Glass bead system to study mycotoxin production of Aspergillus spp. on corn and rice starches.

Mycotoxin production by aflatoxin B1 (AFB1) -producing Aspergillus flavus Zt41 and sterigmatocystin (ST) -hyperproducer Aspergillus creber 2663 mold strains on corn and rice starch, both of high purity and nearly identical amylose-amylopectin composition, as the only source of carbon, was studied. Scanning electron microscopy revealed average starch particle sizes of 4.54 ± 0.635 µm and 10.9 ± 2.78 µm, corresponding to surface area to volume ratios of 127 1/µm for rice starch and 0.49 1/µm for corn starch. Thus, a 2.5-fold difference in particle size correlated to a larger, 259-fold difference in surface area. To allow starch, a water-absorbing powder, to be used as a sole food source for Aspergillus strains, a special glass bead system was applied. AFB1 production of A. flavus Zt41 was determined to be 437.6 ± 128.4 ng/g and 90.0 ± 44.8 ng/g on rice and corn starch, respectively, while corresponding ST production levels by A. creber 2663 were 72.8 ± 10.0 µg/g and 26.8 ± 11.6 µg/g, indicating 3-fivefold higher mycotoxin levels on rice starch than on corn starch as sole carbon and energy sources. KEY POINTS: • A glass bead system ensuring the flow of air when studying powders was developed. • AFB1 and ST production of A. flavus and A. creber on rice and corn starches were studied. • 3-fivefold higher mycotoxin levels on rice starch than on corn starch were detected.

Ecotoxicological Evaluation of Safener and Antimicrobial Additives in Isoxaflutole-Based Herbicide Formulations.

The environmental load by isoxaflutole and its formulated herbicide products has increasingly become apparent because, after the ban of atrazine, isoxaflutole has become its replacement active ingredient (a.i.). Obtaining information regarding the fate of this a.i. in environmental matrices and its ecotoxicological effects on aquatic organisms is essential for the risk assessment of the herbicide. In this study, the effects of Merlin Flexx- and Merlin WG75 formulated isoxaflutole-based herbicide products and two selected additives (cyprosulfamide safener and 1,2-benzisothiazol-3(2H)-one antimicrobial agent) were investigated on Raphidocelis subcapitata in growth inhibition assays. In ecotoxicological tests, two conventional (optical density and chlorophyll-a content) and two induced fluorescence-based (Fv*/Fp: efficiency of the photosystem PSII and Rfd* changes in the observed ratio of fluorescence decrease) endpoints were determined by UV-spectrophotometer and by our FluoroMeter Module, respectively. Furthermore, dissipation of isoxaflutole alone and in its formulated products was examined by an HPLC-UV method. In ecotoxicological assays, the fluorescence-based Rfd* was observed as the most sensitive endpoint. In this study, the effects of the safener cyprosulfamide and the antimicrobial agent 1,2-benzisothiazol-3(2H)-one on R. subcapitata is firstly reported. The results indicated that the isoxaflutole-equivalent toxicity of the mixture of the isoxaflutole-safener-antimicrobial agent triggered lower toxicity (EC50 = 2.81 ± 0.22 mg/L) compared to the individual effect of the a.i. (EC50 = 0.02 ± 0.00 mg/L). The Merlin Flexx formulation (EC50 = 27.04 ± 1.41 mg/L) was found to be approximately 50-fold less toxic than Merlin WG75, which can be explained by the different chemical characteristics and quantity of additives in them. The additives influenced the dissipation of the a.i. in Z8 medium, as the DT50 value decreased by approximately 1.2- and 3.5-fold under light and dark conditions, respectively.

Hormesis, the Individual and Combined Phytotoxicity of the Components of Glyphosate-Based Formulations on Algal Growth and Photosynthetic Activity.

