Low Levels Of Toxins Research Articles

Parameters Optimization for Improving Bioluminescence Inhibition Assay Using Vibrio fischeri Bacteria to Detect Lipopolysaccharide Toxicity in Aquatic Environments.

Bioluminescence inhibition of Vibrio fischeri is a widely used method for toxicity testing in aquatic environments. Certain complex biological contaminants, such as lipopolysaccharide (LPS), can interfere with metabolic pathways during toxicity assays. The standard 15-minute Vibrio fischeri bioluminescence assay has limitations when evaluating and screening water toxicity against complex and emerging chemicals like LPS. To accurately determine the effects of such substances, it is crucial to use a bioassay that encompasses a sufficient cell cycle period. This study tested LPS at varying incubation times (ranging from 60s to 60min) and concentrations (1-1*10- 12 mg/ml) to identify the appropriate incubation time for bioluminescence inhibition and toxicity testing. The results indicated that bioluminescence inhibition begins within 60s and reaches maximum inhibition at 60min. However, at 30 and 45min, the bacterial response to different concentrations of LPS varied, with some concentrations causing increased bioluminescence. The EC50 values at different times (60s, 15, 30, 45, and 60min) were found to be 0.0012, 0.0063, 4.07e + 54, 3.85e-8, and 3.34e-9mg/ml respectively. This study highlights the importance of considering incubation time when using bioluminescence inhibition to detect acute toxicity in aquatic ecosystems. A longer incubation time may enhance the method's sensitivity and improve its ability to detect low levels of toxins, such as LPS, in water resources.

Reliability of the chemical, metabolic, and molecular methods in discriminating aflatoxigenic from non-aflatoxigenic Aspergillus isolates

Aflatoxins, potent carcinogens produced by Aspergillus species (A. flavus and A. parasiticus), are primarily consumed through dietary sources. Aflatoxin management using non-aflatoxigenic Aspergillus to outcompete their aflatoxigenic congeneric to achieve low residual toxin levels is a promising biological control (BC) field intervention. A prerequisite for the BC program is the accurate delineation of aflatoxigenic fungi from their non-aflatoxigenic relatives. In this study, we use three methods: Chemical (Dichlorvos-ammonia), Metabolic (ELISA coupled live cultures), and Molecular (10 aflatoxin biosynthetic genes) to determine the aflatoxigenicity of Aspergillus spp. previously collected from Eastern Kenya, an aflatoxin hotspot. Thirty-six different Aspergillus isolates were identified and studied microscopically from a total of 314 fungal cultures. The chemical method reported 21 aflatoxigenic and 15 non-aflatoxigenic isolates, while the metabolic technique distinguished 20 aflatoxigenic and 16 non-aflatoxigenic isolates. A large proportion (97.22 %) of the isolates harboured the aflR, aflO, and aflM genes, while 91.67 % of the isolates possessed the aflS gene. All isolates (100 %) harboured the aflD, aflQ, aflP, and aflK genes. ELISA-coupled live cultures positively correlated with Dichlorvos-ammonia (p > 0.01), only differing in one sample, indicating a strong concurrence between the two methods at segregating toxigenic A. flavus isolates. Cluster analysis based on Multiple Correspondence revealed that the non-aflatoxigenic group exhibited similar characteristics correlating with the absence of aflR, aflS, aflI, and aflO genes. These findings indicate the reliability of the Chemical and Metabolic methods and can function as confirmatory assays to complement the molecular detection approach in determining aflatoxigenicity among Aspergillus spp.

First Results on the Presence of Mycotoxins in the Liver of Pregnant Fallow Deer (Dama dama) Hinds and Fetuses.

Reproductive abnormalities have been observed in fallow deer populations in Hungary. We supposed mycotoxin contamination to be one of the possible causes because multi-mycotoxin contamination is known to be dangerous even at low toxin levels, especially for young animals. We investigated the spatial pattern of mycotoxin occurrences and the relationship between maternal and fetal mycotoxin levels. A total of 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. We analyzed Aflatoxin (AF), Zearalenone (ZEA), Fumonizin B1 (FB1), DON, and T2-toxin concentrations in maternal and fetal livers by ELISA. AF was present in 70% and 82%, ZEA in 41% and 96%, DON in 90% and 98%, T2-toxin in 96% and 85%, and FB1 in 84% and 3% of hind and fetus livers, respectively. All mycotoxins passed into the fetus, but only Fumonizin B1 rarely passed. The individual variability of mycotoxin levels was extremely high, but the spatial differences were moderate. We could not prove a relation between the maternal and fetal mycotoxin concentrations, but we found an accumulation of ZEA and DON in the fetuses. These results reflect the possible threats of mycotoxins to the population dynamics and reproduction of wild fallow deer.

