TiO2-MWCNT nanohybrid: Cytotoxicity, protein corona formation and cellular internalisation in RTG-2 fish cell line.

Titanium dioxide nanoparticles-multiwalled carbon nanotubes (TiO2-MWCNT) nanohydrid has an enhanced photocatalytic activity across the visible light with promising applications in environmental remediation, solar energy devices and antimicrobial technologies. However, it is necessary to evaluate the toxicological effects of TiO2-MWCNT towards safe and sustainable development of nanohybrids. In this work, we studied the cytotoxicity, protein corona formation and cellular internalisation of TiO2-MWCNT on fibroblasts derived from gonadal rainbow trout tissue (RTG-2) for the first time. This nanohydrid did not show any toxicity effect on RTG-2 cells up to 100 mg L-1 after 24 h of exposure as monitored by alamar blue, neutral red and trypan blue assays (in presence or absence of foetal bovine serum, FBS). Futhermore, cryo-transmission electron microscopy analysis demonstrated that TiO2 particles is attached on nanotube surface after FBS-protein corona formation in cell culture medium. Raman spectroscopy imaging showed that TiO2-MWCNT can be internalised by RTG-2 cells. This work is a novel contribution towards better understanding the nanobiointeractions of nanohydrids linked to their in vitro effects on fish cells in aquatic nanoecotoxicology.

Coating carbon nanotubes with humic acid using an eco-friendly mechanochemical method: Application for Cu(II) ions removal from water and aquatic ecotoxicity.

In this work, industrial grade multi-walled carbon nanotubes (MWCNT) were coated with humic acid (HA) for the first time by means of a milling process, which can be considered an eco-friendly mechanochemical method to prepare materials and composites. The HA-MWCNT hybrid material was characterized by atomic force microscopy (AFM), scanning electron microscopies (SEM and STEM), X-ray photoelectron spectroscopy (XPS), termogravimetric analysis (TGA), and Raman spectroscopy. STEM and AFM images demonstrated that the MWCNTs were efficiently coated by the humic acid, thus leading to an increase of 20% in the oxygen content at the nanotube surface as observed by the XPS data. After the milling process, the carbon nanotubes were shortened as unveiled by SEM images and the values of ID/IG intensity ratio increased due to shortening of the nanotubes and increasing in the number defects at the graphitic structure of carbon nanotubes walls. The analysis of TGA data showed that the quantity of the organic matter of HA on the nanotube surface was 25%. The HA coating was responsible to favor the dispersion of MWCNTs in ultrapure water (i.e. -42mV, zeta-potential value) and to improve their capacity for copper removal. HA-MWCNTs hybrid material adsorbed 2.5 times more Cu(II) ions than oxidized MWCNTs with HNO3, thus evidencing that it is a very efficient adsorbent material for removing copper ions from reconstituted water. The HA-MWCNTs hybrid material did not show acute ecotoxicity to the tested aquatic model organisms (Hydra attenuata, Daphnia magna, and Danio rerio embryos) up to the highest concentration evaluated (10mgL-1). The results allowed concluding that the mechanochemical method is effective to coat carbon nanotubes with humic acid, thus generating a functional hybrid material with low aquatic toxicity and great potential to be applied in environmental nanotechnologies such as the removal of heavy metal ions from water.

Nanotoxicity of graphene oxide: Assessing the influence of oxidation debris in the presence of humic acid.

This study sought to evaluate the toxicological effects of graphene oxide (GO) through tests with Danio rerio (zebrafish) embryos, considering the influence of the base washing treatment and the interaction with natural organic matter (i.e., humic acid, HA). A commercial sample of GO was refluxed with NaOH to remove oxidation debris (OD) byproducts, which resulted in a base washed GO sample (bw-GO). This process decreased the total oxygenated groups in bw-GO and its stability in water compared to GO. When tested in the presence of HA, both GO and bw-GO stabilities were enhanced in water. Although the embryo exposure showed no acute toxicity or malformation, the larvae exposed to GO showed a reduction in their overall length and acetylcholinesterase activity. In the presence of HA, GO also inhibited acid phosphatase activity. Our findings indicate a mitigation of material toxicity after OD removal. The difference in the biological effects may be related to the materials' bioavailability and biophysicochemical interactions. This study reports for the first time the critical influence of OD on the GO material biological reactivity and HA interaction, providing new data for nanomaterial environmental risk assessment and sustainable nanotechnology.

