Evaluation of the ecological risk of pesticide residues from the European LUCAS Soil monitoring 2018 survey.

The 2018 LUCAS (Land Use and Coverage Area frame Survey) Soil Pesticides survey provides a European Union (EU)-scale assessment of 118 pesticide residues in more than 3473 soil sites. This study responds to the policy need to develop risk-based indicators for pesticides in the environment. Two mixture risk indicators are presented for soil based, respectively, on the lowest and the median of available No Observed Effect Concentration (NOECsoil,min and NOECsoil,50) from publicly available toxicity datasets. Two further indicators were developed based on the corresponding equilibrium concentration in the aqueous phase and aquatic toxicity data, which are available as species sensitivity distributions. Pesticides were quantified in 74.5% of the sites. The mixture risk indicator based on the NOECsoil,min exceeds 1 in 14% of the sites and 0.1 in 23%. The insecticides imidacloprid and chlorpyrifos and the fungicide epoxiconazole are the largest contributors to the overall risk. At each site, one or a few substances drive mixture risk. Modes of actions most likely associated with mixture effects include modulation of acetylcholine metabolism (neonicotinoids and organophosphate substances) and sterol biosynthesis inhibition (triazole fungicides). Several pesticides driving the risk have been phased out since 2018. Following LUCAS surveys will determine the effectiveness of substance-specific risk management and the overall progress toward risk reduction targets established by EU and UN policies. Newly generated data and knowledge will stimulate needed future research on pesticides, soil health, and biodiversity protection. Integr Environ Assess Manag 2024;00:1-15. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Identification of phosphodiesterase type-5 (PDE-5) inhibitors in herbal supplements using a tiered approach and associated consumer risk.

The use of herbal supplements for improved sexual performance is a common practice amongst the youth and some senior citizens in Ghana. These products are considered 'natural' and greatly preferred over synthetic alternatives due to the assurance of little to no adverse effects by producers. However, the high rate of adulteration often compromises their safety. Forty herbal supplements, of which 25 were previously shown to result in medium to high intake of phosphodiesterase type-5 (PDE-5) inhibitors using a PDE-Glo bioassay, were further investigated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the reliability of the bioassay and whether the observed higher responses could be ascribed to inherent plant constituents or adulterants. Results showed significant amounts of vardenafil, tadalafil and especially sildenafil, in 2, 1 and 10 samples, respectively, with total concentration levels resulting in estimated daily intakes (EDIs) above 25 mg sildenafil equivalents with six supplements even having EDIs above 100 mg sildenafil equivalents. Only one sample contained a natural ingredient (icariin), but its concentration (0.013 mg g-1) was too low to explain the observed potency in the bioassay. The estimated concentrations of PDE-5 inhibitors in 35 supplements, according to the bioassay, were in line with those of the LC-MS/MS analysis. However, discrepancies were observed for five supplements. Further examination of one of the latter supplements using the PDE-Glo bioassay to select the positive fraction and further examination with LC-MS/MS and 1H-NMR revealed the presence of hydroxythiohomosildenafil, a sildenafil analogue not yet included in the liquid chromatography-mass spectrometry reference library. This study demonstrates the significance of applying a tiered approach, where the use of a bioassay is followed by chemical analysis of bioactive samples in order to identify unknown bioactive compounds.

Controlled Production of Zearalenone-Glucopyranoside Standards with Cunninghamella Strains Using Sulphate-Depleted Media.

In recent years, conjugated mycotoxins have gained increasing interest in food safety, as their hydrolysis in human and animal intestines leads to an increase in toxicity. For the production of zearalenone (ZEN) glycosides reference standards, we applied Cunninghamellaelegans and Cunninghamella echinulata fungal strains. A sulphate-depleted medium was designed for the preferred production of ZEN glycosides. Both Cunninghamella strains were able to produce zearalenone-14-ß-D-glucopyranoside (Z14G), zearalenone-16-ß-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S). In a rich medium, Cunninghamellaelegans preferably produced Z14S, while Cunninghamellaechinulata preferably produced Z14G. In the sulphate-depleted medium a dramatic change was observed for Cunninghamellaelegans, showing preferred production of Z14G and Z16G. From 2 mg of ZEN in sulphate-depleted medium, 1.94 mg of Z14G and 0.45 mg of Z16G were produced. Following preparative Liquid Chromatography-Mass Spectrometry (LC-MS) purification, both fractions were submitted to 1H and 13C NMR and High-Resolution Mass Spectrometry (HRMS). These analyses confirmed that the purified fractions were indeed Z14G and Z16G. In conclusion, the presented research shows that a single Cunninghamella strain can be an effective and efficient tool for the controlled biotransformation of ZEN glycosides and other ZEN metabolites. Additionally, the biotransformation method was extended to zearalanone, ß-zearalenol and other mycotoxins.

