Katherine Muñoz

Simultaneous analysis of ochratoxin A and its major metabolite ochratoxin alpha in plasma and urine for an advanced biomonitoring of the mycotoxin.

Ochratoxin A (OTA) is a frequent mycotoxin contaminant found worldwide in foods and feedstuffs. Biomonitoring has been used to assess internal OTA exposure resulting from dietary intake and from other sources. Mycotoxin levels in blood and/or urine provide good estimates of past and recent exposure since OTA binds to serum proteins and is also partly excreted via the kidney. But, measuring OTA alone does not reflect its biotransformation. In light of scarce data on its metabolites in humans, it was the aim of this study to develop a method that allows analysis of OTA and its detoxication product ochratoxin alpha (OTα) in urine and in blood plasma. The method involves enzymatic hydrolysis of conjugates, liquid-liquid extraction, and analysis of sample extracts by liquid chromatography with fluorescence detection. Application of the validated method in a pilot study with 13 volunteers revealed the presence of OTA and OTα in all samples (limit of quantification: 0.05 ng/mL in urine, and 0.1 ng/mL in plasma). In line with negative findings of others, an OTA glucuronide was not detected, neither in urine nor in plasma. By contrast, conjugates of OTα (glucuronide and/or sulfate) are major products in these samples. This was confirmed by mass spectrometry detection. As OTα represents a large fraction of ingested mycotoxin, we propose to include analyses of this metabolite in future biomonitoring studies, also in light of the observed variations for urine OTα-levels that suggest different interindividual abilities for OTA-detoxification in humans.

Plastic mulching in agriculture. Trading short-term agronomic benefits for long-term soil degradation?

Plastic mulching has become a globally applied agricultural practice for its instant economic benefits such as higher yields, earlier harvests, improved fruit quality and increased water-use efficiency. However, knowledge of the sustainability of plastic mulching remains vague in terms of both an environmental and agronomic perspective. This review critically discusses the current understanding of the environmental impact of plastic mulch use by linking knowledge of agricultural benefits and research on the life cycle of plastic mulches with direct and indirect implications for long-term soil quality and ecosystem services. Adverse effects may arise from plastic additives, enhanced pesticide runoff and plastic residues likely to fragment into microplastics but remaining chemically intact and accumulating in soil where they can successively sorb agrochemicals. The quantification of microplastics in soil remains challenging due to the lack of appropriate analytical techniques. The cost and effort of recovering and recycling used mulching films may offset the aforementioned benefits in the long term. However, comparative and long-term agronomic assessments have not yet been conducted. Furthermore, plastic mulches have the potential to alter soil quality by shifting the edaphic biocoenosis (e.g. towards mycotoxigenic fungi), accelerate C/N metabolism eventually depleting soil organic matter stocks, increase soil water repellency and favour the release of greenhouse gases. A substantial process understanding of the interactions between the soil microclimate, water supply and biological activity under plastic mulches is still lacking but required to estimate potential risks for long-term soil quality. Currently, farmers mostly base their decision to apply plastic mulches rather on expected short-term benefits than on the consideration of long-term consequences. Future interdisciplinary research should therefore gain a deeper understanding of the incentives for farmers and public perception from both a psychological and economic perspective in order to develop new support strategies for the transition into a more environment-friendly food production.

Ochratoxin A and its metabolites in urines of German adults—An assessment of variables in biomarker analysis

Ochratoxin A (OTA), a mycotoxin known for its nephrotoxic and carcinogenic properties, is a worldwide occurring contaminant in a variety of food commodities. Biomonitoring (i.e. analysis in biological fluids) can serve to assess human internal exposure from all consumed foods and beverages. We now determined the concentration of OTA and its metabolite ochratoxin alpha (OTα) in plasma and in urine of two male volunteers with different food habits, in order to assess intra-individual temporal fluctuations and inter-individual differences in their biomarker levels. Moreover, the urinary levels of both OTA and OTα were analyzed in a cohort of German adults (23 males, 27 females) on their regular diet. All samples were subjected to an enzymatic hydrolysis of biomarker conjugates prior to clean-up by liquid-liquid extraction and HPLC-FD analysis. The profile in the first individual showed small fluctuations over time: mean levels in plasma were 0.42 and 0.45ng/mL for OTA and OTα, respectively, and in urine means of 0.06ng/mL for both analytes. The other individual had mean levels of 1.64 and 0.20ng/mL for OTA and OTα in plasma, and 0.24 and 2.22ng/mL for these analytes in urine. It is concluded that inter-individual differences in biomarker levels reflect dissimilar dietary exposure and/or disposition of ingested mycotoxin, with an apparently more efficient detoxification of OTA to OTα in the second individual. In the German cohort (n=50), analytes were detected in 100% (OTA: range 0.02-1.82ng/mL mean level 0.21±0.31ng/mL) and 78% (OTα: range 0.01-14.25ng/mL, mean level 1.33±2.63ng/mL) of all urines. Parameters such as gender, age and body mass index did not show a significant association with urine biomarker levels. This study indicates frequent exposure to OTA among German adults. The new results are discussed in the context of biomarker data from other countries and some methodological issues.