The occurrence of the market-leading glyphosate active ingredient in surface waters is a globally observed phenomenon. Although co-formulants in pesticide formulations were considered inactive components from the aspects of the required main biological effect of the pesticide, several studies have proven the high individual toxicity of formulating agents, as well as the enhanced combined toxicity of the active ingredients and other components. Since the majority of active ingredients are present in the form of chemical mixtures in our environment, the possible combined toxicity between active ingredients and co-formulants is particularly important. To assess the individual and combined phytotoxicity of the components, glyphosate was tested in the form of pure active ingredient (glyphosate isopropylammonium salt) and herbicide formulations (Roundup Classic and Medallon Premium) formulated with a mixture of polyethoxylated tallow amines (POEA) or alkyl polyglucosides (APG), respectively. The order of acute toxicity was as follows for Roundup Classic: glyphosate < herbicide formulation < POEA. However, the following order was demonstrated for Medallon Premium: herbicide formulation < glyphosate < APG. Increased photosynthetic activity was detected after the exposure to the formulation (1.5-5.8 mg glyphosate/L and 0.5-2.2 mg POEA/L) and its components individually (glyphosate: 13-27.2 mg/L, POEA: 0.6-4.8 mg/L), which indicates hormetic effects. However, decreased photosynthetic activity was detected at higher concentrations of POEA (19.2 mg/L) and Roundup Classic (11.6-50.6 mg glyphosate/L). Differences were demonstrated in the sensitivity of the selected algae species and, in addition to the individual and combined toxicity of the components presented in the glyphosate-based herbicides. Both of the observed inhibitory and stimulating effects can adversely affect the aquatic ecosystems and water quality of surface waters.

Current Trends in Mycotoxin Detection with Various Types of Biosensors.

One of the most important tasks in food safety is to properly manage the investigation of mycotoxin contamination in agricultural products and foods made from them, as well as to prevent its occurrence. Monitoring requires a wide range of analytical methods, from expensive analytical procedures with high-tech instrumentation to significantly cheaper biosensor developments or even single-use assays suitable for on-site monitoring. This review provides a summary of the development directions over approximately a decade and a half, grouped according to the biologically sensitive components used. We provide an overview of the use of antibodies, molecularly imprinted polymers, and aptamers, as well as the diversity of biosensors and their applications within the food industry. We also mention the possibility of determining multiple toxins side by side, which would significantly reduce the time required for the analyses.

Investigating the impacts of heavy metal(loid)s on ecology and human health in the lower basin of Hungary's Danube River: A Python and Monte Carlo simulation-based study.

This study aimed to determine the environmental and health risks of the heavy metal levels in the Danube River in Hungary. The metals, including Fe, Mn, Zn, Cu, Ni, Cr, Pb, and As, were measured in the period from 2013 to 2019. The Spearman correlation and heatmap cluster analysis were utilized to determine the origin of pollution and the factors that control surface water quality. Several indices, such as the heavy metal pollution index (HPI), metal index (MI), hazard quotient oral and dermal (HQ), hazard index oral and dermal (HI), and carcinogenic risk (CR), were conducted to evaluate the potential risks for the environment and human health. The values of the HPI were between the range of 15 < HPI < 30, which indicated moderate pollution; however, the MI results showed high pollution in Dunaföldvár and Hercegszántó cities. The ecological risk (RI < 30) and HI values (< 1) showed low environmental risks and non-carcinogenic impacts of the existing metals, either on adults or children. The mean CR value of oral arsenic was 2.2E-04 and 2.5E-04 during April-September and October-March, respectively, indicating that children were the most vulnerable to arsenic-carcinogenic oral effects. While lead's CR oral values for children during April-September exceeded the threshold of 1.0E-04, chromium's oral and dermal CR values for both adults and children were 2.08E-04, 6.11E-04, 1.97E-04, and 5.82E-04 during April-September and October-March, respectively. These results demonstrate the potential carcinogenic risks related to chromium exposure within the two pathways in Hungary and highlight the need for effective measures to mitigate these risks.

Application of Highly Sensitive Immunosensor Based on Optical Waveguide Light-Mode Spectroscopy (OWLS) Technique for the Detection of the Herbicide Active Ingredient Glyphosate.

