Probabilistic modeling and risk characterization of the chronic aflatoxin M1 exposure of Hungarian consumers.

Aflatoxin contamination can appear in various points of the food chain. If animals are fed with contaminated feed, AFB1 is transformed-among others-to aflatoxin M1 (AFM1) metabolite. AFM1 is less toxic than AFB1, but it is still genotoxic and carcinogenic and it is present in raw and processed milk and all kinds of milk products. In this article, the chronic exposure estimation and risk characterization of Hungarian consumers are presented, based on the AFM1 contamination of milk and dairy products, and calculated with a probabilistic method, the two-dimensional Monte-Carlo model. The calculations were performed using the R plugin (mc2d package) integrated into the KNIME (Konstanz Information Miner) software. The simulations were performed using data from the 2018-2020 food consumption survey. The AFM1 analytical data were derived from the Hungarian monitoring survey and 1,985 milk samples were analyzed within the framework of the joint project of the University of Debrecen and the National Food Chain Safety Office of Hungary (NÉBIH). Limited AFM1 concentrations were available for processed dairy products; therefore, a database of AFM1 processing factors for sour milk products and various cheeses was produced based on the latest literature data, and consumer exposure was calculated with the milk equivalent of the consumed quantities of these products. For risk characterization, the calculation of hazard index (HI), Margin of Exposure, and the hepatocellular carcinoma incidence were used. The results indicate that the group of toddlers that consume a large amount of milk and milk products are exposed to a certain level of health risk. The mean estimated daily intake of toddlers is in the range of 0.008-0.221 ng kg-1 bw day-1; the 97.5th percentile exposure of toddlers is between 0.013 ng kg-1 bw day-1 and 0.379 ng kg-1 bw day-1, resulting in a HI above 1. According to our study, the exposure of older age groups does not pose an emergent health risk. Nevertheless, the presence of carcinogenic compounds should be kept to a minimum in the whole population.

Detection of Aflatoxins in Different Matrices and Food-Chain Positions.

Aflatoxins, produced mainly by filamentous fungi Aspergillus flavus and Aspergillus parasiticus, are one of the most carcinogenic compounds that have adverse health effects on both humans and animals consuming contaminated food and feed, respectively. Aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) as well as aflatoxin G1(AFG1) and aflatoxin G2 (AFG2) occur in the contaminated foods and feed. In the case of dairy ruminants, after the consumption of feed contaminated with aflatoxins, aflatoxin metabolites [aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2)] may appear in milk. Because of the health risk and the official maximum limits of aflatoxins, there is a need for application of fast and accurate testing methods. At present, there are several analytical methods applied in practice for determination of aflatoxins. The aim of this review is to provide a guide that summarizes worldwide aflatoxin regulations and analytical methods for determination of aflatoxins in different food and feed matrices, that helps in the decision to choose the most appropriate method that meets the practical requirements of fast and sensitive control of their contamination. Analytical options are outlined from the simplest and fastest methods with the smallest instrument requirements, through separation methods, to the latest hyphenated techniques.

Occurrence of Aflatoxin M1 in Raw Milk Marketed in Italy: Exposure Assessment and Risk Characterization.

The current study is based on the AFM1 contamination of milk determined from April 2013 to December 2018 in the framework of a self-control plan of six milk processing plants in Italy. These data - together with the consumption data of milk consumers - were evaluated and used for the calculation of the Estimated Daily Intake (EDI), the Hazard Index (HI), and the fraction of hepatocarcinoma cases (HCC) due to AFM1 exposure in different population groups. Altogether a total of 31,702 milk samples were analyzed, representing 556,413 tons of milk, which is an outstanding amount compared to published studies. The results indicate the monthly fluctuation of AFM1 levels through a period of nearly 6 years. The EDI of AFM1 in different population groups was in the range of 0.025-0.328 ng kg-1 body weight (bw) per day, based on the average consumption levels and weighted mean contamination of the milk in the study period. Considering average consumptions, in the groups of infants and toddlers, the HI calculation resulted in 1.64 and 1.4, respectively, while for older age groups, it was <1. The estimated fractions of HCC incidences attributable to the AFM1 intakes were 0.005 and 0.004 cases per 100,000 individuals in the 0-0.9 and 1-2.9-year age groups, respectively, and below 0.004 cases in the other age categories. The monthly average AFM1 contamination of tested milk consignments ranged between 7.19 and 22.53 ng kg-1. Although the results of this extensive investigation showed a low risk of HCC, the variability of climatic conditions throughout years that influence AFB1 contamination of feed and consequently AFM1 contamination of milk justifies their continuous monitoring and update of the risk assessment.

