Lessons from the historical dynamics of environmental law enforcement in the Brazilian Amazon.

Here, we analyze critical changes in environmental law enforcement in the Brazilian Amazon between 2000 and 2020. Based on a dataset of law enforcement indicators, we discuss how these changes explain recent Amazon deforestation dynamics. Our analysis also covers changes in the legal prosecution process and documents a militarization of enforcement between 2018 and 2022. From 2004 to 2018, 43.6 thousand land-use embargoes and 84.3 thousand fines were issued, targeting 3.3 million ha of land, and totaling USD 9.3 billion in penalties. Nevertheless, enforcement relaxed and became spatially more limited, signaling an increasing lack of commitment by the State to enforcing the law. The number of embargoes and asset confiscations dropped by 59% and 55% in 2019 and 2020, respectively. These changes were accompanied by a marked increase in enforcement expenditure, suggesting a massive efficiency loss. More importantly, the creation of so-called conciliation hearings and the centralization of legal processes in 2019 reduced the number of actual judgments and fines collected by 85% and decreased the ratio between lawsuits resulting in paid fines over filed ones from 17 to 5%. As Brazil gears up to crack-down on illegal deforestation once again, our assessment suggests urgent entry points for policy action.

Multiresidue Determination of Growth Promoters in Bovine Serum using QuEChERS and UHPLC-Q-ORBITRAP: Validation and In Vivo Study.

A reliable method using a QuEChERS approach and liquid chromatography coupled to Q-Orbitrap mass spectrometry was optimized and validated for the quantification of 20 growth promoters in bovine serum. The recoveries ranged from 91.4-114.1%, relative standard deviations varied between 0.3-4.0%, and CCα values were between 0.023-0.350 µg L-1. The developed method was applied in an in vivo study using steers, which were intramuscularly treated with commercial injections containing stanozolol. A rapid metabolization was observed, with a detection window ranging from 3 to 10 days. The stability of incurred stanozolol was confirmed after 240 days at -20 °C and also after 5 freeze-thaw cycles. To the best of our knowledge, this is the first work in which an in vivo study was performed to support the monitoring of stanozolol in bovine serum. In addition, the use of Q-Orbitrap high-resolution mass spectrometry allows for retrospective analysis from a surveillance perspective.

Determination of the lactose content in low-lactose milk using Fourier-transform infrared spectroscopy (FTIR) and convolutional neural network.

Demand for low lactose milk and milk products has been increasing worldwide due to the high number of people with lactose intolerance. These low lactose dairy foods require fast, low-cost and efficient methods for sugar quantification. However, available methods do not meet all these requirements. In this work, we propose the association of FTIR (Fourier Transform Infrared) spectroscopy with artificial intelligence to identify and quantify residual lactose and other sugars in milk. Convolutional neural networks (CNN) were built from the infrared spectra without preprocessing the data using hyperparameter adjustment and saliency map. For the quantitative prediction of the sugars in milk, a regression model was proposed, while for the qualitative assessment, a classification model was used. Raw, pasteurized and ultra-high temperature (UHT) milk was added with lactose, glucose, and galactose in six concentrations (0.1-7.0 mg mL-1) and, in total, 432 samples were submitted to convolutional neural network. Accuracy, precision, sensitivity, specificity, root mean square error, mean square error, mean absolute error, and coefficient of determination (R2) were used as evaluation parameters. The algorithms indicated a predictive capacity (accuracy) above 95% for classification, and R2 of 81%, 86%, and 92% for respectively, lactose, glucose, and galactose quantification. Our results showed that the association of FTIR spectra with artificial intelligence tools, such as CNN, is an efficient, quick, and low-cost methodology for quantifying lactose and other sugars in milk.

In Vivo Administration of Stanozolol in Cattle: Depletion and Stability Studies Using UHPLC-Q-Orbitrap.

An in vivo study was performed in order to evaluate the depletion time of stanozolol and its main metabolites using naturally incurred urine sample collected after the administration of intramuscular injections in 12 steers. A stability study was also carried out to investigate the influence of the storage period and the freeze-thaw cycles. A fast parent drug metabolization was observed, because within 6 h after drug administration, the signal of the metabolite 16ß-hydroxystanozolol was predominant. After the second drug administration, a detection window of 17 days was obtained. The stability was studied using ANOVA, in which a storage condition of -20 °C proved stable during 240 days, which was also confirmed after 5 freeze-thaw cycles.

A novel strategy for the detection of boldenone undecylenate misuse in cattle using ultra-high performance liquid chromatography coupled to high resolution orbitrap mass spectrometry: From non-targeted to targeted.