The herbicide active ingredient glyphosate is the most widely applied herbicidal substance worldwide. Currently it is the market-leading pesticide, and its use is projected to further grow 4.5-fold between 2022 and 2029. Today, glyphosate use exceeds one megaton per year worldwide, which represents a serious environmental burden. A factor in the overall boost in the global use of glyphosate has been the spread of glyphosate-tolerant genetically modified (GM) crops that allow post-emergence applications of the herbicide on these transgenic crops. In turn, cultivation of glyphosate-tolerant GM crops represented 56% of the glyphosate use in 2019. Due to its extremely high application rate, xenobiotic behaviour and a water solubility (11.6 mg/mL at 25 °C) unusually high among pesticide active ingredients, glyphosate has become a ubiquitous water pollutant and a primary drinking water contaminant worldwide, presenting a threat to water quality. The goal of our research was to develop a rapid and sensitive method for detecting this herbicide active ingredient. For this purpose, we applied the novel analytical biosensor technique optical waveguide light-mode spectroscopy (OWLS) to the label-free detection of glyphosate in a competitive immunoassay format using glyphosate-specific polyclonal antibodies. After immobilising the antigen conjugate in the form of a glyphosate conjugated to human serum albumin for indirect measurement, the sensor chip was used in a flow-injection analyser system. For the measurements, an antibody stock solution was diluted to 2.5 µg/mL. During the measurement, standard solutions were mixed with the appropriate concentration of antibodies and incubated for 1 min before injection. The linear detection range and the EC50 value of the competitive detection method were between 0.01 and 100 ng/mL and 0.60 ng/mL, respectively. After investigating the indirect method, we tested the cross-reactivity of the antibody with glyphosate and structurally related compounds.

Application of Electrochemical Biosensors for Determination of Food Spoilage.

Food security is significantly affected by the mass production of agricultural produce and goods, the growing number of imported foods, and new eating and consumption habits. These changed circumstances bring food safety issues arising from food spoilage to the fore, making food safety control essential. Simple and fast screening methods have been developed to detect pathogens and biomarkers indicating the freshness of food for safety. In addition to the traditional, sequential, chemical analytical and microbiological methods, fast, highly sensitive, automated methods suitable for serial tests have appeared. At the same time, biosensor research is also developing dynamically worldwide, both in terms of the analytes to be determined and the technical toolkit. Consequently, the rapid development of biosensors, including electrochemical-based biosensors, has led to significant advantages in the quantitative detection and screening of food contaminants. These techniques show great specificity for the biomarkers tested and provide adequate analytical accuracy even in complex food matrices. In our review article, we summarize, in separate chapters, the electrochemical biosensors developed for the most important food groups and the food safety issues they can ensure, with particular respect to meat and fish products, milk and dairy products, as well as alcoholic and non-alcoholic beverages.

Development of an Immunofluorescent Capillary Sensor for the Detection of Zearalenone Mycotoxin.

A capillary-based immunofluorescence sensor was developed and incorporated in a flow injection analysis system. The light-guiding capillary was illuminated axially by a 473 nm/5 mW solid state laser through a tailored optofluidic connector. High sensitivity of the system was achieved by efficiently collecting and detecting the non-guided fluorescence signal scattered out along the wall of the capillary. The excitation was highly suppressed with bandpass and dichroic filters by simultaneously exploiting the guiding effect inside the capillary. The glass capillary used as a measuring cell was silanized in liquid phase by 3-aminopropyltriethoxysilane (APTS), and the biomolecules were immobilized using glutaraldehyde inside the capillary. The applicability of the developed system was tested with a bovine serum albumin (BSA)-anti-BSA-IgG model-molecule pair, using a fluorescently labeled secondary antibody. Based on the results of the BSA-anti-BSA experiments, a similar setup using a primary antibody specific for zearalenone (ZON) was established, and a competitive fluorescence measurement system was developed for quantitative determination of ZON. For the measurements, 20 µg/mL ZON-BSA conjugate was immobilized in the capillary, and a 1:2500 dilution of the primary antibody stock solution and a 2 µg/mL secondary antibody solution were set. The developed capillary-based immunosensor allowed a limit of detection (LOD) of 0.003 ng/mL and a limit of quantification (LOQ) of 0.007 ng/mL for ZON in the competitive immunosensor setup, with a dynamic detection range of 0.01-10 ng/mL ZON concentrations.

