Surveillance and dietary risk assessment of endocrine-disrupting pesticides in eggplant/brinjal and cauliflower in Pakistan.

Several pesticides (used in vegetable production) have recently been identified as potential endocrine disruptors. The current study aimed to determine the consumer exposure risk associated with eating contaminated vegetables. The European Union-citrate buffered QuEChERS extraction protocol, validated in accordance with the European Union guidelines, was used to monitor selected endocrine-disrupting pesticides in eggplant/brinjal (Solanum melongena L.) and cauliflower (Brassica oleracea) marketed in Rawalpindi/Islamabad, Pakistan. A total of 80 and 69 percent of eggplant/brinjal (n = 25) and cauliflower (n = 26) samples were found contaminated, respectively. Sixty-five percent of cauliflower samples were found non-contaminated with both European Union (EU) and Codex Alimentarius Commission (CAC) maximum residue limits (MRL), while 20 % of brinjal samples were found to be non-compliant with EU-MRL. Both vegetables contained high levels of the androgen antagonist chlorpyrifos and the thyroid hormone inhibitor cyhalothrin-lambda. The estimated acute health risk associated with dietary exposure to chlorpyrifos and cyhalothrin-lambda in cauliflower for both males and females was found to be greater than 200 % of the FAO/WHO Joint Meeting on Pesticide Residues' (JMPR) established acute reference dose. The estimated chronic health risk for all studied endocrine-disrupting pesticides (0-5.27 %) indicates zero to very low health risk for studied population groups.

Antimicrobial Activity of Pinus wallachiana Leaf Extracts against Fusarium oxysporum f. sp. cubense and Analysis of Its Fractions by HPLC.

Fusarium wilt has ruined banana production and poses a major threat to its industry because of highly virulent Fusarium oxysporum f. sp. cubense (Foc) race 4. The present study focused on the efficacy of Pinus wallachiana leaf extracts and its organic fractions against Foc in in vitro and greenhouse experiments. The presence of polyphenols in the fractions was also investigated using high performance liquid chromatography (HPLC). The in vitro tests carried out for the leaf extract of P. wallachiana showed its inhibitory effect on the mycelial growth and, based on this evidence, further characterization of fractions were done. Complete mycelial inhibition and the highest zone of inhibition against Foc was observed for the n-butanol fraction in vitro, while the n-hexane and dichloromethane fractions showed lower disease severity index (DSI) in greenhouse experiments. The fractions were further analysed by HPLC using nine polyphenolic standards, namely quercitin, myrecitin, kaempferol, rutin, gallic acid, trans-ferulic acid, coumeric acid, epicatechin and catechin. The highest content of polyphenols, based on standards used, was quantified in the n-butanol fraction followed by the ethyl acetate fraction of the leaf extract. This is the first report of antimicrobial activity of Pinus wallachiana extracts against Foc to the best of our knowledge.

Method optimization and validation for the routine analysis of multi-class pesticide residues in Kinnow Mandarin and fruit quality evaluation.

The present study describes the selection of a sensitive multi-residue method that can be used for the routine testing of pesticides in Kinnow Mandarin. The citrate-buffered QuEChERS extraction followed by primary secondary amines and C18 clean-up was found suitable for the analysis of fifty four pesticides. The limit of quantification for the selected pesticides was lower than maximum residue limits (MRLs) set by European Union, Codex Alimentarius Commission (CAC), and twelve other countries. The method's accuracy ranged from 74.4 to 112% and expanded uncertainty ranged from 7.5 to 49.6%. The validated method was applied to Kinnow Mandarin samples, collected from 22 export units of district Sargodha, Pakistan. Almost 27% of the samples (n = 22) were exceeding the CAC-MRLs. The index of quality for residues (IqR), for 64% of the samples, was considered adequate. The study indicates the need for regular monitoring to protect public health and ensure safe and consistent trade.

Determination of pesticide residues in dates using UHPLC-QqQ-MS/MS: method development and validation.