In this work multivariate strategies were employed in order to highlight new potential biomarkers of interest to detect the exogenous treatment of steers intramuscularly treated with boldenone undecylenate. Serum samples collected from treated (n = 4) and control (n = 8) crossbred animals of varying ages and weights were extracted using a simple sample preparation procedure based on salt assisted liquid-liquid extraction. Data acquisition was performed using liquid chromatography and Q-Exactive™ Orbitrap mass spectrometry. Data processing and treatment were performed using two non-targeted workflows: (1) Compound Discoverer software and (2) XCMS package on the open-source R software combined with MetaboAnalyst. Three potential biomarkers were highlighted taking into account the chromatographic shapes, the feature location on the generated s-plots, the fold change, the adjusted p values, the coefficient of variation in the QC samples and the area under the ROC curves. Predicted formulas based on mass accuracy, structural composition and spectra similarity were proposed. A robust statistical model to predict the boldenone treatment was further developed based on the weighted abundances of the selected biomarkers. The requirements for screening methods were successfully fulfilled, together with a wider detection window in comparison with the monitoring of the deconjugated metabolite boldenone, although biomarker identification studies are still ongoing.

Can Serum be a Match for Urine in the Regulatory Analysis of Boldenone in Cattle? A Systematic Comparison Between Detection Window, Stability, and Enzymatic Hydrolysis.

This work involved a systematic comparison between serum and urine for the monitoring of anabolic androgenic steroids in livestock. Incurred samples were collected over 120 days from crossbred steers treated with intramuscular injections containing boldenone undecylenate. Independent high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods were used for the assessment of the respective detection windows, which were larger for serum samples. Both matrices presented adequate performance in terms of long-term stability, assessed using an isochronous approach during 196 days at -20 °C and for five freeze-thaw cycles. The effectiveness of the enzymatic hydrolysis reaction using Helix pomatia juice was also compared. The calculated concentrations in serum samples were not statistically influenced by the deconjugation reaction. On the other hand, urine hydrolysis conditions were studied using a 33 Box-Behnken Design, in which a central point condition led to a satisfactory deconjugation performance. It could be observed that serum exhibited equivalent or better performance than urine for most of the evaluated criteria; thus, its inclusion in the regulatory analysis of boldenone in cattle is supported.

Determination of Steroids in Bovine Serum: Validation of a Reliable LC-MS/MS Method and In Vivo Studies with Boldenone Undecylenate and Testosterone Propionate.

Serum analysis has received much attention in regulatory analysis of food-producing animals, especially for anabolic steroids. The possibility of confirming the parent drugs with minimum metabolization enables the detection of intact steroid esters, whose identification represents unequivocal proof of drug administration. This work involved the development and validation of a quantitative LC-MS/MS method to determine 30 steroids and steroid esters in bovine serum. Sensitivity was improved using microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was successfully conducted in accordance with the Decision 657/2002/EC guidelines. An in vivo experiment was performed on 12 crossbred steers in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections. The samples were collected over a period of 120 days, in which both intact esters were identified within 11 days postadministration. 17ß-Boldenone was observed after 92 days for 2 steers and 56 days for the other animals. The applicability of a cut-off level to the ratio between 17ß-testosterone and epitestosterone was evaluated in an attempt to differentiate testosterone abuse from endogenous production. It could be observed that a calculated ratio above this level is strong evidence of drug administration, although a high false-negative rate was obtained.

Determination of steroids in bovine hair: Validation of a microwave-assisted chemical derivatization method using liquid chromatography-tandem mass spectrometry and in vivo studies.

Hair analysis has attracted great attention in the regulatory analysis of food-producing animals, particularly due to the wider detection window of veterinary drugs in this matrix and also the possibility of confirming parent drugs with minimum metabolization. This work involved the development and validation of a quantitative liquid chromatography-tandem mass spectrometry method to determine 25 steroids and steroid esters in bovine hair. Sensitivity was improved using a fast and effective microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was conducted in accordance with the Decision 657/2002/EC guidelines. An animal experimentation procedure was performed on 12 bovine animals in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections on the neck. The samples were collected for 78 days in which the detection of the administrated analytes was only observed near the application sites. For some of the monitored days, no analyte was detected on the neck area. Since the migration of the analytes was not observed in areas other than the application site, false-negative results should be carefully considered when monitoring animal hair samples.

Viability of Campylobacter spp. in frozen and chilled broiler carcasses according to real-time PCR with propidium monoazide pretreatment.

