Production and characterisation of environmentally relevant microplastic test materials derived from agricultural plastics

Soil environments across the globe, particularly in agricultural settings, have now been shown to be contaminated with microplastics. Agricultural plastics – such as mulching films – are used in close or direct contact with soils and there is growing evidence demonstrating that they represent a potential source of microplastics. There is a demand to undertake fate and effects studies to understand the behaviour and potential long-term ecological risks of this contamination. Yet, there is a lack of test materials available for this purpose. This study describes the manufacture and characterisation of five large (1–40 kg) batches of microplastic test materials derived from agricultural mulching films. Batches were produced from either polyethylene-based conventional mulching films or starch-polybutadiene adipate terephthalate blend mulching films that are certified biodegradable in soil. Challenges encountered and overcome during the micronisation process provide valuable insights into the future of microplastic test material generation from these material types. This includes difficulties in micronising virgin polyethylene film materials. All five batches were subjected to a thorough physical and chemical characterisation - both of the original virgin films and the subsequent microplastic particles generated - including a screening for the presence of chemical additives. This is a critical step to provide essential information for interpreting particle fate or effects in scientific testing. Trade-offs between obtaining preferred particle typologies and time and cost constraints are elucidated. Several recommendations emerging from the experiences gained in this study are put forward to advance the research field towards greater harmonisation and utilisation of environmentally relevant test materials

A microfluidic paper-based analytical device (mu PAD) with smartphone readout for chlorpyrifos-oxon screening in human serum

Acute intoxication incidents due to neurotoxic organophosphate (OP) insecticides are occasionally reported, related either to suicidal attempts or occupational exposure due to the misuse of protective equipment. Among them, chlorpyrifos is a compound related to great controversy, which is still authorized and easily accessible in many countries around the world. However, to screen for its exposure markers, instrumental methods are commonly applied, which cannot enable rapid monitoring at an early stage of an intoxication. Therefore, in this study, a microfluidic paper-based analytical device (mu PAD) able to rapidly screen for chlorpyrifos-oxon, the toxic chlorpyrifos metabolite, in human serum was developed and fully validated. The mu PAD combines wax-printed butyrylcholinesterase (BChE) paper sensors, a lab-on-a-chip (LOC) prototype injector and a smartphone as the analytical detector. In principle, the wax-printed strips with adsorbed BChE are embedded into LOC injectors able to deliver samples and reagents on-demand. A smartphone reader was used to monitor the color development on the strips providing binary qualitative results. mu PAD method performance characteristics were thoroughly evaluated in terms of specificity, detection capability (CC beta) and ruggedness. The developed analytical platform is rapid (results within 10 min), cost-efficient (0.70 (sic)), potentially applicable at the point-of-need and attained a low CC beta (10 mu g L-1 in human serum). Finally, mu PAD characteristics were critically compared to wellestablished methods, namely an in-house BChE microplate assay and liquid chromatography tandem mass spectrometry.Funding Agencies|European Unions Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grantEuropean Union (EU) [720325]; METROFOOD-CZ research infrastructure project (MEYS Grant) [LM2018100]; [A2_FPBT_2020_013]</p

Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food

Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC50 of 0.3 µg L-1 using a monoclonal antibody (Mab22F12) against BaP, similar to the IC50 of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography–mass spectrometry (GC–MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are under development

Multiplex Screening of Persistent Organic Pollutants in Fish Using Spectrally Encoded Microspheres

Persistent organic pollutants (POPs) are environmental and food-related contaminants of global public health concern and known to be carcinogenic and endocrine disruptors. Their monitoring is essential, and an easy-to-use, rapid, and affordable multianalyte screening method with simplified sample preparation can be a valuable tool prior to instrumental analysis. For this purpose, a flow cytometric immunoassay (FCIA), based on a spectrally encoded microbeads technology, was developed for the multiplex detection of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (BDEs) in buffer and fish extracts. The sensitivities of the assays in the three-plex FCIA format were similar to the individual FCIAs for the marker compounds benzo[a]pyrene (BaP), 3,3',4,4'-tetrachlorobiphenyl (PCB77), and 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in buffer with IC50 values of 0.4, 20, and 2 µg L–1, respectively. Apart from the three markers, we could detect at least 14 other POPs. Extracts of fish with different fat content, prepared with a simplified extraction and cleanup procedure, had an insignificant influence on the overall three-plex FCIA performance, with the exception of some impact on the PAHs detection. The performance of the three-plex FCIA, in combination with the simple extraction procedure, is adequate for regulatory control in accordance with the required limits