Global Harmonization of Maximum Residue Limits for Pesticides.

International trade plays an important role in national economics. The Codex Alimentarius Commission develops harmonized international food standards, guidelines, and codes of practice to protect the health of consumers and to ensure fair practices in the food trade. The Codex maximum residue limits (MRLs) elaborated by the Codex Committee on Pesticide Residues are based on the recommendations of the FAO/WHO Joint Meeting on Pesticides (JMPR). The basic principles applied currently by the JMPR for the evaluation of experimental data and related information are described together with some of the areas in which further developments are needed.

Analysis of industry-generated data. Part 2: Risk-based sampling plan for efficient self-control of aflatoxin M1 contamination in raw milk.

Aflatoxin M1 (AFM1) contamination in 21,969 milk samples taken in Italy during 2005-08 and 2010 provided the basis for designing an early warning self-control plan. Additionally, 4148 AFM1 data points from the mycotoxin crisis (2003-04) represented the worst case. No parametric function provided a good fit for the skewed and scattered AFM1 concentrations. The acceptable reference values, reflecting the combined uncertainty of AFM1 measured in consignments consisting of milk from one to six farms, ranged from 40 to 16.7 ng kg(-1), respectively. Asymmetric control charts with these reference values, 40 and 50 ng kg(-1) warning and action limits are recommended to assess immediately the distribution of AFM1 concentration in incoming consignments. The moving window method, presented as a worked example including 5 days with five samples/day, enabled verification of compliance of production with the legal limit in 98% of the consignments at a 94% probability level. The sampling plan developed assumes consecutive analyses of samples taken from individual farms, which makes early detection of contamination possible and also immediate corrective actions if the AFM1 concentration in a consignment exceeds the reference value. In the latter case different control plans with increased sampling frequency should be applied depending on the level and frequency of contamination. As aflatoxin B1 increases in feed at about the same time, therefore a coordinated sampling programme performed by the milk processing plants operating in a confined geographic area is more effective and economical then the individual ones. The applicability of the sample size calculation based on binomial theorem and the fast response rate resulting from the recommended sampling plan were verified by taking 1000-10,000 random samples with replacement from the experimental databases representing the normal, moderately and highly contaminated periods. The efficiency of the control plan could be substantially enhanced if the dairy farms used feed with a tolerable level of AFB1.

Analysis of industry-generated data. Part 1: a baseline for the development of a tool to assist the milk industry in designing sampling plans for controlling aflatoxin M1 in milk.

The presence of aflatoxin M1 (AFM1) in milk was assessed in Italy in the framework of designing a monitoring plan actuated by the milk industry in the period 2005-10. Overall, 21,969 samples were taken from tankers collecting milk from 690 dairy farms. The milk samples were representative of the consignments of co-mingled milk received from multiple (two to six) farms. Systematic, biweekly sampling of consignments involved each of the 121 districts (70 in the North, 17 in the Central and 34 in the South regions of Italy). AFM1 concentration was measured using an enzyme-linked immunoassay method (validated within the range of 5-100 ng kg(-1)) whereas an HPLC method was used for the quantification of levels in the samples that had concentrations higher than 100 ng kg(-1). Process control charts using data collected in three processing plants illustrate, as an example, the seasonal variation of the contamination. The mean concentration of AFM1 was in the range between 11 and 19 ng kg(-1). The 90th and 99th percentile values were 19-34 and 41-91 ng kg(-1), respectively, and values as high as 280 ng kg(-1) were reached in 2008. The number of non-compliant consignments (those with an AFM1 concentration above the statutory limit of 50 ng kg(-1)) varied between 0.3% and 3.1% per year, with peaks in September, after the maize harvest season. The variability between different regions was not significant. The results show that controlling the aflatoxins in feed at farm level was inadequate, consequently screening of raw milk prior to processing was needed. The evaluation of the AFM1 contamination level observed during a long-term period can provide useful data for defining the frequency of sampling.