Evidence of ochratoxin A conjugates in urine samples from infants and adults

Ochratoxin A (OTA), a mycotoxin with nephrotoxic and carcinogenic properties, is an important contaminant of food and feed. Analysis of OTA in human biological fluids (blood, urine, or breast milk) has documented frequent exposure to this mycotoxin, yet at quite variable levels in different population groups across the world. Urine is the preferred matrix in biomonitoring since sample collection is non-invasive and better accepted by study participants. As only a small fraction of the ingested OTA is excreted in urine, determination of urinary OTA requires sensitive analytical techniques, and phase-II-metabolites should be also considered as biomarkers of exposure. Yet, data published so far on the presence of OTA-glucuronide/sulfate in human urine have been contradictory. In this study, urines (n = 38) from two groups of breastfed infants (German and Turkish) and from German adults were now analysed for the presence of OTA glucuronides or sulfates by an indirect method, i.e. by comparing the levels of OTA (aglycone) in urines without and after enzymatic hydrolysis with ß-glucuronidase/arylsulfatase. Additionally, ochratoxin A-8-β-glucuronide and open lactone ochratoxin A-8-β-glucuronide were synthesized to serve as reference materials for metabolite analysis. Attempts for definitive confirmation of glucuronides of OTA via direct identification in LC–MS/MS analysis were hampered by the lower ionizability of the conjugates compared to the parent compound. Considerable increases in OTA levels were found after enzymatic hydrolysis in several (not all) urine samples and provide clear evidence for the excretion of OTA-conjugates. The latter observation is of importance, since OTA phase-II-metabolites may escape detection when direct methods are applied for urinary biomarker analysis. In conclusion, enzymatic hydrolysis of urine samples is highly advisable in order to avoid an underestimation of the OTA-exposure.

Does nutrient enrichment compensate fungicide effects on litter decomposition and decomposer communities in streams

Nutrient and pesticide pollution are widespread agricultural stressors. Fungicides may affect freshwater fungi, which play an important role in litter decomposition (LD), whereas moderate nutrient enrichment can stimulate LD. We examined potential interaction effects of nutrients and fungicides on decomposer communities and LD in a 14-day two-factorial (fungicide and nutrient treatments) mesocosm experiment. Fungicide exposure was limited to 4days to simulate episodic contamination. Only the microbial community responded significantly to the experimental factors, though non-significant increases >20% were found for invertebrate decomposer weight gain and LD under high-nutrient conditions. Fungal community structure responded more strongly to fungicides than sporulation. Sporulation responded strongest to nutrients. Bacterial community structure was affected by both factors, although only nutrients influenced bacterial density. Our results suggest effects from fungicides at field-relevant levels on the microbial community. Whether these changes propagate to invertebrate communities and LD remains unclear and should be analysed under longer and recurrent fungicide exposure.

Daily uptake of mycotoxins – TDI might not be protective for nursed infants

Exclusive breast feeding is recommended by international bodies for the first six months of life. Because of the presence of contaminants, breast feeding might lead to toxicologically relevant exposure of the nursed child. Exposure towards mycotoxins is of specific interest because of their widespread occurrence in food and of their toxicological profile. We calculated the relationship between maternal intake at the level of the existing TDIs and the exposure in the nursed infants of several mycotoxins to evaluate whether maternal exposure at the TDI is also safe for the nursed infant. If published information was not available we used in silico methods for estimating toxicokinetic parameters and the lactational transfer. A single dose and a continuous daily intake scenario were considered. Maternal intake at the TDI exceeds the age-adjusted TDI (TDI/3) values for infants in case of deoxynivalenol and patulin in the single dose scenario. Exceedance is particularly pronounced for ochratoxin A in the continuous daily intake scenario (29.2 fold above the child adjusted TDI). According to published data in infants impaired kidney function may result from this exceedance. When setting a TDI, the safety of the exclusively nursed infant should be considered in the continuous daily intake scenario.