Analysis of chlormequat residue in milk by UPLC-MS/MS

Chlormequat is a plant growth regulator generally used to promote lateral branching and flowering of ornamental plants. It is also frequently used to improve yields for different types of cereals in Europe. These cereals are often processed into animal feed. Consequently chlormequat residues could potentially be found in matrices of animal origin. The MRL in milk were fixed at 0.05 mg/kg in annex I part B of the regulation 396/2005. Milk is a very complex matrix composed of different proteins and fat. Therefore, intensive clean-up is needed before injection to prevent interference or clogging during LC analysis. For the extraction, 4ml of water is added to 1g of milk just after the addition of chlormequat D4 as internal standard. The cleanup strategy selected was the use of dichloromethane andacetic acid to eliminate fat and precipitate protein, respectively. This step was followed by a solid phase extraction (SPE) of the aqueous phase on a Weak Cation Exchange cartridge. The chlormequat was eluted by using ammonium acetate, before injection on an UPLC-MS/MS system (Waters premier). Due to his ionic characteristic, no retention is observed on classical C18 column. The reverse phase polymethacrylate Shodex RSpak DE413 (150 × 4.6 mm) recommended in the CEN method prEN15054 has been selected. Isocratic condition (water/acetonitrile/ methanol/acetic acid - 71.25+17.5+7.5+3.75 V+V+V+V 50 mM ammonium acetate are used at a flow of 0.4 ml/min. The observed a retention time is 4.0 min. The analytical method has a limit of quantification of 0.025 mg/kg (limit of detection of 0.0125 mg/kg) and was validated following SANCO/12495/2011 criteria at the LOQ and at a higher level (10 times LOQ - 0.25 mg/kg). Results show a recovery of approximately 100% (between 100.6 and 103.0) with a repeatability and in-house reproducibility below 10%. These very good validation data are correlated to the robustness of the method and to the use of an isotopic surrogate.</p

Occurrence of perfluorinated alkylated substances in cereals, salt, sweets and fruit items collected in four European countries

In the context of a European project, 12 perfluoroalkyl acids (PFAAs) were determined in 14 food items collected in four European countries representing northern, southern, eastern and western Europe. This study presents the results of PFAAs measured in fruit, cereals, sweets and salt. Out of the 12 PFAAs, 10 PFAAs were detected in 67% of the samples. Overall, PFOA was the most abundant compound and the highest concentrations were found for PFOS but all were less than 1 ng g−1. When comparing the four countries, highest levels and detection frequencies were observed in Belgium (Western Europe), followed by the Czech Republic (Eastern Europe), Italy (Southern Europe) and finally Norway (Northern Europe). Comparison of profiles and levels is difficult due to variations in constitution of the food categories in the investigated countries and countries of origin of the food items. Dietary intake assessments for PFOS and PFOA show that the daily intake of PFAAs is far below the existing tolerable levels. However, they contribute to the total dietary intake and should therefore be included in future dietary exposure assessments

Effect of Polycyclic Aromatic Hydrocarbons Exposure on Cognitive Development in 5 Years Old Children

Objectives: To analyze the impact of polycyclic aromatic hydrocarbons (PAHs) in ambient air at the time of delivery and five years of age on cognitive development in five year old children. Materials and Methods: Two cohorts of children born in the years 2013 and 2014 from Karvina (Northern Moravia, n = 70) and Ceske Budejovice (Southern Bohemia, n = 99) were studied at the age of five years for their cognitive development related to the exposure to PAHs, determined in the ambient air as the concentration of benzo[a]pyrene (B[a]P) and OH&ndash;PAH (hydroxy-PAH) metabolites in urine of the newborns at the time of delivery. As psychological tests, the Bender Visual Motor Gestalt Test (BG test) and the Raven Colored Progressive Matrices (RCPM test) were used. Results: Concentrations of B[a]P in the third trimester of mother&rsquo;s pregnancies were 6.1 &plusmn; 4.53 ng/m3 in Karvina, and 1.19 &plusmn; 1.28 ng/m3 (p &lt; 0.001) in Ceske Budejovice. Neither the outcome of the RCPM test nor the BG test differed between children in Karvina vs. Ceske Budejovice, or boys vs. girls. Cognitive development in five year old children was affected by the higher exposure to PM2.5 during the third trimester in girls in Karvina. Conclusions: We did not observe any significant effect of prenatal PAH exposure on psychological cognitive tests in five year old children

Effect of household and industrial processing on levels of some pesticide residues in carrots

Pesticides are widely used in conventional agriculture to obtain a better yield for crops. They can cause toxic effects (headaches, cancer, reproductive harm and endocrine disruption). The main exposure to pesticides for humans is via food (especially by fruit and vegetables). Processing food can affect the level of pesticide residues and in some special case, more toxic by-products can be formed during processing. Different studies have been related to measure concentrations of pesticide residues after home or industrial processing. However, many processing factors (residues level in processed commodity/residue level in raw commodity) remain unknown and are necessary to estimate the level of pesticide exposure at the point of consumption after processing. To reach this objective, the combination carrots/pesticides (boscalid, chlorpyrifos, difenoconazole, dimethoate, linuron and tebuconazole) was chosen in collaboration with the Belgian Federal Agency for Safety of the Food Chain based on the number of non compliant samples, frequency and concentration level observed and on the toxicity of the pesticides. Treated carrots were grown in Belgium by Redebel sa. The amount of the pesticide residues observed in the raw material varied due to the different treatments, application times, weather conditions, the physico-chemical properties of each pesticide. Food processing (washing, peeling, blanching, microwave cooking, pasteurization and sterilization) was carried out on industrial pilot scale. Pesticides and the degradation products formed during processing were analyzed with GC–MS/MS and UHPLC–MS/MS. The washing step allowed decreasing the concentration of residues for all pesticides up to ~90%. The second process, peeling, results in a reduction comparable to washing. The blanching step, combining heat with a large quantity of water, enhanced the elimination of residues. Even residual concentrations were below 5 ppb, it was observed that microwave cooking did not reduce the level of residues while in-pack sterilization decreased most of the pesticide residues still present except difenoconazole. To conclude, most of the processing factors could be explained in terms of water solubility, the log-octanol-water-partitioning coefficients, the systemic properties of the pesticides studied and the agricultural practices.</p