Vermistabilization of excess sludge employing Eisenia fetida: Earthworm histopathological alterations and phytotoxicity evaluation.

The aim of this work was to stabilize excess sludge (ES) coming from a wastewater treatment plant (WWTP) by vermistabilization and to evaluate ecotoxicological effects over the earthworm species Eisenia fetida. Three mixtures were made up in triplicate using different volume ratios of ES and soil (S) (100% ES, 70:30% ES:S and 30:70% ES:S in wet weight basis). Earthworms were added in order to compare vermicomposting vs. natural stabilization. The mixtures were monitored over 130 days through physical, chemical, pathological and biological analysis, following quality standards depicted in the US EPA 40 CFR Part 503, local regulations and background studies. Histopathological samples were processed as biomarkers of acute and chronic toxicity on earthworms, and germination assays were performed at the end of the experiment to assess phytotoxicity. In terms of pathogen depletion comparing initial and final values from each treatment, the mixtures with higher ES proportions (70 and 100%) with earthworms were the most efficient ones registering 64.8 and 75.5% of reduction of fecal coliforms (FC) respectively, while the lowest ES proportion with earthworms (30%) showed 54.7%. Final pathogens content in all the treatments with earthworms were significantly lower (ranged from 1360 to 1760 MPN g total solids-1) than the values registered in treatments without earthworms (ranged from 2400 to 4000 MPN g total solids-1) (p < 0.05). However, none of the treatments attained class A categorization (FC ≤ 1000 MPN g total solids-1) in terms of FC. Also, values of mean cocoon production and hatched juveniles along time were significantly higher in the treatments with 100 and 70% ES (p < 0.05), while the higher mean adult biomass was detected in the treatment with 30% ES. Volatile solids decrease ranged between 8.45 and 22.34% in treatments with earthworms and all values of specific oxygen uptake rate were below 1.5 mg O2 h -1 g total solids -1. There were not negative effects over behavior or reproduction of E. fetida adults, nor the presence of external and internal injuries. Final products from mixtures with earthworms presented a humus-like structure, were odorless and reached maturity values -presenting no phytotoxicity-with significant differences between germination index values of treatments with and without earthworms (p < 0.05). Vermistabilization is a successful eco-technology to sanitize excess sludge, acquiring an added-value material and contributing to its revalorization as organic amendments or fertilizers in soils within the circular economy framework and the United Nations' Sustainability Development Goals.

Ecotoxicological studies of direct and indirect genotoxicity with Artemia: a integrative review.

Artemia is a brine shrimp genus adapted to extreme habitats like ranges salinity from 5-25 g/L and in temperatures from 9 to 35 °C. It is widely distributed and used as an environmental quality biomarker. Artemia franciscana and Artemia salina species are commonly used in ecotoxicological studies and genotoxicity assays due to their short life cycle, high fecundity rate, easy culture, and availability. Thus, considering the importance of these tests in ecotoxicological studies, the present study aimed to present Artemia genus as a biological model in genotoxicity research. To this end, we reviewed the literature, analyzing data published until July 2023 in the Web of Science, SCOPUS, Embase, and PubMed databases. After screening, we selected 34 studies in which the genotoxicity of Artemia for various substances. This review presents the variability of the experimental planning of assays and biomarkers in genotoxicity using Artemia genus as a biological model for ecotoxicological studies and show the possibility of monitoring biochemical alterations and genetic damage effects. Also highlight innovative technologies such as transcriptomic and metabolomic analysis, as well as studies over successive generations to identify changes in DNA and consequently in gene expression.

Stabilization of residual mercury from gold mining as metacinnabar and cinnabar in ball mills on a pilot scale.