Yessotoxins in Mollusks of the Galician Coast from 2014 to 2022: Variability, Biotransformation, and Resistance to Alkaline Hydrolysis.

The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg-1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July-August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed.

Survey of the enniatins and beauvericin in raw and UHT cow's milk in Poland.

The enniatins A, A1, B and B1 (ENNs) and beauvericin (BEA) are structurally related compounds produced by Fusarium species. They occur as contaminants in cereals, such as wheat, barley and maize. They are called "emerging mycotoxins", because they have been reported in feed and food and their toxic effects are not fully known. Data on their levels in food (especially in milk) are limited. The study aimed to evaluate the occurrence of ENNs and BEA in milk. A total of 103 bovine milk samples (76 of raw milk and 27 of UHT milk) were collected from different parts of Poland and analysed using liquid chromatography-tandem mass spectrometry. Among the 76 raw milk samples, 31 (41%) and 15 (20%) samples were contaminated with ENN B and with BEA, respectively. No contamination with other enniatins was found. The highest concentration of BEA was found in raw milk and was 6.17 μg kg-1. Out of the 27 samples of UHT milk, 16 (59%) were contaminated with ENN B at concentrations ranging from 0.157 μg kg-1 to 0.587 μg kg-1 (limit of quantification (LOQ) 0.098 μg kg-1). Beauvericin was detected in 9 UHT milk samples (33%) at concentrations ranging from 0.101 μg kg-1 to 1.934 μg kg-1 (LOQ 0.095 μg kg-1). This study demonstrated constant but low milk contamination in Poland with ENN B and BEA. The analysis of milk samples revealed that the emerging mycotoxins ENN B and BEA were measured in trace amounts. It does not suggest any immediate risk to milk consumers; however, it is unknown whether long-term exposure to low levels of toxins may be harmful.

A Predictive Model for the Bioaccumulation of Okadaic Acid in Mytilus galloprovincialis Farmed in the Northern Adriatic Sea: A Tool to Reduce Product Losses and Improve Mussel Farming Sustainability

Over the last decades, harmful dinoflagellate (Dinophysis spp.) blooms have increased in frequency, duration, and severity in the Mediterranean Sea. Farmed bivalves, by ingesting large amounts of phytoplankton, can become unsafe for human consumption due to the bioaccumulation of okadaic acid (OA), causing Diarrhetic Shellfish Poisoning (DSP). Whenever the OA concentration in shellfish farmed in a specific area exceeds the established legal limit (160 μg·kg−1 of OA equivalents), harvesting activities are compulsorily suspended. This study aimed at developing a machine learning (ML) predictive model for OA bioaccumulation in Mediterranean mussels (Mytilus galloprovincialis) farmed in the coastal area off the Po River Delta (Veneto, Italy), based on oceanographic data measured through remote sensing and data deriving from the monitoring activities performed by official veterinarian authorities to verify the bioaccumulation of OA in the shellfish production sites. LightGBM was used as an ML algorithm. The results of the classification algorithm on the test set showed an accuracy of 82%. Further analyses showed that false negatives were mainly associated with relatively low levels of toxins (<100 μg·kg−1), since the algorithm tended to classify low concentrations of OA as negative samples, while true positives had higher mean values of toxins (139 μg·kg−1). The results of the model could be used to build up an online early warning system made available to shellfish farmers of the study area, aimed at increasing the economic and environmental sustainability of these production activities and reducing the risk of massive product losses.

