Proposal for a tiered regulatory framework for the aquatic risk assessment of pesticides in Brazil.

Aquatic risk assessment is essential to guarantee the sustainable use of pesticides and the conservation of water resources near agricultural fields. This article discusses a proposal for a tiered regulatory framework for the aquatic risk assessment of pesticides in Brazil. The first step is problem formulation, which includes establishing general and specific protection goals. In the exposure assessment, the Estimated Environmental Concentrations in water should be calculated based on realistic worst-case assumptions regarding application rate and frequency, the entry into the edge-of-field water body, and fate in the water body, using scenario-dependent models suggested by the Brazilian Environmental Agency. These calculations can be refined by including Efate studies with variable exposures to reflect realistic environmental conditions accurately and include mitigation measures that impact the modeling. In the hazard assessment, ecotoxicological data for toxicity to fish, aquatic invertebrates, algae, and aquatic plants should be required for all pesticides based on standardized protocols and species. Tier 2 has several refinement options, including incorporating toxicity data from additional test species and effect modeling. In Tier 3, population- and community-level effects are evaluated using semi-field studies. Considering the case study in Brazil, Tier 1 demonstrated that, from the 12 pesticides that were assessed, seven (58%) failed based on the value of the Risk Quotient. In Tier 2, when exposure refinement options and mitigation measures such as buffer zones are considered, all seven pesticides, for which Tier 1 indicated risk, still failed the assessment. The risk for four of these seven pesticides could be refined by considering toxicity information from additional species. Refinement options and mitigation measures that could be applied to the agricultural scenario in Brazil were discussed. In conclusion, the proposed tiered risk assessment is a feasible way to evaluate whether a pesticide will pose an unacceptable risk to aquatic organisms. Integr Environ Assess Manag 2024;00:1-15. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Use of dry bean fields by birds and mammals in Brazil: Insights from a field study and its use in pesticide risk assessment.

The relationship between agriculture and wildlife can be both synergetic and challenging, as the increased surface of agricultural land makes it increasingly important for agriculture and wildlife to coexist. This study aims to describe the use of freshly drilled dry bean fields by birds and mammals in Brazilian Cerrado and Atlantic Forest sites and their diversity and abundance within in-crop and off-crop areas (with native permanent vegetation and other surrounding crop fields) at four different farms. A comprehensive survey was conducted, using various sampling methods, including point counts, foraging counts, trail cameras, and occasional encounters. In all, contacts for 12 518 birds across 306 species and 313 mammals across 34 species were registered. The off-crop areas exhibited greater species richness, abundance, and diversity than the in-crop areas on all farms. For birds, 47 species were recorded in-crop, of which 15 were classified as insectivores, 15 as granivores, seven as omnivores, seven as carnivores, and three as frugivores. The number of in-crop observations per species was small. The abundance off-crop was greater for 31 species observed in-crop, indicating that dry bean fields are probably not a preferred habitat for those species. Species classified as granivorous are most likely to feed on dry bean seeds. However, almost all granivorous species observed in-crop areas are too small to be able to feed on dry bean seeds. For mammals, nine species were recorded in-crop, of which four were classified as carnivores, three as omnivores, one as insectivore, and one as granivore. Additionally, despite the considerable effort in this study, no evidence was found that birds and mammals feed on dry bean seeds. The results highlight the importance of off-crop areas in dry bean fields, characterized by a more diverse and abundant bird community than in-crop. Integr Environ Assess Manag 2024;20:864-874. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Qualitative analysis of the most locally relevant runoff and erosion parameters for constructing Brazilian scenarios.

Estimating exposure is one of the most important steps in an environmental risk analysis of crop-protection products to nontarget organisms. Regulatory agencies such as the US Environmental Protection Agency (USEPA), Pest Management Regulatory Agency (PMRA), and European Food Safety Authority (EFSA) all use mathematical exposure models in their regulatory assessment process. Brazil has been discussing the adoption of the Pesticide in Water Calculator (PWC) to be applied in aquatic pesticide risk assessment. Therefore, a qualitative sensitivity analysis (Morris OAT method) was performed to understand which are the most important local parameters in the PWC to estimate environmental concentrations in surface water (EECSW ). In addition, an exercise made up of two corn scenarios in two Brazilian regions was developed (Uberlândia [UDI] and Arapoti [ARA]). Two herbicides with different soil-binding properties and modes of action were selected to estimate the EECSW . The results demonstrated that the parameters of importance were different for each site, probably the result of different soil characteristics and meteorological patterns. This outcome suggests that regulatory agencies should consider developing more than one scenario to account for different agricultural regions. For Herbicide 1, the EECSW for UDI were similar to US scenarios, whereas for ARA they were lower. For Herbicide 2, the EECSW for the UDI site was higher than most of the US scenarios, whereas at the ARA site, EECs were similar to four US scenarios and lower than the other six. Local data were used as a refinement, resulting in the decrease in the EECSW for both herbicides in the UDI site. For the ARA site, Herbicide 1 displayed a similar EECSW value, whereas for Herbicide 2, it was lower after the refinement. Overall, these results demonstrated the importance of developing local scenarios to provide more realism to estimate pesticide exposure from its agricultural use and may help regulators to determine and recommend mitigations regarding the use of crop-protection products. Integr Environ Assess Manag 2023;19:1374-1384. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Applying a tiered environmental risk assessment framework to estimate the risk of pesticides to soil organisms in Latin America.