A health risk for consumers: the presence of adulterated food supplements in the Netherlands.

The use of food supplements is increasing. They are marketed as beneficial for health, well-being, physical or mental condition and performance, or to prevent diseases. Producers add synthetic compounds or illicit herbal material to food supplements to claim desired effects. Claims made to support marketing without scientific evidence are, however, illegal. Intake of adulterated food supplements may lead to serious adverse effects. The aim of this paper is to report the results of analyses of (adulterated) food supplements conducted by the Netherlands Food and Consumer Product Safety Authority between October 2013 and October 2018. In total, 416 supplements were analysed of which 264 (64%) contained one or more pharmacological active substances or plant toxins, such as caffeine, synephrine, sildenafil, icariin, sibutramine, higenamine, hordenine, phenethylamine, methylsynephrine, DMAA, phenolphthalein, octopamine and ephedrine. When compared to dose levels that are considered safe, daily doses of the substances in the food supplements were sometimes much higher, causing a risk for consumers who are unaware of the presence of these pharmacologically active substances. In many cases, neither food nor medicines legislation (easily) enables enforcement actions. This means that some products containing pharmacologically active substances (i.e. synthetic medicines and their illicit analogues), stay available on the market. An undesirable situation because for many of these substances no detailed toxicity data are available.

Tolerance and Excretion of the Mycotoxins Aflatoxin B1, Zearalenone, Deoxynivalenol, and Ochratoxin A by Alphitobius diaperinus and Hermetia illucens from Contaminated Substrates.

This study aimed to investigate the potential accumulation of mycotoxins in the lesser mealworm (Alphitobius diaperinus, LMW) and black soldier fly (Hermetia illucens, BSF) larvae. Feed was spiked with aflatoxin B1, deoxynivalenol (DON), ochratoxin A or zearalenone, and as a mixture of mycotoxins, to concentrations of 1, 10, and 25 times the maximum limits set by the European Commission for complete feed. This maximum limit is 0.02 mg/kg for aflatoxin B1, 5 mg/kg for DON, 0.5 mg/kg for zearalenone and 0.1 mg/kg for ochratoxin A. The mycotoxins and some of their metabolites were analysed in the larvae and residual material using a validated and accredited LC-MS/MS-based method. Metabolites considered were aflatoxicol, aflatoxin P1, aflatoxin Q1, and aflatoxin M1, 3-acetyl-DON, 15-acetyl-DON and DON-3-glycoside, and α- and ß-zearalenol. No differences were observed between larvae reared on mycotoxins individually or as a mixture with regards to both larvae development and mycotoxin accumulation/excretion. None of the mycotoxins accumulated in the larvae and were only detected in BSF larvae several orders of magnitude lower than the concentration in feed. Mass balance calculations showed that BSF and LMW larvae metabolized the four mycotoxins to different extents. Metabolites accounted for minimal amounts of the mass balance, except for zearalenone metabolites in the BSF treatments, which accounted for an average maximum of 86% of the overall mass balance. Both insect species showed to excrete or metabolize the four mycotoxins present in their feed. Hence, safe limits for these mycotoxins in substrates to be used for these two insect species possibly could be higher than for production animals. However, additional analytical and toxicological research to fully understand the safe limits of mycotoxins in insect feed, and thus the safety of the insects, is required.

Mycotoxin profiling of 1000 beer samples with a special focus on craft beer.