The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin

After binding to the surface of a target cell, cholera toxin (CT) moves to the endoplasmic reticulum (ER) by retrograde transport. In the ER, the catalytic CTA1 subunit dissociates from the rest of the toxin and is transferred to the cytosol where it is degraded by a ubiquitin-independent proteasomal mechanism. However, CTA1 persists long enough to induce excessive cAMP production through the activation of Gsα. It is generally believed that only one or a few molecules of cytosolic CTA1 are necessary to elicit a cytopathic effect, yet no study has directly correlated the levels of cytosolic toxin to the extent of intoxication. Here, we used the technology of surface plasmon resonance to quantify the cytosolic pool of CTA1. Our data demonstrate that only 4% of surface-bound CTA1 is found in the cytosol after 2 h of intoxication. This represented around 2600 molecules of cytosolic toxin per cell, and it was sufficient to produce a robust cAMP response. However, we did not detect elevated cAMP levels in cells containing less than 700 molecules of cytosolic toxin. Thus, a threshold quantity of cytosolic CTA1 is required to elicit a cytopathic effect. When translocation to the cytosol was blocked soon after toxin exposure, the pool of CTA1 already in the cytosol was degraded and was not replenished. The cytosolic pool of CTA1 thus remained below its functional threshold, preventing the initiation of a cAMP response. These observations challenge the paradigm that extremely low levels of cytosolic toxin are sufficient for toxicity, and they provide experimental support for the development of post-intoxication therapeutic strategies.

Occurrence and Exposure Assessment of Deoxynivalenol and Its Acetylated Derivatives from Grains and Grain Products in Zhejiang Province, China (2017-2020).

Deoxynivalenol (DON) together with its acetylated derivatives cause detrimental effects on human health, and the purpose of this study was to assess the prevalence of DON and its acetylated derivatives from grains and grain products in Zhejiang province, China, and to assess the risk of DON and its acetylated derivatives due to multiple consumptions of grains and grain products among the Zhejiang population. Food samples numbering 713 were collected, and the LC-MS/MS method was used to determine the toxins. The levels of toxins from grains and grain products were relatively low: DON was the toxin at the highest levels. The food frequency questionnaire was used to collect food consumption data. The result of exposure assessments showed that the population was overall at low levels of toxin exposure. The probable mean group daily intake of toxins was 0.21 μg/kg bw/day, which was far from the group provisional maximum tolerable daily intake of 1 μg/kg bw/day, but 0.71% of participants were at high exposure levels. Rice and dried noodles (wheat-based food) were the main sources of toxin exposure, and reducing the consumption of rice and dried noodles while consuming more of other foods with lower levels of toxins is recommended.

Dynamics of Deoxynivalenol and Nivalenol Glucosylation in Wheat Cultivars Infected with Fusarium culmorum in Field Conditions─A 3 Year Study (2018-2020).

Fusarium head blight (FHB) caused by pathogenic species of Fusarium fungi is one of the most important diseases of cereal plants and a factor contributing to losses in plant production. The growth of FHB-associated species is often accompanied by biosynthesis of secondary metabolites─mycotoxins, which serve as a virulence factor. The aim of the study was to evaluate the ratios between deoxynivalenol (DON) and nivalenol (NIV) and their derivatives in the ears of six cultivars of winter wheat with varying resistance to FHB, taking into account a range of factors (weather conditions, location, cultivar, and year) after inoculation with Fusarium culmorum, during a 3 year field experiment, 2018-2020. The presence of toxins in the ears was measured within 21 days of inoculation. The toxins were found in the ears as soon as on the third day from the start of the experiment, whereas relative humidity higher than 80% was a decisive factor for FHB incidence. All wheat cultivars showed the ability to biotransform DON and NIV present in the ears to glucosides, that is, deoxynivalenol-3-glucoside (DON-3G) and nivalenol-3-glucoside (NIV-3G). The levels of these metabolites showed significant correlation with the levels of their basic analogues. In most cases, higher levels of DON and NIV in wheat ears and higher levels of their metabolites were observed, but the relative levels of DON-3G/DON and NIV-3G/NIV at relatively high levels of toxins were lower compared to the ear samples with relatively low toxin levels. The presented results are the first studies, which systematically correlate a variety of wheat cultivars with their extent to glucosylate trichothecenes.

Comparison of laxative effects of fermented soybeans (Cheonggukjang) containing toxins and biogenic amines against loperamide-induced constipation mouse model.