This work investigates the application of a tiered risk assessment scheme for soil organisms based on the risk quotient (RQ) and the toxicity exposure ratio (TER). Forty-five pesticides registered in Latin America were chosen and the ecotoxicological endpoints for earthworms, Collembola, and microorganisms were collated. Tier I assessment was made on conservative assumptions in which no refinements were applied. There, 14 pesticides (31%) exceed the RQ regulatory trigger indicating unacceptable risk, whereas 27 (60%) indicate unacceptable risk on the TER approach. In a Tier II evaluation when refinement options such as foliar interception, field half-life, and the dissipation following the peak estimated environmental concentration are considered, eight (18%) pesticides indicate unacceptable risk based on the RQ, and 15 (33%) indicate unacceptable risk based on the TER. A nonmetric multidimensional scaling evaluation was performed to understand the relevant characteristics involved in how each pesticide poses a risk to soil organisms. Based on the outcome of this analysis, we observed that, for a given pesticide, the combination of high persistence, low or no crop interception, and high toxicity are likely to require higher tier risk assessment. Refinement options can consider either or both the exposure and/or the effect side of the framework. Exposure refinements are potentially simpler and can be conducted with data already available to risk assessors, whereas effect refinements involving further testing with the organisms potentially at risk are still under discussion for intermediate and higher tiers. A sensitive, simple, and logical environmental risk assessment framework can be used to adequately identify risks based on the relevant protection goals that, in turn, will help to protect the desired soil multifunctionality of the ecosystem. We encourage academia and industry to further investigate these topics to provide the most scientifically robust and evidence-based information to decision makers. Integr Environ Assess Manag 2023;19:446-460. © 2022 SETAC.

Estimating pesticide environmental concentrations in Latin America: The importance of developing local scenarios.

Data to assess pesticide exposure in soil and water are scarce and unevenly distributed in Latin America, especially due to the size of the region and the vast agricultural landscape. This makes it difficult to assess associated environmental risks. We suggest that the lack of pesticide exposure or monitoring data can be addressed by using validated models to provide estimated pesticide exposure concentrations in soil and water bodies. This exposure modeling approach has been used by regulatory agencies in other countries and regions such as the United States, Canada, and the European Union. In order to properly estimate pesticide exposure concentrations, we advocate for the development of local scenarios containing local weather, soil, and crop data to be used in the existing models. A sensitivity analysis of the models can be performed to determine parameters that are sensitive and therefore inputs to these parameters are derived locally. We believe the development of local scenarios in the region is attainable and can be a pragmatic approach for developing a more comprehensive picture of potential pesticide exposure in the region. Integr Environ Assess Manag 2021;17:901-904. © 2021 Syngenta Proteção de Cultivos Ltda.

Conformational properties of angiotensin II and its active and inactive TOAC-labeled analogs in the presence of micelles. Electron paramagnetic resonance, fluorescence, and circular dichroism studies.

The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents--negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)--was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC1-AII and inactive TOAC3-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOAC-promoted intramolecular fluorescence quenching was more pronounced for TOAC3-AII because of the proximity between the nitroxide and Tyr4. CD spectra showed that although both AII and TOAC1-AII presented flexible conformations in water, TOAC3-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for AII and TOAC1-AII, different conformations were acquired by TOAC3-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC3-AII is unable to acquire conformations similar to those of native AII and partially active TOAC1-AII is probably the explanation for its lack of biological activity.

Sticholysins I and II interaction with cationic micelles promotes toxins' conformational changes and enhanced hemolytic activity.