Comparative Assessment of the Inhibitory Potential of the Herbicide Glyphosate and Its Structural Analogs on RGD-Specific Integrins Using Enzyme-Linked Immunosorbent Assays.

Transmembrane glycoprotein integrins play crucial roles in biochemical processes, and by their inhibition or activation, different signal pathways can be disrupted, leading to abnormal physiological functions. We have previously demonstrated the inhibitory effect of glyphosate herbicide's active ingredient on cell adhesion and its αvß3 integrin antagonist effect. Therefore, it appeared particularly exciting to investigate inhibition of glyphosate and its metabolites on a wider range of Arg-Gly-Asp (RGD) binding integrins, namely αvß3, α5ß1 and αllbß3. Thus, the purpose of this study was to assess how extended the inhibitory effect observed for glyphosate on the integrin αvß3 is in terms of other RGD integrins and other structurally or metabolically related derivatives of glyphosate. Five different experimental setups using enzyme-linked immunosorbent assays were applied: (i) αvß3 binding to a synthetic polymer containing RGD; (ii) αvß3 binding to its extracellular matrix (ECM) protein, vitronectin; (iii) α5ß1 binding to the above polymer containing RGD; (iv) αllbß3 binding to its ECM protein, fibrinogen and (v) αvß3 binding to the SARS-CoV-2 spike protein receptor binding domain. Total inhibition of αvß3 binding to RGD was detected for glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), as well as for acetylglycine on α5ß1 binding to RGD.

Utilization of a Novel Immunofluorescence Instrument Prototype for the Determination of the Herbicide Glyphosate.

An enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the quantitative analytical determination of the herbicide active ingredient glyphosate in environmental matrices (surface water, soil, and plant tissues). Glyphosate, as a ubiquitous agricultural pollutant, is a xenobiotic substance with exposure in aquatic and terrestrial ecosystems due its extremely high worldwide application rate. The immunoassay developed in Project Aquafluosense is part of a fluorescence-based instrumentation setup for the in situ determination of several characteristic water quality parameters. The 96-well microplate-based competitive immunoassay method applies fluorescence signal detection in the concentration range of 0-100 ng/mL glyphosate. Application of the fluorescent signal provides a limit of detection of 0.09 ng/mL, which is 2.5-fold lower than that obtained with a visual absorbance signal. Beside the improved limit of detection, determination by fluorescence provided a wider and steeper dynamic range for glyphosate detection. No matrix effect appeared for the undiluted surface water samples, while plant tissues and soil samples required dilution rates of 1:10 and 1:100, respectively. No cross-reaction was determined with the main metabolite of glyphosate, N-aminomethylphosphonic acid, and related compounds.

Cytotoxic effects of Roundup Classic and its components on NE-4C and MC3T3-E1 cell lines determined by biochemical and flow cytometric assays.

Cytotoxic effects of the market leading broad-spectrum, synthetic herbicide product Roundup Classic, its active ingredient glyphosate (in a form of its isopropylamine (IPA) salt) and its formulating surfactant polyethoxylated tallowamine (POE-15) were determined on two murine cell lines, a neuroectodermal stem cell-like (NE-4C) and a high alkaline phosphatase activity osteoblastic cell line (MC3T3-E1). Cytotoxicity, genotoxicity, effects on cell viability and cell cycles were examined in five flow cytometry tests, the two former of which were compared by the enzymatic-assay and the alkaline single cell gel electrophoresis (Comet) assay. All of the tests indicated the NE-4C cells being more sensitive, than the MC3T3-E1 cell line to the treatments with the target compounds. Higher sensitivity differences were detected in the viability test by flow cytometry (7-9-fold), than by the MTT assay (1.5-3-fold); in the genotoxicity test by the Comet assay (3.5-403-fold), than by the DNA-damage test (9.3-158-fold); and in the apoptosis test by the Annexin V dead cell kit (1.1-12.7-fold), than by the Caspase 3/7 kit (1-6.5-fold). Cell cycle assays indicated high count of cells (~70%) in the G0/G1 phase for MC3T3-E1 cells, than in NE-4C cell (~40%) after 24 h. The order of the inhibitory potency of the target substances has unequivocally been POE-15 > Roundup Classic > > glyphosate IPA salt.