A modified, efficient, and sensitive acetate-buffered QuEChERS extraction method was developed for the quantitative study of 16 commonly applied multiclass pesticides on date palm fruit. The date palm fruit samples were rehydrated by adding water during comminution. Samples were extracted with acidified acetonitrile, buffered with acetate salt. To minimize the matrix interferences, clean-up of the rehydrated samples was optimized by comparison with different sorbents (alumina, silica gel, florisil, primary secondary amine (PSA), and chitosan). The method validation parameters were evaluated as per European Union (EU) guidelines (SANTE/12682/2019). For 16 pesticides, % recovery of 69 to 121.8% with an associated precision (RSD ≤ 20%) was achieved at the fortification levels that were 0.5 to 2 times of European Union maximum residue limits (EU-MRLs). The validated method was successfully employed for the analysis of date palm fruit samples (n = 20) collected from various markets. Forty percent (40%) of samples (n = 8) were found to be contaminated with various pesticides. The most frequently detected residues were carbofuran, carbaryl, metalaxyl, tebuconazole, triazophos, and pyriproxyfen. The concentration of all the detected pesticides in real samples was below the EU-MRLs.

Zn2+ and Cd2+ assisted photo-catalytic degradation of chlorpyrifos in soil.

The Cd2+ and Zn2+ assisted photo-catalytic degradation of soil incorporated chlorpyrifos (CLP) was reported in current study. The soil samples fortified with CLP and metals were irradiated in photo-reactor for different time intervals to check maximum degradation. Soil samples extracted with acetonitrile were analyzed by HPLC. The results of the study revealed a complete mineralization of insecticide from soil that followed first-order Langmuir-Hinshelwood (L-H) kinetic model. The CLP degradation rate in soil was higher in photoreactor than control with variation in half-life from 41 days to 20 days. The degradation of CLP in photoreactor was 5 fold augmented after Zn2+ fortification of soil while Cd2+ had negligible effect on CLP photodegradation. Thus Zn2+ fortification of soil will not only replenish the important nutrient for plant growth but will also help in alleviating the harmful effects of CLP on soil flora and fauna by enhancing its rate of photodegradation.

Screening of multiclass pesticide residues in honey by SPE-GC/MSD: a pilot study.

Analytical method for the monitoring of residues of multiclass pesticides (variable chemical structure and chromatographic behavior) in honey has been optimized and in-house validated in the present study. Chemical confirmation of 35 selected pesticides (in-hive-treated pesticides and pesticides applied for agricultural practices in vicinity of apiaries) has been successfully achieved with the acetonitrile extraction/partitioning and cleanup by modified US EPA solid-phase extraction (SPE) protocol following analysis on the GC/MS DRS Pesticide Screener. The applied extraction procedure has given acceptable recoveries with an associated precision (RSD) for selected pesticides within the range as suggested by SANTE at MQL of 10 µg kg-1. Potential matrix effect for selected analytes was calculated by using honey from five different floral sources. The optimized method was used to determine levels of pesticide residues in honey samples randomly collected from 26 different apiaries in Pakistan. Residues of nine selected pesticide (dichlorvos, mevinphos, ethalfluralin, trifluralin, lindane, chlorpyrifos-methyl, dieldrin, profenofos, 4,4-DDE) were frequently detected in the ranges of 3-48.8 µg kg-1 in 26.9% of analyzed samples (n = 26) and 15.3% of the studied samples exceeded maximum residue limits (MRLs). In-hive-treated acaricides, i.e., coumaphos, tau-fluvalinate, and malathion, were not detected in any of the analyzed honey samples.

Extraction and quantification of polyphenols from kinnow (Citrus reticulate L.) peel using ultrasound and maceration techniques.