Currently beer is booming, mainly due to the steady rise of craft breweries worldwide. Previous surveys for occurrence of mycotoxins in beer, were mainly focussed on industrial produced beer. The present survey reports the presence of mycotoxins in craft beer and how this compares to industrial produced beer. More than 1000 beers were collected from 47 countries, of which 60% were craft beers. A selection of 1000 samples were screened for the presence of aflatoxin B1, ochratoxin A (OTA), zearalenone (ZEN), fumonisins (FBs), T-2 and HT-2 toxins (T-2 and HT-2) and deoxynivalenol (DON) using a mycotoxin 6-plex immunoassay. For confirmatory analysis, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and applied. The 6-plex screening showed discrepancies with the LC-MS/MS analysis, possibly due to matrix interference and/or the presence of unknown mycotoxin metabolites. The major mycotoxins detected were DON and its plant metabolite deoxynivalenol-3-ß-D-glucopyranoside (D3G). The 6-plex immunoassay reported the sum of DON and D3G (DON+D3G) contaminations ranging from 10 to 475 µg/L in 406 beers, of which 73% were craft beers. The popular craft beer style imperial stout, had the highest percentage of samples suspected positive (83%) with 29% of all imperial stout beers having DON+D3G contaminations above 100 µg/L. LC-MS/MS analysis showed that industrial pale lagers from Italy and Spain, predominantly contained FBs (3-69 µg/L). Besides FBs, African traditional beers also contained aflatoxins (0.1-1.2 µg/L). The presence of OTA, T-2, HT-2, ZEN, ß-zearalenol, 3/15-acetyl-DON, nivalenol and the conjugated mycotoxin zearalenone 14-sulfate were confirmed in some beers. This study shows that in 27 craft beers, DON+D3G concentrations occurred above (or at) the Tolerable Daily Intake (TDI). Exceeding the TDI, may have a health impact. A better control of brewing malts for craft beer, should be put in place to circumvent this potential problem.

Survey of moniliformin in wheat- and corn-based products using a straightforward analytical method.

A straightforward analytical method was developed and validated to determine the mycotoxin moniliformin in cereal-based foods. Moniliformin is extracted with water and quantified with liquid chromatography tandem mass spectrometry, and its presence confirmed with liquid chromatography-Orbitrap-high-resolution mass spectrometry. The method was validated for flour, bread, pasta and maize samples in terms of linearity, matrix effect, recovery, repeatability and limit of quantification. Quantification was conducted by matrix-matched calibration. Positive samples were confirmed by standard addition. Recovery ranged from 77 to 114% and repeatability from 1 to 14%. The limit of quantification, defined as the lowest concentration tested at which the validation criteria of recovery and repeatability were fulfilled, was 10 µg/kg. The method was applied to 102 cereal-based food samples collected in the Netherlands and Germany. Moniliformin was not detected in bread samples. One of 22 flour samples contained moniliformin at 10.6 µg/kg. Moniliformin occurred in seven out of 25 pasta samples at levels around 10 µg/kg. Moniliformin (MON) was present in eight out of 23 maize products at levels ranging from 12 to 207 µg/kg.

Persistence of glyphosate and aminomethylphosphonic acid in loess soil under different combinations of temperature, soil moisture and light/darkness.

The dissipation kinetics of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) were studied in loess soil, under biotic and abiotic conditions, as affected by temperature, soil moisture (SM) and light/darkness. Nonsterile and sterile soil samples were spiked with 16mgkg-1 of glyphosate, subjected to three SM contents (20% WHC, 60% WHC, saturation), and incubated for 30days at 5°C and 30°C, under dark and light regimes. Glyphosate and AMPA dissipation kinetics were fit to single-first-order (SFO) or first-order-multicompartment (FOMC) models, per treatment combination. AMPA kinetic model included both the formation and decline phases. Glyphosate dissipation kinetics followed SFO at 5°C, but FOMC at 30°C. AMPA followed SFO dissipation kinetics for all treatments. Glyphosate and AMPA dissipation occurred mostly by microbial activity. Abiotic processes played a negligible role for both compounds. Under biotic conditions, glyphosate dissipation and AMPA formation/dissipation were primarily affected by temperature, but also by SM. Light regimes didn't play a significant role. Glyphosate DT50 varied between 1.5 and 53.5days, while its DT90 varied between 8.0 and 280days, depending on the treatment. AMPA persisted longer in soil than glyphosate, with its DT50 at 30°C ranging between 26.4 and 44.5days, and its DT90 between 87.8 and 148days. The shortest DT50/DT90 values for both compounds occurred at 30°C and under optimal/saturated moisture conditions, while the largest occurred at 5°C and reaching drought stress conditions. Based on these results, we conclude that glyphosate and AMPA dissipate rapidly under warm and rainy climate conditions. However, repeated glyphosate applications in fallows or winter crops in countries where cold and dry winters normally occur could lead to on-site soil pollution, with consequent potential risks to the environment and human health. To our knowledge, this study is the first evaluating the combined effect of temperature, soil moisture and light/dark conditions on AMPA formation/dissipation kinetics and behaviour.