Waste liquid mercury generated as a by-product of the Merrill-Crowe process in gold mining and recovered from mercury-containing waste must be stabilized for secure storage or disposal. This study developed a procedure for mercury stabilization. A ball mill with a 0.5 m3 capacity and a rotational speed of 43 rpm was used to stabilize the residual mercury with sulfur. The treatments were conducted for 30, 60, and 90 min at mercury: sulfur molar ratios of 1.0, 0.8, and 0.67. The ball loading ratio was 7.0 with residual mercury, and the temperature was below 40 °C. The treatment efficiency was evaluated by measuring the concentrations of mercury and other metals using the Toxicity Characteristic Leaching Procedure (TCLP), examining the stabilized residual mercury by X-ray diffraction, and conducting bioassays on Daphnia magna and Lactuca sativa. Principal component analysis (PCA) was performed on the aforementioned variables. The 90-min treatment, with a mercury-to-sulfur molar ratio of 0.67, stabilized mercury mainly as cinnabar compared with the other treatments and presented leachate mercury values below the detection limit <0.003. The leachate from the treatments also showed values of 21.28-38.44 toxic units, classified as very toxic, and generated toxicity, particularly for D. magna, because of the presence of other metals such as Al, Ba, B, Ca, Cu, Cr, Fe, Mn, Ni, and Zn. The variability of the residues in the PCA analysis was explained by the treatment effect and the presence of other metals in the residual mercury. The stabilized residual mercury obtained was classified as non-hazardous and could be stored or disposed of as ordinary waste in a security landfill.

Is pearl millet (Pennisetum glaucum) a good plant species for ecotoxicological tests?

Various plant species can be selected for environmental testing, including pearl millet (Pennisetum glaucum (L.) R. Br), a globally significant cereal crop. This study aims to assess millet's suitability as a species for ecotoxicological tests, examining (1) germination and initial development dynamics, (2) the minimum seed quantity for reliable sampling, (3) optimal experimental design with replication numbers, (4) suitability of positive control, and (5) the effectiveness of the protocol in evaluating toxic effects of environmental pollutants. Millet exhibited rapid and uniform germination as well as consistent initial seedling development. To establish the minimum number of seeds required for reliable experimentation, germination, and seedling growth were compared across plots containing 10, 25, and 50 seeds. Consequently, 10 seeds per plot were chosen for subsequent experiments to reduce labor and costs while maintaining reliability. To validate the selected experimental design, and to establish a positive control for assays, aluminum was used as a toxic element at concentrations ranging from 10-2 to 10-6 M. While aluminum did not affect the final percentage of germinated seeds, it did exhibit an impact on the Germination Speed Index (GSI). Significant differences in root and aerial growth, and with fresh weight, were observed. The 10-3M concentration was chosen as the positive control as the 10-2 concentration showed extreme toxicity. To assess the applicability of the established protocol in determining the toxic effects of environmental pollutants, millet roots were exposed to the toxic agents atrazine, cadmium, methyl methane sulfonate (MMS), and Spent pot liner (SPL). Millet demonstrated sensitivity and efficiency in response to these tests. In conclusion, millet proves to be an effective species for the toxicological risk assessment of environmental pollutants.

Ecotoxicological effects of the emerging contaminant ivermectin on Rhinella arenarum: A comparative study of active ingredient and commercial formulation.

Ivermectin (IVM) is a broad-spectrum veterinary antiparasitic used worldwide in cattle breeding. The aim of this study was to evaluate the lethal effects of the active ingredient and a commercial formulation of IVM (1 % active ingredient) in the embryonic stage (S. 4-6) and larval stage (S. 25) of the South American amphibian Rhinella arenarum through chronic standardized bioassays. Also, behavior analysis and oxidative stress and cholinergic effects biomarkers were analyzed at 1, 10 and 100 µg IVM/L concentrations. For the embryonic stage, the active ingredient (96 h- LC50: 15900 µg/L) was more toxic than the commercial formulation (96 h-LC50: 51230 µg/L) during the acute period, while at chronic exposure the commercial formulation was more toxic (504 h-LC50: 10.25 µg/L), compared to the active ingredient (504 h-LC50: 312.80 µg/L). For the larval stage, in acute exposure, the active ingredient (96 h-LC50: 800 µg/L) was more toxic than the commercial formulation (96 h-LC50: 1550 µg/L). In the chronic exposure, the commercial formulation (504 h-LC50: 77.33 µg/L) was more toxic than the active ingredient (504 h-LC50: 195.25 µg/L). Overall, larvae exhibited greater sensitivity to both the active ingredient and the commercial formulation. However, during chronic exposure, embryos were more sensitive to the commercial formulation than larvae. The commercial formulation primarily induced oxidative stress, and both forms of the compound affected behavior and cholinergic effect biomarkers, even at low environmentally relevant concentrations (1 µg/L). These results highlight the potential impact of IVM on aquatic ecosystems.

