Evaluation and validation of two different chromatographic methods (HPLC and LC-MS/MS) for the determination and confirmation of ochratoxin A in pig tissues

This study was undertaken to compare two different analytical methods for the determination and confirmation of ochratoxin A (OTA) in blood serum, kidney and liver of pigs. Sample clean-up was based on liquid-liquid phase extraction. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with fluorescence detection (FL) or electro spray ionization (ESI+) tandem mass spectrometry (MS–MS). Comparative method evaluation was based on the investigation of 90 samples of blood serum, kidney and liver per animal originating from different regions of Serbia. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with (LC–MS–MS) liquid chromatography electro spray tandem mass spectro metry detection offered comparable good results at the sub-ppb concentration level. The results indicate that the liquid chromatography electro spray tandem mass spectrometric (LC-MS/MS) method was more specific and sensitive for the analysis and confirmation of ochratoxin A in pig tissues then high pressure liquid chromatography (HPLC) method after methylation of OTA.     

Bacterial diversity in paclobutrazol applied agricultural soils

The aim of this study was to investigate the bacterial communities on paclobutrazol [(2RS, 3RS)-1-(4-Chlorophenyl)-4, 4-dimethyl-2-(1H-1,2,4-triazol-1-yl) pentan-3-ol]–applied agricultural soils by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rDNA gene fragments. Three different agricultural soil samples were collected from paclobutrazol applied mango and waxapple orchards, peanut fields and untreated rice fields as a control for DGGE analysis. The DGGE pattern of PCR- generated 16S rDNA gene fragments indicated that the bacterial populations from four paclobutrazol–applied soils of peanut fields were closely related to each other and two paclobutrazol–applied soils of mango and waxapple orchards harbored closely related bacterial communities. But, paclobutrazol–free agricultural soils comprised relatively a different bacterial group. However, the bacterial populations of mango and waxapple orchard are completely different from the bacterial communities of peanut field. Further purification and sequence analysis of 40 DGGE bands followed by phylogenetic tree assay showed similar results that soil bacteria from paclobutrazol applied mango and waxapple orchard are phylogenetically related. Based on the phylogenetic analysis, the clone M-4 was clad 100 % (bootstrap value) with Mycobacterium sp. The Mycobacterium sp. has been proved to degrade the phenolic compounds such as phenol, 4-chlorphenol, 2,4-dichlorophenol and paclobutrazol molecule containing chlorobenzene ring.     

Alterations in the rabbit lymphoid tissue after bendiocarb administration

Various pesticides have immuno-suppressive effects, and thus the organisms become responsive to viral, bacterial and parasitic diseases and neoplasm. The aim of the study was to observe the structure of the small intestine (height of enterocytes and crypts), mucosal lymphoid tissue (Payer's patches, lymphocytes in lamina propria) and a lymph node after administration of bendiocarb (2,2-dimethyl-1,3-benzodioxol-4-yl-methylcarbamate) on days 3, 10, 20, 30 and 60 of the experiment. The height of the observed enterocytes showed an increasing tendency. On days 20, 30 and 60 we also observed an increase in diameter of crypts located in intestinal epithelium. The number of cells in lamina propria mucosae was significantly reduced on days 20 and 30 after administration of bendiocarb. Observations of the lymph node showed that on days 10 and 20 there was a significant increase in relative volume of medulla at the expense of the relative volume of the cortex and a decrease in the number of lymphocytes. However, we recorded an increase in diameter of lymphocytes. The intracellular parasite Toxoplasma gondii (T. gondii) belongs to the most common pathogenic parasites in the world and it can cause serious health complications in pregnant and immunodeficient individuals. DNA isolation, standard polymerase chain reaction (PCR) and visualization in a 2.5 % agarose gel, the presence of DNA T. gondii was detected in no examined rabbit brain samples. Using real time PCR T. gondii DNA was detected and quantified in the three rabbit brain samples (10 %).     