Limitations in the determination of maximum residue limits and highest residues of pesticides: Part I.

The pesticide usages are controlled by comparing residue concentrations in treated commodities to legally permitted maximum levels (MRLs) determined based on supervised trials designed to reflect likely maximum residues occurring in practice following authorised use. The number of trials available may significantly affect the accuracy of estimated maximum residues. We conducted a study with synthetic lognormal distributions with mean of 1 and standard deviations of 0.8 and 1.0, which reflect the residue distributions observed in practice. The likely residues in samples were modelled by drawing random samples of size 3, 5, 10 and 25 from the synthetic populations. The results indicate that the estimations of highest residues (HR), used for calculation of short-term intake, and the MRLs, serving as legal limits, are very uncertain based on 3-5 trials indicated by the calculated HR0.975/HR0.025 and MRL0.975/MRL0.025 ratios of 12 and 9, and 13 and 10, respectively, which question the suitability of such trials for the intended purpose. As the 95% range of HR and MRL rapidly decreases with number of trials, ideally ≥15 but minimum 6-8 trials should be used for estimation of HR and MRL according to the current typical practice of Codex Alimentarius.

Validation of an efficient method for the determination of pesticide residues in fruits and vegetables using ethyl acetate for extraction.

In this study, a version of the "quick, easy, cheap, effective, rugged, and safe" (QuEChERS) method was modified to use ethyl acetate (EtOAc) rather than acetonitrile (MeCN) for extraction in the determination of multiple pesticide residues in fruits and vegetables. EtOAc is better suited than MeCN for gas chromatographic (GC) analysis with electron capture detection (ECD) and nitrogen-phosphorus detection (NPD). The method entailed extraction of 30 g chopped sample plus 5 g NaHCO(3) and 30 g anhydrous Na(2)SO(4) with 60 mL EtOAc using a probe blender. After a centrifugation step, removal of residual water and cleanup were performed using dispersive solid-phase extraction (dispersive-SPE) with MgSO(4) and primary secondary amine (PSA) sorbent. (14)C-labeled chlorpyrifos with liquid scintillation counting was used to assist in optimizing and characterizing the method, and GC-ECD and GC-NPD were used for analysis of 24 selected pesticides. The method was validated using tomato, apple and frozen green bean matrices spiked at 0.05, 0.5, and 5 mg/kg. For 22 of the analytes, recoveries averaged 93% for all three commodities over the validation range with a relative standard deviation of 10% (n = 1182). Lower recoveries of dichlorvos were obtained with the method and iprodione determination was compromised in the green beans by an interfering peak. Typical limits of detection were 0.005-0.01 mg/kg with the method.

Development and single-laboratory validation of a new gas chromatographic multi-pesticide method of analysis of commercial emulsifiable concentrate formulations containing alachlor, chlorpyrifos methyl, fenthion and trifluralin as active ingredients.

A multi-pesticide (MP) method was developed and single-laboratory validated for the quality control of commercial pesticide products containing alachlor, chlorpyrifos methyl, fenthion and trifluralin as active ingredients (a.i.). A capillary gas chromatographic system with flame ionization detection (FID) and a programmable temperature vaporising split injector was used. The performance characteristics (specificity, linearity, precision and repeatability) of the method satisfied international acceptability criteria.

Application of a system suitability test for quality assurance and performance optimisation of a gas chromatographic system for pesticide residue analysis.

In pesticide residue analysis, screening for over 150 compounds has to be performed on a daily basis. As part of the quality control measures it is crucial to verify that the chromatographic system fits the purpose, or if any deterioration occurred during its previous use. The operation conditions of the chromatographic system can be best monitored with properly selected system suitability test (SST) mixtures, which provide information with one injection on the characteristic performance parameters of the whole system from the injector to the detectors. We developed SST mixtures that are also suitable for use with electron-capture, nitrogen-phosphorus and pulse flame photometric detectors. These SST mixtures were applied over 3 years to monitor the system performance parameters, such as the number of effective theoretical plates, resolution, asymmetry, detection limit and selectivity. The applicability and advantages of these tests are illustrated and discussed.