Assessments of Motor Abnormalities on the Grid-Walking and Foot-Fault Tests From Undernutrition in Wistar Rats.

This study was designed to verify whether different lactation conditions influenced nervous system development. The authors used motor tasks to verify changes in exploratory activity and muscle strength of weaned rats from different litter sizes and evaluated the applicability of the grid-walking test for assessing motor abnormalities caused by undernutrition. Alterations in litter size during the suckling period perturbed the nutritional status of pups, which exhibited body weight differences between the groups. Large-litter (L) pups showed significant delays in achieving developmental milestones and neurological reflexes compared to the small-litter (S) and medium-litter (M) pups. The S, M, and L group pups exhibited similar exploratory responses and muscle strength. In the grid-walking and foot-fault tests, the L group pups traveled shorter distances and, consequently, had less footsteps. However, the percentages of foot faults in the L group were higher than S and M groups. These results reflect delayed maturation of structures responsible for sensorimotor responses, such as the cerebellum, because much cerebellar maturation takes place postnatally. This is the first study to report that early undernutrition in pups resulted in suboptimal performances on the grid-walking and foot-fault tests and that the former test was sensitive to alterations caused by nutritional deficiency.

Avaliação da toxicidade aguda depiraclostrobin, epoxiconazol e suamistura em Daphnia similis / Toxicity of formulations based on piraclostrobin, epoxiconazole and its combination to Daphnia similis

No presente trabalho avaliou-se o efeito toxicológico (inibição damobilidade) de formulações fungicidas à base de piraclostrobin eepoxiconazol, isoladamente e em formulação conjugada sobreDaphnia similis, mediante determinação da Concentração EfetivaMédia (CE50-48h) de cada uma das formulações. Também foideterminado o possível efeito sinérgico ou antagônico que ouso conjugado dos dois fungicidas poderia exercer em relaçãoà sua toxicidade. Foram encontrados valores de CE50-48h de18,36 μg.L-1 para a formulação à base de piraclostrobin, de89,98 μg.L-1 para a formulação à base de epoxiconazol e de23,50 μg.L-1 para a formulação conjugada. Das três formulaçõesestudadas, o piraclostrobin mostrou-se o mais tóxico para osorganismos em estudo. A análise isobolográfi ca e a determinação doÍndice de Aditividade (IA) sugerem que a mistura dos princípios ativosexerce apenas efeito toxicológico aditivo sobre o organismo-alvo.

Avaliação da toxicidade de Piraclostrobin, epoxiconazol e sua mistura em alga Pseudokirchneriella subcapitata / Toxicity of formulations basedof Piraclostrobin, epoxiconazole and its combination on algae Pseudokirchneriella subcapitata

Estudou-se o efeito toxicológico (inibição de crescimento) deformulações fungicidas à base de piraclostrobin e epoxiconazol,isoladamente e em formulação conjugada, sobre a algaPseudokirchneriella subcapitata mediante determinação daConcentração Efetiva Média (CE50-72 h) dos princípios ativos decada uma das formulações. Foram encontrados valores de CE50-72 h de 5,57 mg.L-1 para a formulação à base de piraclostrobin,de 1,14 mg.L-1 para a formulação à base de epoxiconazol e de0,20 mg.L-1 para a formulação conjugada. O piraclostrobin apresentoumenor toxicidade às algas em relação aos dados encontrados naliteratura, o epoxiconazol resultados semelhantes e a mistura maiortoxicidade. A análise isobolográfi ca e a determinação do Índice deAditividade (IA) sugerem forte sinergismo da mistura quanto à suaação tóxica sobre o organismo-teste. Esse fato realça a necessidadede revisão dos padrões limitantes desses fungicidas em corpos deágua, os quais tendem a subestimar os efeitos adversos resultantesda combinação dos agentes tóxicos.

Assessing the safety of Pseudomonas putida introduction in the environment: an overview of ecotoxicological tests.