A multi-approach analysis of the toxicity of a commercial formulation of monensin on Rhinella arenarum embryos and larvae.

Monensin, an antibacterial commonly used in animal fattening, can enter aquatic ecosystems and harm non-target organisms. Since there are no previous studies about the effects of monensin on amphibians, the aim of the present study was to evaluate the lethal and sublethal toxicity of a commercial formulation of monensin (CFM) through standardized bioassays with embryos and larvae of the amphibian Rhinella arenarum. Oxidative stress (catalase and glutathione S-transferase activities, and reduced glutathione and lipid peroxidation levels), cholinesterasic effect (acetylcholinesterase and butyrylcholinesterase activities) and mutagenicity (micronuclei frequency) biomarkers were evaluated. The CFM produced teratogenic effects, with a teratogenic index of 6.21. Embryos (504 h-LC50: 273.33 µg/L) were more sensitive than larvae, as no significant mortality was observed on larvae exposed up to 3000 µg/L for 504 h. However, oxidative stress, cholinesterasic effect and mutagenicity biomarkers were altered on larvae exposed for 96 h to environmentally relevant concentrations (4, 12 and 20 µg/L of monensin active ingredient). The CFM caused adverse effects on the exposed organisms, primarily on embryos, leading to lethal and sublethal effects, which could impact the wildlife when it reaches aquatic ecosystems.

Unveil the sugar diet and associated environmental compounds in the crop of the mosquito Culex pipiens.

Culex pipiens (Linnaeus, 1758) mosquitoes search plant sources of sugars to cope with the energetic demand of various physiological processes. The crop as part of the digestive system is devoted to the storage of sugar-based meal obtained from various nectars sources. The profiling of sugars and metabolites in the Culex pipiens' crop is scarce, and only few studies used Liquid Chromatography - Mass Spectrometry (LC-MS), which provides broad detection for biomonitoring environmental substances and even contaminants in the sugar diet of mosquitoes populations. Therefore, sugar and metabolite profiling were performed on crops obtained from mosquitoes exposed to plant nectar under laboratory or natural conditions by Ultra High-Performance LC-MS (UHPLC-MS). This method allowed us a precise quantitative and qualitative identification of sugar diet and associated environmental compounds in the crop of the mosquito C. pipiens. Under laboratory condition, mosquitoes were allowed to feed on either glucose solution, commercially-available flowers or field collected flowers. In addition, we collected mosquitoes from the field to compare those crop metabolomes with metabolome patterns occurring after nectar feeding in the lab. The sugar quantities and quality obtained from the crops of mosquitoes collected in the field were similar to those crops obtained from mosquitoes that fed on commercially-available flowers and from field collected flowers with a limit of detection of 10 µg/L for sucrose, glucose and sucrose. Next to sugar compounds, we identified 2 types of amino acids, 12 natural products, and 9 pesticides. Next to the diversity of sugar compounds, we could confirm that secondary metabolites and environmental pollutants are typically up taken from floral nectar sources by C. pipiens. The in-depth knowledge on mosquito-plant interactions may inspire the development and further optimization of mosquito trap systems and arboviral surveillance systems.

High toxicity of agro-industrial wastewater on aquatic fauna of a South American stream: Mortality of aquatic turtles and amphibian tadpoles as bioindicators of environmental health.