Transfer of pesticides to the brew during mate drinking process and their relationship with physicochemical properties

In order to evaluate the extraction of pesticide residues that are transferred to the brew during mate drinking process of P.U.1 yerba mate leaves (Ilex paraguariensis), a special device to simulate the way in which mate is drunk in Uruguay was developed. The transfer to the brew of 12 organophosphates, 5 synthethic pyrethroids and one organochlorine pesticide from spiked samples was studied. The relationship between the transfer data thus obtained and physicochemical properties like water solubility (Ws), octanol-water coefficient (Kow) and Henry's constant (H) was evaluated. The extractability of the pesticide residues from yerba mate can be correlated with log Ws and log Kow. These transfer values allowed the calculation of ARLs (acceptable residue level) for the pesticides following Food and Agriculture Organization (FAO), World Health Organizaion (WHO) guidelines. These results can help the future establishment of maximum residue levels (MRLs)     

Pesticide dissipation curves in peach,pear and tomato crops in Uruguay*

Dissipation curves of azoxystrobin and of the neonicotinoids acetamiprid and thiacloprid in peach; azinphos-methyl and carbaryl in pear and azoxystrobin, chlorfenapyr and chlorpyrifos in high-tunnel tomato crops were studied in the Southern region of Uruguay. An analytical methodology based on solid phase extraction (SPE) and detection by High Performance Liquid Chromatography with Diode Array Detector (HPLC/DAD) was used for acetamiprid and thiacloprid. Coupled SPE and detection by Gas Chromatography with Mass Selective Detector (GC/MSD) was used for the detection of azinphos-methyl, azoxystrobin, carbaryl, chlorfenapyr and chlorpyrifos residues. Curves were modeled mathematically with Solver program of Microsoft Excel®. The best fit for acetamiprid and thiacloprid in peach was achieved with the exponential model (r²=0.961 and 0.944, respectively). In the case of peach fruits there is not a Maximum Residue Limit (MRL) for acetamiprid in the Codex Alimentarius, while 0.5 mg/kg is the value rated for thiacloprid. The MRLs accepted by the European Union (EU) are 0.1 mg/kg for acetamiprid and 0.3 mg/kg for thiacloprid. According to the curves determined in these experiments, thiacloprid residues 10 to 12 days after application (daa) were below the MRLs established by both sources. In the case of acetamiprid, 25 daa would be required, according to the exponential mathematical model, to get residues levels below the MRL values established by the EU. For azinphos methyl in pear, the residues detected were mathematically fitted to an exponential model (r²=0.999). According to it, residue levels under the MRL established by the EU (0.05 mg/kg) are gotten in our conditions in 20 daa. In plastic tunnel tomato chlorfenapyr residues were not detected from 16 daa, having the dissipation curve an exponential trend. In the same condition, there was not a decay of the azoxystrobin concentration during a 24-day trial, being it around 0.40 ± 0.05 mg/kg.     

Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B1 adsorption ability for use in poultry feedstuffs

In this study the aflatoxin B₁ (AFB₁) removal capacity, the tolerance to salivary and gastrointestinal conditions, autoaggregation and coaggregation with pathogenic bacteria of Saccharomyces cerevisiae strains isolated from broiler feces, were evaluated. Only four of twelve isolated strains were identified as Saccharomyces cerevisiae using molecular techniques. The results obtained in AFB₁ binding studies indicated that the amount of AFB₁ removed was both strain and mycotoxin-concentration dependent. Therefore, a theoretical model was applied in order to select the most efficient strain to remove AFB₁ in a wide range of mycotoxin concentration. The results indicated that S. cerevisiae 08 and S. cerevisiae 01 strains were the most efficient microorganisms in the mycotoxin removal. Viability on simulated salivary and gastrointestinal conditions was investigated and S. cerevisiae 08 strain showed the best results, achieving 98% of total survival whereas S. cerevisiae 01 reached only 75%. Autoaggregation and coaggregation assays showed S. cerevisiae 08 as the most appropriate strain, mainly because it was the unique strain able to coaggregate with the four bacterial pathogens assayed. Consequently, S. cerevisiae 08 is the best candidate for future in vivo studies useful to prevent aflatoxicosis. Further quantitative in vitro and in vivo studies are required to evaluate the real impact of yeast-binding activity on the bioavailability of AFB₁ in poultry. However, this study could be useful in selecting efficient strains in terms of AFB₁ binding and provide an important contribution to research into microorganisms with potential probiotic effects on the host.     