Dietary supplement for energy and reduced appetite containing the ß-agonist isopropyloctopamine leads to heart problems and hospitalisations.

In 2013 the Dutch authorities issued a warning against a dietary supplement that was linked to 11 reported adverse reactions, including heart problems and in one case even a cardiac arrest. In the UK a 20-year-old woman, said to have overdosed on this supplement, died. Since according to the label the product was a herbal mixture, initial LC-MS/MS analysis focused on the detection of plant toxins. Yohimbe alkaloids, which are not allowed to be present in herbal preparations according to Dutch legislation, were found at relatively high levels (400-900 mg kg(-1)). However, their presence did not explain the adverse health effects reported. Based on these effects the supplement was screened for the presence of a ß-agonist, using three different biosensor assays, i.e. the validated competitive radioligand ß2-adrenergic receptor binding assay, a validated ß-agonists ELISA and a newly developed multiplex microsphere (bead)-based ß-agonist assay with imaging detection (MAGPIX(®)). The high responses obtained in these three biosensors suggested strongly the presence of a ß-agonist. Inspection of the label indicated the presence of N-isopropyloctopamine. A pure standard of this compound was bought and shown to have a strong activity in the three biosensor assays. Analysis by LC-full-scan high-resolution MS confirmed the presence of this 'unknown known' ß3-agonist N-isopropyloctopamine, reported to lead to heart problems at high doses. A confirmatory quantitative analysis revealed that one dose of the preparation resulted in an intake of 40-60 mg, which is within the therapeutic range of this compound. The case shows the strength of combining bioassays with chemical analytical techniques for identification of illegal pharmacologically active substances in food supplements.

Decay characteristics and erosion-related transport of glyphosate in Chinese loess soil under field conditions.

The decay characteristics and erosion-related transport of glyphosate and aminomethylphosphonic acid (AMPA) were monitored for 35 d at different slope gradients and rates of application in plots with loess soil on the Loess Plateau, China. The initial glyphosate decayed rapidly (half-life of 3.5d) in the upper 2 cm of soil following a first-order rate of decay. AMPA content in the 0-2 cm soil layer correspondingly peaked 3d after glyphosate application and then gradually decreased. The residues of glyphosate and AMPA decreased significantly with soil depth (p<0.05) independently of the slope inclination and application rate. About 0.36% of the glyphosate initially applied was transported from plots after one erosive rain 2d after the application. Glyphosate and AMPA concentrations in runoff were low while the contents in the sediment were much higher than in the upper 2 cm of the soil. CAPSULE: Although the rate of glyphosate decay is rapid in Chinese loess soil, the risks of glyphosate and AMPA need to be taken into account especially in the area with highly erosive rainfall.

Short-term transport of glyphosate with erosion in Chinese loess soil--a flume experiment.