BACKGROUND/OBJECTIVES Cheonggukjang is a traditional fermented soybean paste with significant health-promoting effects. On the other hand, there have been insufficient studies on the safety and efficacy of Cheonggukjang, which is produced using traditional methods containing toxins and biogenic amines (BAs). This study compared the laxative effect of Cheonggukjang, containing high or low levels of toxins and BAs (HTBC or LTBC) in a loperamide (Lop)-induced constipation mouse model.MATERIALS/METHODSTo induce constipation, Lop (5 mg/kg) was administered orally to ICR mice twice a day for 4 days, and the dose was increased to 8 mg/kg after a 3-day rest period. Cheonggukjang (500 mg/kg, HTBC, or LTBC respectively) was administered for four weeks before the Lop treatment.RESULTSThe number of stools, fecal weight, water contents, gastrointestinal transit, and histological alterations were recovered significantly in the HTBC or LTBC groups. HTBC and LTBC administration did not induce significant changes in body weight, dietary intake, and behavior. The opioid-receptor downstream signaling pathway in colon tissues was also evaluated. The c-Kit, stem cell kinase, and mitogen-activated protein kinases subfamilies, including extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinases, and p38, were all downregulated in the HTBC or LTBC-administered mice colon compared to the Lop group.CONCLUSIONThese results show that Cheonggukjang, containing high levels of toxins and BAs, have a similar laxative effect in a mouse model of Lop-induced constipation.

Host Plant Species Mediates Impact of Neonicotinoid Exposure to Monarch Butterflies.

Simple SummaryNeonicotinoids are the most widely used insecticides in North America and many studies document the negative effects of neonicotinoids on bees. Monarch butterflies are famous for their long-distance migrations, and for their ability to sequester toxins from their milkweed host plants. The neonicotinoids imidacloprid and clothianidin were suggested to correlate with declines in North American monarchs. We examined how monarch development, survival, and flight were affected by exposure to neonicotinoids, and how these effects depend on milkweed host plant species that differ in their cardenolide toxins. Monarch survival and flight were unaffected by low and intermediate neonicotinoid doses. At the highest dose, neonicotinoids negatively affected monarch pupation and survival, for caterpillars that fed on the least toxic milkweed. Monarchs fed milkweed of intermediate toxicity experienced moderate negative effects of high insecticide doses. Monarchs fed the most toxic milkweed species had no negative consequences associated with neonicotinoid treatment. Our work shows that monarchs tolerate low neonicotinoid doses, but experience detrimental effects at higher doses, depending on milkweed species. To our knowledge, this is the first study to show that host plant species potentially reduce the residue of neonicotinoid insecticides on the leaf surface, and this phenomenon warrants further investigation.Neonicotinoids are the most widely used insecticides in North America. Numerous studies document the negative effects of neonicotinoids on bees, and it remains crucial to demonstrate if neonicotinoids affect other non-target insects, such as butterflies. Here we examine how two neonicotinoids (imidacloprid and clothianidin) affect the development, survival, and flight of monarch butterflies, and how these chemicals interact with the monarch’s milkweed host plant. We first fed caterpillars field-relevant low doses (0.075 and 0.225 ng/g) of neonicotinoids applied to milkweed leaves (Asclepias incarnata), and found no significant reductions in larval development rate, pre-adult survival, or adult flight performance. We next fed larvae higher neonicotinoid doses (4–70 ng/g) and reared them on milkweed species known to produce low, moderate, or high levels of secondary toxins (cardenolides). Monarchs exposed to the highest dose of clothianidin (51–70 ng/g) experienced pupal deformity, low survival to eclosion, smaller body size, and weaker adult grip strength. This effect was most evident for monarchs reared on the lowest cardenolide milkweed (A. incarnata), whereas monarchs reared on the high-cardenolide A. curassavica showed no significant reductions in any variable measured. Our results indicate that monarchs are tolerant to low doses of neonicotinoid, and that negative impacts of neonicotinoids depend on host plant type. Plant toxins may confer protective effects or leaf physical properties may affect chemical retention. Although neonicotinoid residues are ubiquitous on milkweeds in agricultural and ornamental settings, commonly encountered doses below 50 ng/g are unlikely to cause substantial declines in monarch survival or migratory performance.