The aim of this study was to characterize an aquatic system of Santa Fe province (Argentina) receiving wastewater from agro-industrial activities (mainly dairy) by in situ assessment (fauna mortality, physicochemical, microbiological, and pesticide residues measurement), and ecotoxicity bioassays on amphibian tadpoles. Water and sediment samples were obtained from the Los Troncos Stream (LTS), previous to the confluence with the "San Carlos" drainage channel (SCC), and from the SCC. Biological parameters (mortality and sublethal biomarkers) were used to evaluate ecotoxicity during 10-day exposure of Rhinella arenarum tadpoles to LTS and SCC samples. Nine pesticides were detected in both LTS and SCC. Chemical and biochemical oxygen demand, ammonia, and coliform count recorded in SCC greatly exceeded limits for aquatic life protection. At SCC and LTS after the confluence with SCC, numerous dying and dead aquatic turtles (Phrynops hilarii) were recorded. In the ecotoxicity assessment, no mortality of tadpoles was observed in LTS treatment, whereas total mortality (100%) was observed in SCC treatments in dilution higher than 50% of water and sediment. For SCC, median lethal concentration and the 95% confidence limits was 18.30% (14.71-22.77) at 24 h; lowest-observed and no-observed effect concentrations were 12.5% and 6.25%, respectively. Oxidative stress and neurotoxicity were observed in tadpoles exposed to 25% SCC dilution treatment. In addition, there was a large genotoxic effect (micronuclei test) in all sublethal SCC dilution treatments (6.25%, 12.5%, and 25%). These results alert about the high environmental quality deterioration and high ecotoxicity for aquatic fauna of aquatic ecosystems affected by agro-industrial wastewater. PRACTITIONER POINTS: Great mortality of turtles was observed in a basin with a high load of agro-industrial wastewater. San Carlos Channel (SCC), where effluents are spilled, is environmentally deteriorated. The water-sediment matrix of SCC caused 100% lethality in tadpoles. SCC dilutions caused neurotoxicity, oxidative stress, and genotoxicity on tadpoles.

Caenorhabditis elegans as a suitable model to evaluate the toxicity of water from Rolante River, southern Brazil.

Urbanization and agricultural activities increased environmental contaminants. Integrated analysis of water parameters and bioassays represents an essential approach to evaluating aquatic resource quality. This study aimed to assess water quality by microbiological and physicochemical parameters as well as the toxicological effects of water samples on the Ames test and Caenorhabditis elegans model. Samples were collected during (collection 1) and after (collection 2) pesticide application in the upper (S1), middle (S2), and lower (S3) sections of the Rolante River, southern Brazil. Metals were determined by GFAAS and pesticides by UPLC-MS/MS. Bioassays using the Ames test and the nematode C. elegans were performed. Levels of microbiological parameters, as well as Mn and Cu were higher than the maximum allowed limits established by legislation in collection 2 compared to collection 1. The presence of pesticide was observed in both collections; higher levels were found in collection 1. No mutagenic effect was detected. Significant inhibition of body length of C. elegans was found in collection 1 at S2 (P < 0.001) and S3 (P < 0.001) and in collection 2 at S2 (P = 0.004). Comparing the same sampling site between collections, a significant difference was found between the site of collection (F(3,6)=8.75, P = 0.01) and the time of collection (F(1,2)=28.61, P = 0.03), for the S2 and S3 samples. C. elegans model was useful for assessing surface water quality/toxicity. Results suggest that an integrated analysis for the surface water status could be beneficial for future approaches.

A comprehensive overview on solid waste leachate effects on terrestrial organisms.