Repeated applications of glyphosate may contaminate the soil and water and threaten their quality both within the environmental system and beyond it through water erosion related processes and leaching. In this study, we focused on the transport of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) related to soil erosion at two slope gradients (10 and 20°), two rates of pesticide with a formulation of glyphosate (Roundup®) application (360 and 720 mg m(-2)), and a rain intensity of 1.0 mm min(-1) for 1 h on bare soil in hydraulic flumes. Runoff and erosion rate were significantly different within slope gradients (p<0.05) while suspended load concentration was relatively constant after 15 min of rainfall. The glyphosate and AMPA concentration in the runoff and suspended load gradually decreased. Significant power and exponent function relationship were observed between rainfall duration and the concentration of glyphosate and AMPA (p<0.01) in runoff and suspended load, respectively. Meanwhile, glyphosate and AMPA content in the eroded material depended more on the initial rate of application than on the slope gradients. The transport rate of glyphosate by runoff and suspended load was approximately 14% of the applied amount, and the chemicals were mainly transported in the suspended load. The glyphosate and AMPA content in the flume soil at the end of the experiment decreased significantly with depth (p<0.05), and approximately 72, 2, and 3% of the applied glyphosate (including AMPA) remained in the 0-2, 2-5, and 5-10 cm soil layers, respectively. The risk of contamination in deep soil and the groundwater was thus low, but 5% of the initial application did reach the 2-10 cm soil layer. The risk of contamination of surface water through runoff and sedimentation, however, can be considerable, especially in regions where rain-induced soil erosion is common.

Rapid detection of pesticides not amenable to multi-residue methods by flow injection-tandem mass spectrometry.

Flow injection combined with tandem mass spectrometry (MS/MS) was investigated for the rapid detection of highly polar pesticides that are not amenable to multi-residue methods because they do not partition into organic solvents and require dedicated chromatographic conditions. The pesticides included in this study were amitrole, chlormequat, cyromazine, daminozide, diquat, ethephon, fosetyl-Al, glufosinate, glyphosate and its metabolite aminomethylphosphonic acid, maleic hydrazide, mepiquat and paraquat. The composition of the flow-injection solvent was optimized to achieve maximum MS/MS sensitivity. Instrumental limits of detection varied between <0.05 and 1 pg. Fruit, vegetable, cereal, milk and kidney samples were extracted with water (1% formic acid in case of paraquat/diquat) and ten times diluted in either methanol/0.1% formic acid, methanol/0.1% ammonia or acetonitrile/0.1% ammonia, depending on the pesticide. The ion suppression observed depended strongly on both the matrix and the pesticide. This could be largely compensated for by matrix-matched calibration, but more accurate quantification was obtained by using isotopically labelled standards (commercially available for most of the pesticides studied). The method detection limits ranged from 0.02 mg/kg for chlormequat and mepiquat to 2 mg/kg for maleic hydrazide and were 0.05-0.2 mg/kg for most other pesticide/matrix combinations. This was sufficiently low to test compliance with EU maximum residue limits for many relevant pesticide/commodity combinations. The method substantially reduces the liquid chromatography-MS/MS capacity demand which for many laboratories is prohibitive for inclusion of these pesticides in their monitoring and surveillance programmes.

Development and validation of an LC-MS/MS method for the detection of phomopsin A in lupin and lupin-containing retail food samples from the Netherlands.

Phomopsins (PHO) are mycotoxins produced by the fungus Diaporthe toxica (also referred to as Phomopsis leptostromiformis). Lupin is the most important host crop for this fungus and PHO are suspected as cause of lupinosis, a deadly liver disease, in sheep. Lupin is currently in use to replace genetically modified soy in many food products available on the European market. However, a validated method for analysis of PHO is not available until now. In this work, a dilute-and-shoot LC-MS/MS-based method was developed for the quantitative determination and identification of phomopsin A (PHO-A) in lupin and lupin-containing food. The method involved extraction by a mixture of acetonitrile/water/acetic acid (80/20/1 v/v), dilution of the sample in water, and direct injection of the crude extract after centrifugation. The method was validated at 5 and 25 µg PHO-A kg(-1) product. The average recovery and RSD obtained were 79% and 9%, respectively. The LOQ (the lowest level for which adequate recovery and RSD were demonstrated) was 5 µg PHO-A kg(-1). Identification of PHO-A was based on retention time and two transitions (789 > 226 and 789 > 323). Using the average of solvent standards from the sequence as a reference, retention times were all within ± 0.03 min and ion ratios were within ± 12%, which is compliant with European Union requirements. The LOD (S/N = 3 for the least sensitive transition) was 1 µg PHO-A kg(-1) product. Forty-two samples of lupin and lupin-containing food products were collected in 2011-2012 from grocery stores and internet shops in the Netherlands and analysed. In none of the samples was PHO-A detected.