MO977POTENTIAL INTERACTIONS BETWEEN UREMIC TOXINS AND DRUGS: AN APPLICATION IN KIDNEY TRANSPLANT RECIPIENTS TREATED WITH CALCINEURIN INHIBITORS*

Abstract Background and Aims In addition to affecting glomerular filtration rate, chronic kidney disease (CKD) changes the absorption, distribution and nonrenal clearance of a number of drugs. The uremic toxins that accumulate with as renal function deteriorates can potentially affect drug pharmacokinetics. Given the requirement for therapeutic drug monitoring (TDM) with most immunosuppressants, the latter are good models for understanding the potential impact of uremic toxins on blood concentrations of drugs. We focused on two calcineurin inhibitors (tacrolimus and cyclosporine) with similar pharmacological properties: low but variable oral bioavailability, strong binding to plasma proteins (albumin for tacrolimus and lipoproteins for cyclosporine), metabolism by cytochrome P450 3A4/5, and excretion in the bile. Our starting hypothesis was that uremic toxins influence the exposure of immunosuppressants. The study’s objective was to determine whether plasma concentrations of certain uremic toxins were correlated with blood concentrations of the two immunosuppressants. Method DRUGTOX was a cross-sectional study of 403 adult patients followed up after kidney transplantation at Amiens University Medical Center (Amiens, France) and who had undergone TDM of calcineurin inhibitors between August 4th, 2019, and March 11th, 2020. The patients had been treated daily with oral tacrolimus or cyclosporine. Two groups were constituted: 203 patients with low uremic toxin levels (estimated glomerular filtration rate (eGFR) according to the Modification of Diet in Renal Disease equation >40 mL/min/1.73m2) and 200 patients with high uremic toxin levels (eGFR <40 mL/min/1.73m2). For each patient, immunosuppressant trough concentrations (C0) were measured in whole-blood samples and then normalized against the total daily dose: C0/D ratio. After centrifugation, the sample was assayed for five uremic toxins (urea, trimethylamine N-oxide (TMAO), indole acetic acid (IAA), para-cresylsulfate (PCS), and indoxylsulfate (IxS)) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Univariate and multivariate logistic regressions were used to evaluate the associations between plasma uremic toxin levels and the blood tacrolimus or cyclosporine C0/D ratio. Results The median [interquartile range] age was 56 [48-66] and the median eGFR was 41 [30-57] ml/min/1.73 m2. The majority of the patients (56.5%) were at CKD stage 3. A total of 248 (61.5%) patients were being treated with tacrolimus and 155 (38.5%) were being treated with cyclosporine. The median time between transplantation and study inclusion was 6.5 years. Plasma urea, TMAO, IAA, PCS and IxS levels rose progressively with the CKD stage. In a univariate logistic analysis, age, sex, BMI, urea, IxS and PCS were significantly associated with an increment in the tacrolimus C0/D ratio. A multivariate analysis revealed an independent association with IxS (odds ratio [95% confidence interval]: 1.36 [1.00; 1.85], after adjustment for sex, age and BMI, whereas adjustment for age weakened the association for PCS (OR: 1.22 (0.99; 1.55)) and urea (OR: 1.71 (0.93; 3.16)). In a univariate logistic analysis, age, sex, BMI, and the TMAO level (but neither PCS, IxS, IAA nor urea) were significantly associated with an increment in the cyclosporine C0/D ratio. Conclusion Even though TDM and dose adaptation of immunosuppressants keeps levels within the therapeutic window, elevated exposure to tacrolimus (but not cyclosporine) is associated with an accumulation of PCS, IxS and urea. This might be explained by an interaction between tacrolimus and highly albumin-bound uremic toxins. Our findings may have implications for other drugs that bind with high affinity to albumin (since an elevated free fraction might lead to unexpected exposures, which can alter their efficacy and/or tolerance).

The nearest neighbor nuclei method to objectify analysis of pertussis toxin-induced clustering.

The in vivo histamine sensitization test (HIST) has historically been performed to guarantee the safety of acellular per­tussis vaccine batches. Non-compliance of batches is primarily associated with the presence of low levels of pertussis toxin (PTx). Because of ethical, standardization and scientific reasons, a variety of alternative in vitro approaches have been studied to replace the lethal HIST. A broadly applied and partially accepted method is the CHO cell clustering test, which is based on the clustered growth pattern of CHO cells when exposed to minute amounts of PTx. One of the major hurdles for global application of the CHO clustering test is the manual assessment of the clusters, which is associated with suboptimal reproducibility of test outcomes and is time-consuming. Here, various parameters of CHO cell nuclei were evaluated in search for a reliable, objective read-out parameter. We demonstrate that the distance between each nucleus and its nearest neighbor (3N method) is the most suitable parameter to assess clustered cell growth. This method detects 2.8 mIU PTx/mL and thereby complies with the requirement set for the sensitivity of the CHO clustering test based on visual reading. In commercial acellular pertussis vaccines spiked with PTx, the method detects 45 mIU/mL PTx, which is substantially lower than the 181-725 mIU/mL PTx detected by visual interpretation. The 3N method thus allows objective and sensitive assessment of CHO clustering and thereby encourages broad and global implementation of the in vitro test as an alternative to the HIST.