An investigation was carried out to extract polyphenols from the peel of kinnow (Citrus reticulate L.) by maceration and ultrasound-assisted extraction (UAE) techniques. The antioxidant potential of these polyphenols was evaluated using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and superoxide radical scavenging assays; and their antimicrobial activity was assessed against bacterial strains Staphyloccoccus aureus, Bacillus cereus, and Salmonella typhimurium. The highest extraction yield was obtained through the solvent ethanol at 80% concentration level, whereas UAE was a more efficient technique and yielded comparatively higher polyphenol contents than maceration. Maximum polyphenols were extracted with 80% methanol [32.48 mg gallic acid equivalent (GAE)/g extract] using UAE, whereas minimum phenolics (8.64 mg GAE/g extract) were obtained with 80% ethyl acetate through the maceration technique. Elevated antioxidant activity of kinnow peel extracts was exhibited in three antioxidant assays, where 80% methanolic extracts showed the highest antioxidant activity (27.67±1.11mM/100 g for FRAP) and the highest scavenging activity, 72.83±0.65% and 64.80±0.91% for DPPH and superoxide anion radical assays, respectively. Strong correlations between total polyphenols and antioxidant activity were recorded. Eleven phenolic compounds-including five phenolic acids and six flavonoids-were identified and quantified by high performance liquid chromatography. Ferulic acid and hesperidin were the most abundant compounds whereas caffeic acid was the least abundant phenolic compound in kinnow peel extracts. Maximum inhibition zone was recorded against S. aureus (16.00±0.58 mm) whereas minimum inhibition zone was noted against S. typhimurium (9.00±1.16 mm). It was concluded that kinnow mandarin peels, being a potential source of phenolic compounds with antioxidant and antimicrobial properties, may be used as an ingredient for the preparation of functional foods.

Cu(2+) and Fe(2+) mediated photodegradation studies of soil-incorporated chlorpyrifos.

The influences of Cu(2+) and Fe(2+) on the photodegradation of soil-incorporated chlorpyrifos were investigated in the present study. The soil samples spiked with chlorpyrifos and selected metal ions were irradiated with UV light for different intervals of time and analyzed by HPLC. The unsterile and sterile control soil samples amended with pesticides and selected metals were incubated in the dark at 25 °C for the same time intervals. The results of the study evidenced that photodegradation of chlorpyrifos followed the first-order kinetics. The dissipation t0.5 of chlorpyrifos was found to decrease from 41 to 20 days under UV irradiation. The rate of chlorpyrifos photodegradation was increased in the presence of both metals, i.e., Cu(2+) and Fe(2+). Thus, initially observed t0.5 of 19.8 days was decreased to 4.39 days in the case of Cu(+2) and 19.25 days for Fe(+2). Copper was found to increase the rate of photodegradation by 4.5 orders of magnitude while the microbial degradation of chlorpyrifos was increased only twofold. The microbial degradation of chlorpyrifos was only negligibly affected by Fe(2+) amendment. The studied trace metals also affected the abiotic degradation of the pesticide in the order Cu(2+) > Fe(2+).

Pesticide residues in fruits and vegetables from Pakistan: a review of the occurrence and associated human health risks.

The main objective of the review is to document, assess and analyze the results of the previously reported data on levels of different pesticides in selected fruits and vegetables from Pakistan. The findings of the previous studies clearly indicated that more than 50 % of the samples were contaminated with organophosphate, pyrethroids and organochlorine pesticides. Many studies reported that among fresh fruits and vegetables tomato, apple, melon, mango, grapes, and plum crossed the FAO/WHO permissible limits for these contaminants residual levels. The comparison of other regions showed that observed levels were found above maximum residue limits (MRLs) in 50 % of the samples but were in agreement with the studies from neighboring countries like China and Bangladesh. Higher hazard risk index (HRI) values were calculated for dieldrin, methamidophos, o,p'-DDT, diazinon and p,p'-DDT in apple, mango, banana, melon, potato and onion. The review also highlights that data on pesticide residues in foodstuff is scarce which should be overcome by further extending studies from different areas of Pakistan. In order to ascertain the provision of food suitable for human consumption, it is imperative to monitor pesticides in food commodities by the country's authorities and enforce guidelines based on permissible limits.

