibit the MXR system. The IC50 varied between species and exposure substrates: water (E. albidus - IC50 0.74 ± 0.24 mg/L; E. crypticus - 1.31 ± 0.24 mg/L) or soil (E. albidus - 1.79 ± 0.42 mg/kg; E. crypticus - 1.79 ± 0.17 mg/kg). In conclusion, the tested compounds changed the MXR activity, which confirms the applicability of this method as a valuable complementary biomarker in soil ecotoxicology.

ErIK-a software-based identification key for earthworm species of Croatia.

The need for reliable taxonomic species identification is always present. Correct identification of specimens is based on taxonomic research, scattered in the scientific literature and accessible only for a restricted group of specialists or research institutions with extensive library facilities (Zuquim et al. 2017). Therefore, identification keys that summarize the knowledge of a group of biota are very important; they provide the link between producers and users of taxonomy. Recently, printed identification keys are increasingly being replaced or supplemented by computer-aided keys.