Numbers, neighbors, and hungry predators: What makes chemically defended aposematic prey susceptible to predation?

Many chemically defended aposematic species are characterized by relatively low toxin levels, which enables predators to include them in their diets under certain circumstances. Knowledge of the conditions governing the survival of such prey animals—especially in the context of the co‐occurrence of similar but undefended prey, which may result in mimicry‐like interactions—is crucial for understanding the initial evolution of aposematism. In a one‐month outdoor experiment using fish (the common carp Cyprinus carpio) as predators, we examined the survival of moderately defended aposematic tadpole prey (the European common toad Bufo bufo) with varying absolute densities in single‐species prey systems or varying relative densities in two‐species prey systems containing morphologically similar but undefended prey (the European common frog Rana temporaria). The density effects were investigated in conjunction with the hunger levels of the predator, which were manipulated by means of the addition of alternative (nontadpole) food. The survival of the B. bufo tadpoles was promoted by increasing their absolute density in the single‐species prey systems, increasing their relative density in the two‐species prey systems, and providing ample alternative food for the predator. Hungry predators eliminated all R. temporaria individuals regardless of their proportion in the prey community; in treatments with ample alternative food, high relative B. bufo density supported R. temporaria survival. The results demonstrated that moderately defended prey did benefit from high population densities (both absolute and relative), even under long‐term predation pressure. However, the physiological state of the predator was a crucial factor in the survival of moderately defended prey. While the availability of alternative prey in general should promote the spread and maintenance of aposematism, the results indicated that the resemblance between the co‐occurring defended and undefended prey may impose mortality costs on the defended model species, even in the absence of actual mimicry.

A sensitive and accurate method for simultaneous analysis of algal toxins in freshwater using UPLC-MS/MS and 15N-microcystins as isotopically labelled internal standards

The development of analytical methods for the detection and accurate quantification of algal toxins is of importance to assess the health risk of exposure to algal toxins in freshwater sources. This study established a sensitive and accurate analytical method for the quantification of 13 algal toxins (microcystins and nodularin) based on solid phase extraction (SPE) coupled with UPLC-MS/MS, in which 15N-microcystins were used as surrogate/internal standards. SPE method was optimized to extract the target algal toxins in freshwater samples. Good SPE efficiencies (84–96%) were achieved for the overwhelming majority of the investigated algal toxins when SPE was performed using HLB (500 mg, 6 mL) under alkaline conditions (pH 11). An accurate quantitative analysis of the algal toxins in real freshwater samples was performed by using 15N-labelled microcystins as isotopically labelled internal standards (ILISs), which compensated for the loss of target toxins during the whole analytical process. In addition, ILISs also helped to correct the effects of environmental matrices and instrument fluctuation in UPLC-MS/MS analysis. The limit of method quantification (MQL) for the algal toxins was <2.0 ng/L that is sensitive enough to quantify extremely low levels of target toxins in freshwater samples.

Toxic potential of sewage sludge: Histopathological effects on soil and aquatic bioindicators

Abstract Sewage sludge has been used as a soil amendment because it is rich in plant essential nutrients and can serve as a substitute for chemical fertilizers. However, sewage sludge can contain a complex mixture of pollutants that are harmful to organisms. Considering that the management of the waste should not result in risks to environmental health, the toxic potential of sewage sludge to organisms at different trophic levels should be measured before it final disposal. Diplopod (Rhinocricus padbergi) and fish (Xiphophorus maculatus) were exposed to sewage sludge or sewage sludge treated with lime (biosolid) at different dilutions under laboratory conditions. Semiquantitative histopathological analyses were performed on target organs (midgut and fat body for the diplopod and gills for the fish). Toxins from sludge or biosolid caused tissue damage and activated detoxification processes in both the diplopod and fish. In comparison to other sludge concentrations, the highest sludge concentrations tested were more toxic, and millipedes likely reduced their food intake to avoid toxins, resulting in weight loss. Our data suggest that sewage sludge pathogens promoted specific histological alterations. Chemical analysis revealed that the concentrations of priority pollutants did not exceed the limits established by Brazilian law. Therefore, this outcome indicates that R. padbergi and X. maculatus are sensitive to low levels of toxins from sewage sludge.