Leachate is a highly complex waste with high toxicological potential that poses a significant threat to the terrestrial environment. Determining leachate physicochemical parameters and identifying xenobiotics alone is, however, not enough to determine the real environmental impacts. In this context, the use of terrestrial model organisms has been highlighted as a tool in ecotoxicological leachate assessments and as a guiding principle in risk assessments. In this context, this review aimed to present the most current state of knowledge concerning leachate toxicity and the bioassays employed in this evaluation concerning terrestrial plants and animals. To this end, a literature search on leachate effects on terrestrial organisms was carried out using ten search terms, in 32 different combinations, at the Web of Science and Scopus databases. A total of 74 eligible articles were selected. The retrieved studies analyzed 42 different plant and animal species and employed nine endpoints, namely phytotoxicity, genotoxicity, bioaccumulation, antioxidant system, cytotoxicity, reproduction, physiological changes, behavior and lethality. A frequent association of toxic leachate effects with metals was observed, mainly Pb, Cd, Cr, Mg, Zn and Cr, which can cause antioxidant system alterations and cyto- and genotoxicity. These elements have also been associated to reproductive effects in earthworms and mice. Specifically concerning plants, most of the retrieved studies employed Allium cepa in toxicity assays, reporting phytotoxic effects frequently associated to metals and soil parameter changes. Animal studies, on the other hand, mostly employed mice and evaluated genotoxicity and antioxidant system effects. Even with the description of toxic leachate effects in both plants and animals, a lack of knowledge is still noted concerning reproductive, physiological, cytotoxic, and behavioral effects in terrestrial species. We, thus, suggest that further studies be carried out on other animals, advancing our understanding on potential environmental leachate effects, also allowing for human health risk assessments.

Ecotoxicology Evaluation of a Fenton-Type Process Catalyzed with Lamellar Structures Impregnated with Fe or Cu for the Removal of Amoxicillin and Glyphosate.

Antibiotics and pesticides, as well as various emerging contaminants that are present in surface waters, raise significant environmental concerns. Advanced oxidation processes, which are employed to eliminate these substances, have demonstrated remarkable effectiveness. However, during the degradation process, by-products that are not completely mineralized are generated, posing a substantial risk to aquatic ecosystem organisms; therefore, it is crucial to assess effluent ecotoxicity following treatment. This study aimed to assess the toxicity of effluents produced during the removal of amoxicillin and glyphosate with a Fenton-type process using a laminar structure catalyzed with iron (Fe) and copper (Cu). The evaluation included the use of Daphnia magna, Selenastrum capricornutum, and Lactuca sativa, and mutagenicity testing was performed using strains TA98 and TA100 of Salmonella typhimurium. Both treated and untreated effluents exhibited inhibitory effects on root growth in L. sativa, even at low concentrations ranging from 1% to 10% v/v. Similarly, negative impacts on the growth of algal cells of S. capricornutum were observed at concentrations as low as 0.025% v/v, particularly in cases involving amoxicillin-copper (Cu) and glyphosate with copper (Cu) and iron (Fe). Notably, in the case of D. magna, mortality was noticeable even at concentrations of 10% v/v. Additionally, the treatment of amoxicillin with double-layer hydroxides of Fe and Cu resulted in mutagenicity (IM ≥ 2.0), highlighting the necessity to treat the effluent further from the advanced oxidation process to reduce ecological risks.

Assessing the transformation products and fate of Oxytetracycline by simulated aerobic degradation tests.

Oxytetracycline (OTC) is a widely used broad-spectrum antibiotic, whose presence in water and sediments was reported in various regions of the world. The effects of OTC and other tetracyclines on the environment have been intensively studied although many of their transformation products (TPs) formed in the environment and their impact have not been yet fully characterized. Abiotic and biotic degradation tests under aerobic conditions at two pH values were carried out using OTC in artificial water/sediment systems to assess the effect of these variables on the environmental fate of the pollutant. HPLC-MSn was employed to detect and identify the main degradation products and pathways. Several transformations involved in the process were identified including alcohol oxidation, decarbonylation and hydroxylation. Differences in TPs and kinetics were found among degradation conditions, remarking a faster degradation of both OTC and TPs in the presence of microorganisms and at lower pH values. In summary, a total of 44 TPs were detected and structures were proposed for 20 of them, none of them having been previously reported. Furthermore, OTC degradation generated 24 TPs which remained in either solution or sediment, although none of them displayed higher algae toxicity than OTC. These results might be useful for planning future remediation and monitoring strategies.