Cattle egrets as a biosentinels of persistent organic pollutants exposure.

We investigated selected chlorinated pollutants (ß-HCH, γ-HCH, DDDs, DDEs, o,p'-DDT, p,p'-DDT, heptachlor, aldrin, dieldrin, and endrin) in the Lahore and the Sialkot districts of Pakistan, using eggs of cattle egret (Bubulcus ibis) collected during May and June 2007. The pollutant with highest level and frequency was ΣDDT, followed by ß-HCH, γ-HCH, heptachlor, aldrin, dieldrin, and endrin in descending order. The concentration(s) were significantly higher in Sialkot heronry for all the pollutants (except p,p'-DDT) than in Lahore. The values for DDTs, ß-HCH, γ-HCH, and heptachlor were significantly higher (p < 0.05) in the egg(s) than in sediment(s) and in the chicks' diet, due to biomagnification. Among DDTs analogues, p,p'-DDD was the major contaminant with >60 % of total DDT burden, reflecting the widespread aged as well as recent use of DDT as well as anaerobic degradation (DDD/DDE > 1 in many cases) in the nearby paddy soils. In few samples, p,p'-DDT/(DDD + DDE) > 0.5 suggested the recent emission patterns from surrounding contaminated areas of demolished DDT units and obsolete pesticide stores. The higher levels of HCHs (i.e., ß-HCH) in the samples collected from Sialkot indicate exposure from long-term agricultural use. Overall, concentrations of all studied POPs were less than the threshold levels known to affect reproduction. Nevertheless, total DDTs and/or HCHs burdens in some eggs contained concentrations of greater than what would educe adverse effects on birds. This is among few studies on OCPs exposure to avian species, which provide the evidence of Pakistan's contribution toward the Global POPs emission.

Pesticide exposure and endocrine dysfunction in the cotton crop agricultural workers of southern Punjab, Pakistan.

This study evaluated pesticide effects on reproductive and thyroid hormones of cotton farmers of southern Punjab, Pakistan. A total of 88 cotton farmers (42 spray applicators and 46 cotton pickers) were randomly included with an equal number of age- and sex-matched controls. Sampling was done in high spraying and peak picking seasons. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, prolactin, thyroid-stimulating hormone (TSH), total triiodothyroxine (TT3), and free thyroxine (fT4) were carried out by enzymatic immunoassay. Plasma cholinesterase (PChE) levels were measured by Ellman's method. Serum FSH, LH, and testosterone levels were significantly high in spray applicators (P < .01).Serum FSH and testosterone levels were significantly raised in cotton pickers (P < .01). Serum prolactin was decreased significantly in both groups (P < .01).Serum fT4 was significantly reduced in cotton pickers (P < .01). Pesticide exposure is associated with thyroid and reproductive hormone levels disturbance.

Organochlorine residual concentrations in cattle egret from the Punjab Province, Pakistan.

In this study, residual concentration of organochlorine pesticides (OCPs) in the sediments, prey, and eggs of Bubulcus ibis were measured from three breeding heronries from the Punjab province of Pakistan. Pattern of contamination in eggs followed the order: DDTs>HCHs>heptachlor>aldrin. Overall, pesticide residual concentrations were greater in eggs of cattle egrets collected from heronry on the River Ravi. Among HCHs, γ-HCH was more prevalent in eggs, whereas DDTs followed the order: DDD>DDE>p,p'-DDT>o,p'-DDT. Eggshell thinning was detected which showed negative relationship with residual concentration of DDE. In prey samples, residual concentration of POPs followed the order: DDTs>HCHs>dicofol>heptachlor; however, contamination pattern in sediments followed a slightly different order: DDTs>heptachlor>dicofol>HCHs>dieldrin>aldrin. Concentration of ß-HCH was more prevalent in sediments and comparatively greater concentrations of POPs were measured in sediments collected from the River Ravi. Dicofol was found for the very first time in the biological samples from Pakistan, and its concentration was measured as relatively high in eggs from heronry from the River Chenab. Residual concentrations measured in eggs were below the levels that could affect egret populations. Biomagnification of the total OCPs through the food chain was evident in three breeding heronries. The concentration of DDE measured in eggs of the cattle egret suggests the need for monitoring this contaminant in other bird species at different trophic levels.