Measuring Peripheral and Some Mucosal Immune Cells to Better Understand Immunomodulation by T-2 Toxin in Broilers

SUMMARY The ability of naturally-occurring low levels of food-borne toxins and environmental contaminants to alter immune function in animals has been the subject of increasing interest in recent years. A total of 96 1-d-old broiler chickens were used to evaluate the effect of feeding naturally contaminated rations with low levels (800 ppb) of T-2 toxin and the protective effect of a commercial mycotoxin eliminator on circulating and intestinal immune cells, blood biochemistry, and hematological variables. Although there were no effects on body weight, contamination with T-2 toxin lead to a significant increase in serum levels of total protein (mainly due to increased albumin levels), alkaline phosphatase (AP), and aspartate aminotransferase (AST), and increased jejunal levels of CD3+ and Goblet cells at day 28. T-2 toxin significantly altered immune parameters, as was demonstrated by alterations in the number of circulating macrophages and suppressor macrophages, B-lymphocytes, T-lymphocytes from mucosa, virgin cytotoxic T-lymphocytes, and terminally activated cytotoxic T-lymphocytes compared to uncontaminated control birds. The use of a mycotoxin eliminator partly compensated for the changes in immune cells. The importance of low levels of mycotoxins is still debatable. However, in this research, it is clearly demonstrated that T-2 toxins affects immune responses, possibly due to impaired gut barrier functioning and metabolic changes.

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low \u03b2-L-ODAP grass pea lines

BackgroundGrass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties.ResultsA plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for β-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled β-L-ODAP) allowing accurate quantification of β-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any β-L-ODAP in these species. The LCMS method was also used to quantify β-L-ODAP accurately in different tissues of grass pea.ConclusionsThe plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and β-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of β-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new ‘gold standard’ for β-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-β-L-ODAP genotypes.

2359. Prospective Feasibility Study for Novel Ultrasensitive Multiplexed Immunoassay for Clostridiodes difficile Toxins A and B

BackgroundThe diagnosis of Clostridiodes difficile infection is challenging. A wide array of diagnostic tests are used in practice; however, each available test has important limitations. We examined the feasibility and analytical performance of a novel ultrasensitive multiplexed immunoassay designed by Meso Scale Diagnostics (MSD) compared with five current diagnostic assays for detection of C. difficile toxin A and B.MethodsStool, serum and urine samples from 44 admitted inpatients were collected within 72 hours of a standard of care nucleic acid amplification test (NAAT) result (23 positive, 21 negative). These specimens underwent five standard diagnostic assays: enzyme immunoassay for toxins A and B (EIA), cytotoxin cell assay, bacterial culture isolation, and two different NAATs to determine presence of viable C. difficile cells, toxins, and toxin-encoding genes (Table 1). The concentration (fg/mL) of toxin A and toxin B in all stool samples was then quantified using MSD’s multiplexed immunoassay (Table 1).ResultsAt least one of the five standard diagnostic tests for C. difficile was positive in 16 of the 23 clinically positive patients. The MSD multiplex immunoassay detected toxin A and/or toxin B in 15 of these 16 samples and quantified low levels of toxin A in one clinically positive sample that was negative for all other tests. In contrast, only 2 of the 16 positive samples were positive by EIA, demonstrating the benefits of the ultrasensitive assay over standard immunoassay methods. All clinically negative specimens were negative in all tests. Toxin detection in urine and serum samples was negligible. In stool samples, the MSD test had an estimated sensitivity of 93% (95% CI: 70–99%) and specificity of 93% (95% CI: 78–98%) compared with the clinically used NAAT.ConclusionThe MSD multiplex toxin assay is a feasible test to move forward for further evaluation. Ultimately, future studies should examine the performance of this test compared with standard of care in a prospective randomized trial assessing clinical outcomes. Disclosures All authors: No reported disclosures.

Microbial Toxins in E-Liquid: A Potential New Vaping-Related Exposure to Explore.

Microbial Toxins in E-Liquid: A Potential New Vaping-Related Exposure to Explore.