Development and validation of a confirmative LC-MS/MS method for the determination of ß-exotoxin thuringiensin in plant protection products and selected greenhouse crops.

Bacterial products based on Bacillus thuringiensis are registered in many countries as plant protection products (PPPs) and are widely used as insecticides and nematocides. However, certain B. thuringiensis strains produce harmful toxins and are therefore not allowed to be used as PPPs. The serotype B. thuringiensis thuringiensis produces the beta-exotoxin thuringiensin (ßeT) which is considered to be toxic for almost all forms of life including humans (WHO 1999). The use of a non-registered PPP based on B. thuringiensis thuringiensis called bitoxybacillin was established through the determination of ßeT. First, an analytical reference standard of ßeT was characterized by nuclear magnetic resonance, liquid chromatography-high-resolution mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, a confirmatory quantitative method for the determination of ßeT in PPPs and selected greenhouse crops based on LC-MS/MS was developed and validated. A limit of quantitation of 0.028 mg/kg was established, and average recoveries ranged from 85.6 % to 104.8 % with repeatability (RSDr) of 1.5-7.7 % and within-lab reproducibility (RSD(WLR)) of 17 %. The method was used for analysis of >100 samples. ßeT was found in leaves of ornamentals, but no evidence was found for use in edible crops.

Modification and re-validation of the ethyl acetate-based multi-residue method for pesticides in produce.

The ethyl acetate-based multi-residue method for determination of pesticide residues in produce has been modified for gas chromatographic (GC) analysis by implementation of dispersive solid-phase extraction (using primary-secondary amine and graphitized carbon black) and large-volume (20 muL) injection. The same extract, before clean-up and after a change of solvent, was also analyzed by liquid chromatography with tandem mass spectrometry (LC-MS-MS). All aspects related to sample preparation were re-assessed with regard to ease and speed of the analysis. The principle of the extraction procedure (solvent, salt) was not changed, to avoid the possibility invalidating data acquired over past decades. The modifications were made with techniques currently commonly applied in routine laboratories, GC-MS and LC-MS-MS, in mind. The modified method enables processing (from homogenization until final extracts for both GC and LC) of 30 samples per eight hours per person. Limits of quantification (LOQs) of 0.01 mg kg(-1) were achieved with both GC-MS (full-scan acquisition, 10 mg matrix equivalent injected) and LC-MS-MS (2 mg injected) for most of the pesticides. Validation data for 341 pesticides and degradation products are presented. A compilation of analytical quality-control data for pesticides routinely analyzed by GC-MS (135 compounds) and LC-MS-MS (136 compounds) in over 100 different matrices, obtained over a period of 15 months, are also presented and discussed. At the 0.05 mg kg(-1) level acceptable recoveries were obtained for 93% (GC-MS) and 92% (LC-MS-MS) of pesticide-matrix combinations.

Determination of polar organophosphorus pesticides in vegetables and fruits using liquid chromatography with tandem mass spectrometry: selection of extraction solvent.

A method based on liquid chromatography (LC)-mass spectrometry (MS)/MS was developed for sensitive determination of a number of less gas chromatography (GC)-amenable organophosphorus pesticides (OPs; acephate, methamidophos, monocrotophos, omethoate, oxydemeton-methyl and vamidothion) in cabbage and grapes. For extraction, several solvents were evaluated with respect to the possibility of direct injection, matrix-induced suppression or enhancement of response, and extraction efficiency. Overall, ethyl acetate was the most favourable solvent for extraction, although a solvent switch was required. For some pesticide/matrix combinations, reconstitution of the residue after evaporation required special attention. Extracts were analysed on a C18 column with polar endcapping. The pesticides were ionised using atmospheric pressure chemical ionisation on a tandem mass spectrometer in multiple reaction monitoring mode. The final method is straightforward and involves extraction with ethyl acetate and a solvent switch to 0.1% acetic acid/water without further cleanup. The method was validated at the 0.01 and 0.5 mg/kg level, for both cabbage and grapes. Recoveries were between 80 and 101% with R.S.D. < 11% (n = 5). The limit of quantification was 0.01 mg/kg and limits of detection were between 0.001 and 0.004 mg/kg.