The effect of three cationic surfactants bearing the same polar head group and different chain length (cetyltrimethyl ammonium bromide (CTAB); tetradecyltrimethylammonium bromide (TTAB); dodecyltrimethylammonium bromide (DTAB)) on the conformation and function of the sea anemone pore-forming toxins sticholysins I and II (St I and St II) was studied by fluorescence and circular dichroism spectroscopy and evaluation of hemolytic activity (HA). Preincubation of the toxins with the longer chain surfactants CTAB and TTAB at concentrations slightly above their critical micelle concentration (CMC) leads to an enhancement of their HA. Significant increases in the fluorescence intensity with a slightly red shift in lambda(max) were observed at concentrations close to the surfactants' CMC, suggesting changes in the environment of the tryptophan residues. The changes in the fluorescence intensity are more noticeable and take place at lower surfactant concentrations for St I, irrespective of the surfactant alkyl chain length, although the differences between St I and St II increase as the surfactant alkyl chain length increases. This is evinced not only by the higher fluorescence intensity values and the lower surfactant concentrations required to reach them, but also by the higher acrylamide-quenching constant values (Ksv) for St I. However, the surfactant's effects on the toxins' HA were not found to be directly related to the observed changes in fluorescence intensity, as well as near- and far-UV-CD spectra. In particular, the latter spectra indicate that changes in HA and in fluorescence behavior take place without noticeable modifications in St I and St II secondary and tertiary structures. The results suggest that the interaction with the surfactants induces only subtle conformational changes in the toxins that favor the formation of lytic competent structures.

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II.

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: electron paramagnetic resonance, circular dichroism, and fluorescence studies.

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.

Estudos conformacionais de seqüências hidrofóbicas de domínios de receptores acoplados a proteínas G (GPCR) e da proteína G. Um estudo de CD e espectroscopia de fluorescência / Conformational study of hydrophobic sequences of domains of receivers coupled to proteins G (GPCR) and of the protein G. A study of CD and fluorescence spectroscopy

O trabalho desenvolvido nesta tese doutorado enfocou dois projetos: O primeiro envolveu o estudo conformacional de um peptídeo correspondente à região N-terminal da TM6 do LHR (KMAILIFTDFT, resíduos 570-580) e de dois análogos (D578G e D578Y) envolvidos na síndrome da puberdade precoce masculina familiar (FMPP). A substituição do resíduo de `Asp IND. 578´ por outros aminoácidos como Gly ou Tyr provoca a ativação constitutiva do receptor, levando à FMPP. Os peptídeos foram estudados por CD e fluorescência em solução aquosa, na presença de TEF e na presença de membranas-modelo. Observou-se que os três peptídeos agregavam em solução aquosa e na presença de membranas...

Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces--modulation of conformation and activity, and lipid-protein interaction.

Sticholysins I and II (St I and St II) are water-soluble toxins produced by the sea anemone Stichodactyla helianthus. St I and St II bind to biological and model membranes containing sphingomyelin (SM), forming oligomeric pores that lead to leakage of internal contents. Here we describe functional and structural studies of the toxins aiming at the understanding at a molecular level of their mechanism of binding, as well as their effects on membrane permeabilization. St I and St II caused potassium leakage from red blood cells and temperature-dependent hemolysis, the activation energy of the process being lower for the latter toxin. Protein intrinsic fluorescence measurements provided evidence for toxin binding to model membranes composed of 1:1 (mol:mol) egg phosphatidyl choline (ePC):SM. The fluorescence intensity increased and the maximum emission wavelength decreased as a result of binding. The changes were quantitatively different for both toxins. Circular dichroism spectra showed that both St I and St II exhibit a high content of beta-sheet structure and that binding to model membranes did not alter the toxin's conformation to a large extent. Changing the lipid composition by adding 5 mol% of negatively charged phosphatidic acid (PA) or phosphatidyl glycerol (PG) had small, but detectable, effects on protein conformation. The influence of lipid composition on toxin-induced membrane permeabilization was assessed by means of fluorescence measurements of calcein leakage. The effect was larger for ePC:SM bilayers containing 5 mol% of negative curvature-inducing lipids. Electron paramagnetic resonance (EPR) spectra of intercalated fatty acid spin probes carrying the nitroxide moiety at different carbons (5, 7, 12, and 16) evidenced the occurrence of lipid-protein interaction. Upon addition of the toxins, two-component spectra were observed for the probe labeled at C-12. The broader component, corresponding to a population of strongly immobilized spin probes, was ascribed to boundary lipid. The contribution of this component to the total spectrum was larger for St II than for St I. Moreover, it was clearly detectable for the C-12-labeled probe, but it was absent when the label was at C-16, indicating a lack of lipid-protein interaction close to the lipid terminal methyl group. This effect could be either due to the fact that the toxins do not span the whole bilayer thickness or to the formation of a toroidal pore leading to the preferential interaction with acyl chain carbons closer to the phospholipids head groups.