Acute and subchronic effects of ibuprofen on the ten spotted live-bearer fish Cnesterodon decemmaculatus (Jenyns, 1842).

Ibuprofen (IBP) is an anti-inflammatory drug found in aquatic environments, potentially toxic for the biota. We exposed the test fish C. decemmaculatus to two environmentally relevant concentrations (50 and 100 µg IBP/L) for 4 and 12 d and evaluated the effect on some biomarkers. Micronucleus test, nuclear abnormality test and comet assay indicated cyto-genotoxicity at both concentrations and exposure periods. Oxidative stress and biochemical biomarkers were not affected, excepting muscle AChE activity for 4 d. Muscle metabolic biomarkers showed significant decrease in ETS, lipid and protein content, while carbohydrate content was not affected. The CEA index increased at the lower IBP concentration for 4 and 12 d, possibly due to changes in body energy reserves. A full-factorial GLM performed to assess the effects of IBP and exposure times showed that the metabolic and genotoxicity biomarkers were the most sensitive to IBP toxicity, mainly at 50 µg IBP/L for 4 d.

Preliminary assessment of toxicity of aerosol samples from central-west Brazil using Artemia spp. bioassays.

The present study reports the development of a bioassay using Artemia spp. to analyse the preliminary ecotoxicity of atmospheric aerosols (PM), which can affect the environment and human health. Herein, PM samples were collected in the city of Goiânia (Brazil) in 2016, extracted with ultrapure water and subsequently filtered through membranes with different pore sizes (100, 0.8, and 0.22 µm), and the extracts employed in the bioassays. The mortality rates (endpoint analysed) declined to membranes with smaller pore sizes (15 ± 4%, 47 ± 10% and 43 ± 9% for pore sizes of 100 µm, 0.8 µm and 0.22 µm, respectively). In general, the toxicity of the extract depended on its concentration, except for the sample with a higher negative particle surface charge, which presents a lower affinity for the negatively charged surfaces of cellular membranes. Moreover, although the PM concentration was higher for the sample collected during the dry season (September), the mortality rate was not significantly different to that determined for a sample with similar physical and chemical characteristics collected in the rainy season (December). This result demonstrates the importance of monitoring PM toxicities and their chemical and physical characteristics, in addition to their concentrations. Therefore, the new protocol to provide a preliminary analysis of the toxicity of the extracts of aerosol emerges as a useful, accessible, and fast tool for monitoring possible environmental hazards, and can simplify fieldwork.

Water and sediment quality of an agro-industrial area through different approaches: Physicochemical parameters and ecotoxicity with amphibians.

Amphibians are subject to several stressors in the aquatic and terrestrial environments, and human activities have profoundly impacted this vertebrate group. The aim of the present study was to analyze physicochemical parameters, metals and pesticide residues, and the toxicity of water and sediment samples from an environment with high agricultural activity (S1: Salto stream; S2: drainage channel downstream from S1) by means of bioassays using Rhinella arenarum (Amphibia: Anura) larvae. Metals and pesticides were analyzed in water and sediment samples by fluorescence spectrometer of X-ray by total reflection and ultra-high-performance liquid chromatography-MS/MS, respectively. For lethality bioassays, 10 larvae (in triplicate) were exposed for 504 h to water and sediment samples. Also, 50 larvae were exposed for 96 h (in triplicate) to water and sediment samples for the evaluation of biomarkers of neurotoxicity, oxidative stress, and genotoxicity. Twenty-six different pesticides (mainly herbicides) were detected in both sites, and Cu, Zn, and Pb exceeded the limit for protection of aquatic life. Lethality was observed in larvae exposed to water and sediment samples from both sites at chronic exposure. Oxidative stress was observed in larvae exposed to both sites. In larvae exposed to samples from S1, alterations in the neurotoxicity biomarkers were observed. These results alert about the degradation of the sites and highlight the need to monitor and control the use of pesticides. PRACTITIONER POINTS: Twenty-six pesticides were detected in water and sediment from Salto stream basin. Significant mortality was observed in larvae exposed to samples from all sites. Sublethal effects were observed mainly in larvae exposed to samples from Salto stream. The degraded quality can be associated with the agricultural activities of the area.