Potential Risk of Pollen from Genetically Modified MON 810 Maize Containing Cry1Ab Toxin to Protected Lepidopteran Larvae in the Pannonian Biogeographical Region-A Retrospective View.

A credible risk analysis of maize pollen containing Cry1Ab toxin must include the assessment of (i) pollen production and its Cry1 toxin content; (ii) distribution of the pollen grains in the surroundings; (iii) pollen-catching capacity of the weeds on field edges; (iv) the lifestyle of protected lepidopteran larvae living on weeds; (v) Cry1 toxin sensitivity of non-target caterpillars; and (vi) Cry1 toxin resistance of individual non-target populations. The concentration range of 5-4300 ng Cry1Ab toxin/g dry pollen determined in MON 810 pollen batches is too diverse for handling it as a single set in any mathematical modeling. Within the work carried out mainly with the DK-440 BTY cultivar, the seed samples officially received from the variety owner produced significantly different (250-470 vs. 5-15 ng/g) Cry1Ab toxin concentrations in the pollen. Nymphalis io L1-L3 larvae were nearly six times more sensitive for Dipel than Nymphalis c-album. Feeding on the back side and in a leaf nest, Vanessa atalanta may be subject to lower pollen exposures. N. io larvae may actively attempt to avoid patches with high pollen contamination. Cry1Ab toxin resistance also partially emerged in N. io populations reared in the Pannonian Biogeographical Region (Hungary).

Herbivorous Juvenile Grass Carp (Ctenopharyngodon idella) Fed with Genetically Modified MON 810 and DAS-59122 Maize Varieties Containing Cry Toxins: Intestinal Histological, Developmental, and Immunological Investigations.

Feeding experiments with juvenile grass carp (Ctenopharyngodon idella) fed with genetically modified maize MON 810 or DAS-59122 dried leaf biomass were carried out with 1-, 3- and 6-month exposures. Dosages of 3-7 µg/fish/day Cry1Ab or 18-55 µg/fish/day Cry34Ab1 toxin did not cause mortality. No difference occurred in body or abdominal sac weights. No differences appeared in levels of inorganic phosphate, calcium, fructosamine, bile acids, triglycerides, cholesterol, and alanine and aspartame aminotransferases. DAS-59122 did not alter blood parameters tested after 3 months of feeding. MON 810 slightly decreased serum albumin levels compared to the control, only in one group. Tapeworm (Bothriocephalus acheilognathi) infection changed the levels of inorganic phosphate and calcium. Cry34Ab1 toxin appeared in blood (12.6 ± 1.9 ng/mL), but not in the muscle. It was detected in B. acheilognathi. Cry1Ab was hardly detectable in certain samples near the limit of detection. Degradation of Cry toxins was extremely quick in the fish gastrointestinal tract. After 6 months of feeding, only mild indications in certain serum parameters were observed: MON 810 slightly increased the level of apoptotic cells in the blood and reduced the number of thrombocytes in one group; DAS-59122 mildly increased the number of granulocytes compared to the near-isogenic line.

Physiological and metabolic alterations induced by commercial neonicotinoid formulations in Daphnia magna.