Risk assessment of pesticide exposure on health of Pakistani tobacco farmers.

Tobacco is an important cash crop of Pakistan. Pesticides are commonly used to increase the crop yield, but their health impact has not been studied yet. The objectives of the study were to determine the frequency of pesticide poisoning and to explore the knowledge, attitudes and practices (KAP) towards safety measures among the tobacco farmers in Swabi, Pakistan. One hundred and five tobacco farmers involved in pesticide application were randomly selected from two villages of district Swabi. A structured questionnaire was used for clinical and KAP information. Plasma cholinesterase (PChE) levels were measured by Ellman's method by using GD Italy kits. All tobacco farmers were males with a mean (SD) age of 26 (9) years. The majority of the farmers reported multiple symptoms headache, dizziness, vomiting, shortness of breath, muscle weakness and skin rash correlate with the clinically significant depression of PChE levels. Out of 105 pesticide applicators, 58 (55%) had post-exposure reduction in PChE levels <20% from baseline, 35 (33%) had mild poisoning (20-40% reduction) and 12 (11%) had moderate poisoning (>40% reduction). Most of the farmers did not use any personal protective equipment during pesticide handling. Only a few used shoes (31%), masks (14%) and gloves (9%) during pesticide spray. In conclusion, the tobacco farmers had mild to moderate pesticide poisoning, which was correlated with depression in PChE levels. Moreover, most farmers had little knowledge about the safety measures, casual attitude and unsatisfactory safety practices with regard to the use of basic protective equipments during pesticide applications on the tobacco crop.

Monitoring results for organochlorine pesticides in soil and water from selected obsolete pesticide stores in Pakistan.

This study reports on the concentration and distribution pattern of organochlorine pesticides in soil and water samples collected from obsolete pesticide stores in three provinces of Pakistan and analyzed on capillary gas chromatography/electron capture detection. The data for soil and water samples were highly variable as samplings were done from diversified locations. The soil samples mainly contained DDTs followed by lindane and heptachlor. The contamination levels in North West Frontier Province (NWFP), Punjab, and Sindh were in ranges of 247-9,157 mg kg(-1), 214-10,892 mg kg(-1), and 86-1,139 mg kg(-1), respectively. In water, residue range levels were 0-15.17 (median 0.29) microg L(-1), 0.25-0.78 (median 0.36) microg L(-1), and 0.11-0.83 (median 0.21) microg L(-1) in NWFP, Punjab, and Sindh, respectively.

Applicability of some mass spectrometric criteria for the confirmation of pesticide residues.

This study was initiated to demonstrate the impact of tolerance intervals calculated based on experimental data and standard criteria on the capability to detect and identify pesticide residues by GC-MS in plant extract. The tolerance intervals specified by the current guidelines and standards (EU, EPA, ISO, EC) result in a large number of false negative values. The tolerance intervals calculated based on the actually measured ion ratios at 2 different probability levels did not provide a better estimate. Based on experimental findings a 2-phase procedure for confirmation of pesticide residues is recommended. The first screening phase comprises identification of those pesticide residues that are likely to be present avoiding false negatives as far as possible. Relatively large tolerance intervals, +/-30% of absolute ion abundances ratios, have to be used at this point. When 2 or 3 selected ion ratios are within the established tolerance intervals the residue is confirmed. When the ions detected still indicate the possible presence of a residue the result may be reported as tentatively identified. However, when the result would lead to regulatory action, second confirmatory phase has to be undertaken. This can be achieved with the same GC-MS equipment, by injecting matrix-matched standards of the suspected analyte, in order to compensate for matrix influence on ion ratios. Alternatively, methods using different physico-chemical properties of the compound have to be used. The choice of the technique depends on their availability, time and cost.