Biological Efficacy of Plant Growth-Promoting Bacteria and Arbuscular Mycorrhizae Fungi: Assessments in Laboratory and Greenhouse Conditions.

Utilizing the interactions of microorganisms with plants offers a favorable path to increase crop production and replace the use of synthetic fertilizers. Different bacteria and fungi have been used as biofertilizers to improve agricultural production, yield, and sustainability. Beneficial microorganisms can act as free-living organisms, symbiotes, and endophytes. Soil bacteria called plant growth-promoting bacteria (PGPB) and fungi called arbuscular mycorrhizae fungi (AMF) stimulate the growth and health of plants by direct and indirect mechanisms including nitrogen fixation, phosphorus solubilization, phytohormone production, enzyme production, antibiotic synthesis, and induced systemic resistance. To use these microorganisms as a biofertilizer, it is necessary to assess their efficacy under laboratory and greenhouse conditions. Few reports detail the methods used to develop a test under different environmental conditions, and without these details it is difficult to develop suitable methodologies to evaluate microorganism-plant relationships. We describe four protocols that go from sample preparation to testing in vitro the efficacy of different biofertilizers. Each protocol can be used to test a different biofertilizer microorganism, focusing on bacteria such as Rhizobium sp., Azotobacter sp., Azospirillum sp., Bacillus sp. as well as AMF such as Glomus sp. These protocols can be used in several stages of biofertilizer development, including microorganism selection, microorganism characterization, and in vitro evaluation of efficacy for the registration process. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Evaluating the biological effect of biofertilizer based on PGPB under laboratory conditions Basic Protocol 2: Evaluating the biological effect of biofertilizer based on PGPB under greenhouse conditions Basic Protocol 3: Evaluating the biological effect of biofertilizer based on symbiotic nitrogen-fixing bacteria Basic Protocol 4: Evaluating the biological effect of biofertilizer based on AMF.

Dog hair volatiles attract Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) females.

Rhipicephalus sanguineus sensu lato (s.l.) (Latreille) feeds on domestic dogs worldwide. This tick species uses dog volatiles during host-seeking behavior. In this study, we identified volatile compounds from dog hairs involved in the host location of R. sanguineus s.l. The R. sanguineus s.l. females, but not males, were attracted to hair samples and Super Q extracts from Schnauzer dogs in Y-tube olfactometer bioassays. A total of 54 compounds from dog hair extracts were identified by gas chromatography coupled to mass spectrometry, including hydrocarbons, aldehydes, alcohols, ketones, and carboxylic acids. Screening the identified compounds by the single sensillum recording technique showed that isovaleric acid, hexanal, heptanal, and sucaltone (6-methyl-5-hepten-2-one) significantly stimulated the olfactory receptor neurons of the basiconic, chaeticum, and trichodeum sensilla of female ticks. When synthetic compounds were evaluated alone, or in binary, tertiary, or quaternary mixtures, female ticks were only attracted to isovaleric acid and 1 tertiary mixture (hexanal, heptanal, and isovaleric acid). We conclude that isovaleric acid functions as an attractant for R. sanguineus s.l. These findings contribute to the understanding of the chemical ecology of ticks during host location.

Holistic profiling of the venom from the Brazilian wandering spider Phoneutria nigriventer by combining high-throughput ion channel screens with venomics.

Introduction: Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer, often referred to as the Brazilian wandering spider, banana spider or "armed" spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. Methods: In this study, we explored the neuroactivity and molecular diversity of P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Results: Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Discussion: Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggest that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.

Metals, pesticides, and emerging contaminants on water bodies from agricultural areas and the effects on a native amphibian.