The aim of this study was to evaluate the viability of Campylobacter spp. in frozen and chilled broiler carcasses using real-time PCR with propidium monoazide (PMA) pretreatment. Sixty broiler carcasses were collected: 30 frozen and 30 chilled. Each carcass was submitted to 2 real-time PCR protocols to detect and quantify Campylobacter spp.: one using pretreatment with PMA, which blocks the amplification of DNA from dead bacteria, and the other without PMA. The results showed that PMA-pretreated carcasses, either frozen or chilled, had a lower positivity rate compared to untreated samples (P < 0.001). Regarding storage temperatures, PMA-pretreated frozen carcasses that tested positive were in a lesser number than chilled carcasses (P < 0.05). However, the quantification of total and live bacteria in PMA-pretreated frozen carcasses that tested positive showed no significant difference compared to chilled carcasses. It was concluded that the real-time PCR with PMA pretreatment was a sensitive method for evaluating the viability of Campylobacter spp. in broiler carcasses. Chilled broiler carcasses would represent greater hazard to public health concerning Campylobacter transmission.

Diagnóstico e fatores de risco do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais / Diagnosis and risk factors of bovine taeniasis-cysticercosis complex in Salinas, Minas Gerais, Brazil

Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais, foram coletadas amostras de sangue de 355 bovinos distribuídos em 18 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também um levantamento epidemiológico dos casos de teníase diagnosticados nos laboratórios credenciados pela Secretaria Municipal de Saúde de Salinas, no período de 2007 a 2010. A prevalência de cisticercose bovina foi de 4,70% enquanto as prevalências de teníase, encontradas durante os quatro períodos avaliados, foram de 0,29%, 0,36%, 0,24% e 0,24%. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Foi concluído que a cisticercose bovina está presente no município de Salinas, Minas Gerais, sendo o tratamento térmico ineficiente da carne bovina o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária.(AU)

In order to diagnose the situation of bovine taeniasis-cysticercosis complex in the municipality of Salinas, Minas Gerais, Brazil, blood samples were collected from 355 cattle in 18 randomly selected farms. A socioeconomic questionnaire was filled in each farm for the analysis of factors which favor the maintenance of the taeniasis-cysticercosis complex. An epidemiological survey of human taeniasis was performed through analyses of the Municipal Health Department in the 2007-2010 period. A prevalence of 4.7% for bovine cysticercosis and the frequency of 0.29, 0.36, 0.24 and 0.24% for human taeniasis, during the evaluated period, was found. Among the risk factors, a statistically significant correlation was found between the occurrence of bovine cysticercosis and the ingestion of undercooked meat. It was concluded that bovine cysticercosis is present in the municipality of Salinas, due to inefficient heat treatment of the meat as the main risk factor for maintenance of the taeniasis-cysticercosis complex, reinforcing the need to adopt control measures with continuous epidemiological and health surveillance.(AU)

Dipyrone metabolite 4-MAA induces hypothermia and inhibits PGE2 -dependent and -independent fever while 4-AA only blocks PGE2 -dependent fever.

BACKGROUND AND PURPOSE: The antipyretic and hypothermic prodrug dipyrone prevents PGE2 -dependent and -independent fever induced by LPS from Escherichia coli and Tityus serrulatus venom (Tsv) respectively. We aimed to identify the dipyrone metabolites responsible for the antipyretic and hypothermic effects. EXPERIMENTAL APPROACH: Male Wistar rats were treated i.p. with indomethacin (2 mg·kg(-1) ), dipyrone, 4-methylaminoantipyrine (4-MAA), 4-aminoantipyrine (4-AA) (60-360 mg·kg(-1) ), 4-formylaminoantipyrine, 4-acethylaminoantipyrine (120-360 mg·kg(-1) ) or vehicle 30 min before i.p. injection of LPS (50 µg·kg(-1) ), Tsv (150 µg·kg(-1) ) or saline. Rectal temperatures were measured by tele-thermometry and dipyrone metabolite concentrations determined in the plasma, CSF and hypothalamus by LC-MS/MS. PGE2 concentrations were determined in the CSF and hypothalamus by elisa. KEY RESULTS: In contrast to LPS, Tsv-induced fever was not followed by increased PGE2 in the CSF or hypothalamus. The antipyretic time-course of 4-MAA and 4-AA on LPS-induced fever overlapped with the period of the highest concentrations of 4-MAA and 4-AA in the hypothalamus, CSF and plasma. These metabolites reduced LPS-induced fever and the PGE2 increase in the plasma, CSF and hypothalamus. Only 4-MAA inhibited Tsv-induced fever. The higher doses of dipyrone and 4-MAA also induced hypothermia. CONCLUSIONS AND IMPLICATIONS: The presence of 4-MAA and 4-AA in the CSF and hypothalamus was associated with PGE2 synthesis inhibition and a decrease in LPS-induced fever. 4-MAA was also shown to be an antipyretic metabolite for PGE2 -independent fever induced by Tsv suggesting that it is responsible for the additional antipyretic mechanism of dipyrone. Moreover, 4-MAA is the hypothermic metabolite of dipyrone.