Macrophage activity and histopathology of the lymphohematopoietic organs in male Wistar rats orally exposed to single or mixed pesticides

The noxious effects of low or effective dose exposure to single or mixed pesticides on macrophage activity and the lymphohematopoietic organs were investigated. Male Wistar rats were orally exposed to dichlorvos, dicofol, endosulfan, dieldrin and permethrin, either as single or combined mixtures during a 28-day study containing eight groups: one group received a semipurified diet (non-treated); two groups received a semipurified diet containing low dose mixture (dieldrin 0.025 mg/kg, endosulfan, 0.6 mg/kg, dicofol 0.22 mg/kg, dichlorvos 0.23 mg/kg, permethrin 5 mg/kg) or an effective dose mixture (dichlorvos 2.3 mg/kg, dicofol 2.5 mg/kg, endosulfan 2.9 mg/kg, dieldrin 0.05 mg/kg and permethrin 25.0 mg/kg), respectively; the other five groups received a semipurified diet containing each single pesticide in effective doses. At sacrifice, the thymus, spleen, mesenteric lymph nodes, Payer's patches and bone marrow were removed for histological analysis. Peritoneal macrophages were obtained to determine the phagocytosis and spreading indexes and tumoral necrosis factor alpha (TNF-α), nitric oxide (NO) and H₂O₂ production. Exposure to pesticide mixtures did not alter the percentage of macrophage phagocytosis and spreading, TNF-α production or the NO and H₂O₂ release when compared to the non-treated group. Neither was there any apparent evidence that a pesticide mixture at low or effective doses altered the histological structure of the lymphohematopoietic organs. The findings indicate that short-term treatment with pesticide mixtures did not induce an apparent immunotoxic effect in male Wistar rats.     

Determination of Volatile Organic Compounds in the Atmosphere Using Two Complementary Analysis Techniques

ABSTRACT

During a preliminary field campaign of volatile organic compound (VOC) measurements carried out in an urban area, two complementary analysis techniques were applied to establish the technical and scientific bases for a strategy to monitor and control VOCs and photochemical oxidants in the Autonomous Community of the Basque Country. Integrated sampling was conducted using Tenax sorbent tubes and laboratory analysis by gas chromatography, and grab sampling and in situ analysis also were conducted using a portable gas chromatograph. With the first technique, monocyclic aromatic hydrocarbons appeared as the compounds with the higher mean concentrations. The second technique allowed the systematic analysis of eight chlorinated and aromatic hydrocarbons. Results of comparing both techniques, as well as the additional information obtained with the second technique, are included.     

Impact of long‐term pesticide applications on some soil biological parameters

Abstract

The soil oxidative and anaerobic processes, as well as, the microbial biomass were followed during three years in a cotton farm (Tatuí) where the recommended pesticides have been used for several years, and in an experimental field (São Paulo) treated first time with the same pesticides. The oxidative process was monitored by the dehydrogenase (DHA)‐activity using triphenyltetrazolium chloride (TTC) as substrate. The anaerobic process was followed by the iron‐oxide reduction, and the microbial biomass was estimated by the substrate (glucose)‐indiced respiration. Increases in DHA‐activity and in the microbial biomass occurred only in the farm soil, with concomitant decreases in iron‐reduction. In the experimental field soil, the increases in DHA‐activity were followed only by decreases in iron‐reduction. Soil characteristics were the determining factor for different biological parameters after pesticide inputs. All the pesticides produced at least one clear but transient effect.     

Combined effect of diuron and simazine on photosystem II photochemistry in a sandy soil and soil amended with solid olive-mill waste

Diuron (3-(3,4-dichlorophenyl)- = 1,1-dimethylurea) and simazine (6-chloro-N ², N ⁴-diethyl-1,3,5-triazine-2,4-diamine) are soil-applied herbicides used in olive crops. The objective of this study is to investigate the effect of these herbicides on Photosystem II photochemistry of Olea europaea L., and whether the amendment of soil with an organic waste (OW) from olive oil production industry modifies this effect. For this purpose, herbicide soil adsorption studies, with unamended and OW-amended soil, and chlorophyll fluorescence measurements in adult olive leaves, after one, two and three weeks of soil herbicide treatment and/or OW amendment, were performed. Soil application of these herbicides reduced the efficiency of Photosystem II photochemistry of olive trees due to chronic photoinhibition, and this effect is counterbalanced by the addition of OW to the soil. OW reduces herbicide uptake by the plant due to an increase in herbicide adsorption.