In the Paraná River lower basin, an important agro-productive area of Argentina, crop fields and cattle breeding activities are common and may affect water quality. So, the aim of this study was to analyze the impacts of cattle breeding and agricultural activities on a stream from Buenos Aires, through physicochemical parameters (metals, pesticides, and emerging contaminants) and ecotoxicological parameters with Rhinella arenarum larvae, a native amphibian species. Three sites were selected on an ordinary plain stream that goes through agricultural fields and a cattle breeding establishment (upstream -S1-, near -S2- and downstream -S3- the establishment). Physicochemical parameters were measured in situ (in water) and in laboratory (in water and sediment samples: metals, pesticides, ivermectin and oxytetracycline). A semi-static chronic toxicity bioassay (504 h) was performed with water samples, and neurotoxicity, oxidative stress and genotoxicity biomarkers were measured after acute exposure (96 h). According to the index, a degradation in the water quality was observed in all sites. Ivermectin (8.03 mg/kg) and oxytetracycline (1.9 mg/kg) were detected in sediment samples from S2. Pesticides were detected in all sites, mainly in water samples: S1 presented the highest variability (7 residues) and in S3 AMPA, glyphosate and acetochlor concentrations were higher (10.3, 22.4 and 23.8 µg/L). Also, all sites significantly produced lethality at chronic exposure. Lethality at 504h was 40% for S1, 56.66% for S2 and 93.33% for S3. At acute exposure, the oxidative stress biomarkers were altered on R. arenarum larvae exposed to all sites and the neurotoxicity biomarkers were altered on larvae exposed to S1 and S3. Water quality was severely degraded by the surrounding agricultural and cattle breeding activities, which may represent a threat to the ecosystems.

Sustainable agricultural use of sewage sludge: impacts of high Zn concentration on on Folsomia candida, Enchytraeus crypticus, Lactuca sativa, and Phaseolus vulgaris.

Zinc (Zn) is an essential micronutrient for plants and an important component for maintaining soil quality. Commonly found in the soil due to anthropogenic activities, such as industrialization and application of organic waste as fertilizers, in high concentrations, Zn may induce soil toxicity, affecting important communities, such as edaphic fauna. Despite its high concentrations found in the environment, Zn bioavailability can be affected by the type of soil, organic matter content and pH. In this work, Zn had its toxicity evaluated in a natural tropical soil, sampled in São Paulo-Brazil, for two soil invertebrates (Folsomia candida, Enchytraeus crypticus) and two seeds (Lactuca sativa and Phaseolus vulgaris), through ecotoxicological tests. The invertebrate E. crypticus was exposed to Zn concentrations of 10.0 (T1); 100.0 (T2); 150.0 (T3); 200.0 (T4); 400.0 (T5) mg Zn kg-1 of dry soil, while F. candida, L. sativa and P. vulgaris were exposed to Zn concentrations of 100.0; 200.0; 400.0; 800.0 (t6); 1600.0 (t7); and 2000.0 (t8) mg Zn kg-1 of dry soil. The outcome evaluated were seed germination, for L. sativa and P. vulgaris, and reproduction, for F. candida and E. crypticus. The EC50 obtained for E. crypticus, F. candida, L. sativa, and P. vulgaris were 261.5, 1089.7, 898.5, and 954.5 mg Zn kg-1 of dry soil, respectively, being E. crypticus the most sensitive organism, and only at the highest Zn's concentrations the organisms' reproduction and seeds' germination showed a statistically significant inhibitory effect (p < 0.05). Therefore, this work's results showed that Zn does not present significant toxicity for the tested soil organisms and seeds and that at 100 mg Zn kg-1 of dry soil it can be beneficial to F. candida and E. crypticus' reproduction and L. sativa's germination. These results imply that the presence of Zn in low concentrations, both in soil and biofertilizers, such as sewage sludge, not only is not a concern, but it can even benefit certain crops and functions of edaphic organisms, which may contribute to the engagement of sustainable agricultural practices and the quest for food security.