Risk assessment guidelines for the environmental release of microbial agents are performed in a tiered sequence which includes evaluation of exposure effects on non-target organisms. However, it becomes important to verify whether environmental risk assessment from temperate studies is applicable to tropical countries, as Brazil. Pseudomonas putida is a bacteria showing potential to be used for environmental applications as bioremediation and plant disease control. This study investigates the effects of this bacteria exposure on rodents and aquatic organisms (Daphnia similis) that are recommended to be used as non-target organism in environmental risk assessments. Also, the microbial activity in three different soils under P. putida exposure was evaluated. Rats did not show clinical alterations, although the agent was recovered 16h after the exposure in lung homogenates. The bacteria did not reduce significantly the reproduction and survival of D. similis. The soil enzymatic activities presented fluctuating values after inoculation with bacteria. The measurement of perturbations in soil biochemical characteristics is presented as an alternative way of monitoring the overall effects of the microbial agent to be introduced even in first stage (Tier I) of the risk assessment in tropical ecosystems.

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

There has been some concern about the environmental impact of microbial agents. Pseudomonas may be used as bioremediator and as biopesticide. In this study, we report the use of soil enzyme assays as biological indicator of possible negative effects in soil functioning after the P. putida AF7 inoculation. For that, P. putida AF7 was originally isolated from the rizosphere of rice and was inoculated on three soil types: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). The acid phosphatase, beta-glucosidase and protease enzymes activities were measured for three period of evaluation (7, 14 and 21 days). In general, the enzymatic activities presented variation among the tested soils. The highest activities of beta-glucosidase and acid phosphatase were observed in the RH and AH soils, while the protease activity was higher in the TH soil. Also, the soil characteristics were measured for each plot. The activity of enzymes from the carbon cycle was positively correlated with the N and the P and the enzyme from the nitrogen cycle was negatively correlated with N and C.org. The presented data indicate that soil biochemical properties can be an useful tool for use as an indicator of soil perturbations by microbial inoculation in a risk assessment.

Métodos para avaliaçäo de risco ambiental e ecotoxicológico de biopesticidas / Methodology to evaluate environment riisks and toxicity pathogenicity of biopesticides

Pretendeu-se verificar se a metodologia proposta pelos protocolos internacionais para análise toxicopatológica dos biopesticidas é adequada ao estudo de seu risco de aplicaçäo por pulverizaçäo em florestas no Brasil. Foram realizados testes toxicopatológicos em ratos, para verificaçäo da persistência do microorganismo no animal e a possível ocorrência de danos provocados pela exposiçäo ao Bacillus thuringiensis. A fim de analisar a toxicidade/patogenicidade dos biopesticidas testou-se em animais, a administraçäo do produto pelas vias respiratórias e oral, e realizou-se sua quantificaçäo em órgäos e fezes, além da verificaçäo de lesöes na necropsia. Em relaçäo a avaliaçäo de sua persistência e disseminaçäo no ambiente avaliou-se a presença do microorganismo em amostras de folhas verdes e em detritos no solo, em uma área de reflorestamento de eucaliptos, tratada por aplicaçäo aérea e terrestre. Concluiu-se que a dose proposta nos protocolos internacionais de avaliaçäo de risco do uso de biopesticidas etá muito acima da dose encontrada em campo, entretanto sugere-se que sejam incluídos testes de exposiçäo prolongada a diferentes doses

Consideraçöes metodológicas a respeito da avaliaçäo do desenvolvimento animal / Methodological considerations about the evaluation of animal development

O uso de agroquímicos pode gerar problemas dentro da área de toxicologia. Estas substâncias em contato com fêmeas gestantes podem causar atraso no desenvolvimento físico ou mesmo mental dos filhotes. Contudo, nem sempre há informaçoes precisas a respeito de tais danos. A prevençao de efeitos mutagênicos ou embriofetotóxicos depende muitas vezes de testes laboratoriais que podem evidenciar através da pesquisa o que nem sempre torna-se claro no exame clínico. Assim, o estudo destas substâncias através de metodologia cada vez mais sensível torna-se importante para avaliaçao correta dos danos às populaçoes expostas.