Neonicotinoid insecticides are widely used agents in agriculture to control a broad range of insect pests. Although use of neonicotinoid pesticides has resulted in the widespread contamination of surface waters, sublethal toxicity data of these products in relation to non-target aquatic biota are still poor. Therefore, the objective of this study was to assess the effects of two neonicotinoid pesticides with widespread use on the basic physiological functions: the thoracic limb activity and heart rate of Daphnia magna, and to screen for their potential to affect the cytochrome P450 monooxygenase system (ECOD activity) of daphnids. The considered pesticides were the acetamiprid- and thiacloprid based products Mospilan 20 SG and Calypso 480 SC, respectively. The dose-dependent variation in the three biological endpoints considered were assessed following 24 h exposures. The two neonicotinoid formulations elicited significant depression on the thoracic limb activity and heart rate of daphnids at doses close to the immobility thresholds of formulations (48h-EC50: Mospilan 20 SG = 190 mg L-1; Calypso 480 SC = 120 mg L-1), an effect mainly attributable to the overall drop in the general health status of the organisms. The alterations in the physiological traits were significant at exposures to 190 mg L-1 for Mospilan 20 SG and 48 mg L-1 for Calypso 480 SC. The dose related variation in the ECOD activity of daphnids exposed to the selected neonicotinoid formulations followed a biphasic pattern, with starting effective doses for Mospilan 20 SG of 6.3 mg L-1 (=1/20 of 48h-EC50 for Daphnia neonates), and for Calypso 480 SC of 0.034 mg L-1 (=1/4000 of 48h-EC50). Maximal ECOD activity (2.2 fold increase vs. controls) was induced by Mospilan 20 SG in daphnids exposed to 114 mg L-1 product (=48 h-EC20), and by Calypso 480 SC (1.8 fold increase) at 5.2 mg L-1 dose (=1/20 of 48 h-EC50). Our results outlined significant alterations in the physiological traits and ECOD activity in exposed daphnids at concentrations below the immobility thresholds (48 h-EC50) of the products used as benchmarks to rate their toxicity risks to aquatic biota. Therefore, we think our findings might deserve consideration in the environmental risk evaluation of these products.

Analysis of Fuel Alternative Products Obtained by the Pyrolysis of Diverse Types of Plastic Materials Isolated from a Dumpsite Origin in Pakistan.

The current energy crisis and waste management problems have compelled people to find alternatives to conventional non-renewable fuels and utilize waste to recover energy. Pyrolysis of plastics, which make up a considerable portion of municipal and industrial waste, has emerged as a feasible resolution to both satisfy our energy needs and mitigate the issue of plastic waste. This study was therefore conducted to find a solution for plastic waste management problems, as well as to find an alternative to mitigate the current energy crisis. Pyrolysis of five of the most commonly used plastics, polyethylene terephthalate (PET), high- and low-density polyethylene (HDPE, LDPE), polypropylene (PP), and polystyrene (PS), was executed in a pyrolytic reactor designed utilizing a cylindrical shaped stainless steel container with pressure and temperature gauges and a condenser to cool down the hydrocarbons produced. The liquid products collected were highly flammable and their chemical properties revealed them as fuel alternatives. Among them, the highest yield of fuel conversion (82%) was observed for HDPE followed by PP, PS, LDPE, PS, and PET (61.8%, 58.0%, 50.0%, and 11.0%, respectively). The calorific values of the products, 46.2, 46.2, 45.9, 42.8 and 42.4 MJ/kg for LPDE, PP, HPDE, PS, and PET, respectively, were comparable to those of diesel and gasoline. Spectroscopic and chromatographic analysis proved the presence of alkanes and alkenes with carbon number ranges of C9-C15, C9-C24, C10-C21, C10-C28, and C9-C17 for PP, PET, HDPE, LDPE, and PS, respectively. If implemented, the study will prove to be beneficial and contribute to mitigating the major energy and environmental issues of developing countries, as well as enhance entrepreneurship opportunities by replicating the process at small-scale and industrial levels.

Mycotoxins as Emerging Contaminants. Introduction to the Special Issue "Rapid Detection of Mycotoxin Contamination".

Concerns for human and environmental health regarding mycotoxins are predominantly raised in connection with their occurrence in food and feed (especially in grains) [...].

Biosensors for Deoxynivalenol and Zearalenone Determination in Feed Quality Control.

Mycotoxin contamination of cereals used for feed can cause intoxication, especially in farm animals; therefore, efficient analytical tools for the qualitative and quantitative analysis of toxic fungal metabolites in feed are required. Current trends in food/feed analysis are focusing on the application of biosensor technologies that offer fast and highly selective and sensitive detection with minimal sample treatment and reagents required. The article presents an overview of the recent progress of the development of biosensors for deoxynivalenol and zearalenone determination in cereals and feed. Novel biosensitive materials and highly sensitive detection methods applied for the sensors and the application of these sensors to food/feed products, the limit, and the time of detection are discussed.