An endophytic fungus from Azadirachta indica A. Juss. that produces azadirachtin.

Azadirachtin A and its structural analogues are a well-known class of natural insecticides having antifeedant and insect growth-regulating properties. These compounds are exclusive to the neem tree, Azadirachta indica A. Juss, from where they are currently sourced. Here we report for the first time, the isolation and characterization of a novel endophytic fungus from A. indica, which produces azadirachtin A and B in rich mycological medium (Sabouraud dextrose broth), under shake-flask fermentation conditions. The fungus was identified as Eupenicillium parvum by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Azadirachtin A and B were identified and quantified by LC-HRMS and LC-HRMS(2), and by comparison with the authentic reference standards. The biosynthesis of azadirachtin A and B by the cultured endophyte, which is also produced by the host neem plant, provides an exciting platform for further scientific exploration within both the ecological and biochemical contexts.

What is the spatial-temporal behavior of a low, medium and high adsorptive compound in two contrasting natural sediments in OECD 218/219 test systems?

Artificial sediment used in studies according to OECD 218/219 (Sediment Water Chironomid Toxicity Test Using Spiked Sediment/Water) does not necessarily mirror the characteristics of natural sediments. To investigate the influence of sediment characteristics on the spatial-temporal behaviors of bixafen (KfOM = 2244 mL/g), fluopyram (KfOM = 162 mL/g) and N,N-dimethylsulfamide (KfOM ≈ 0 mL/g), experiments according to OECD 218/219 with two contrasting natural sediments were conducted. The silt loam sediment provided a high content of organic matter (OM) (13.1%), while the OM (0.45%) of the sandy sediment was low. Diffusion into (OECD 219) or out (OECD 218) of the sediment was dependent on the extent of adsorption, which is linked to the model compounds ́ adsorption affinities and the sediments ́ OM. Consequently, N,N-dimethylsulfamide showed unhindered mobility in each experimental set up, while the high adsorption affinities of fluopyram and bixafen limited the diffusion in the respective sediments. Therefore, in experiments with the silt loam sediment, both compounds revealed a limited mobility and either accumulated in the top 5 mm of the sediment (OECD 219) or remained homogenously distributed over the sediment depth (OECD 218). A greater mobility was observed within the sandy sediment.The influence of OM as found in a study using artificial sediment could be confirmed. Moreover, the applicability of a TOXSWA model was reassured to predict the measured concentrations at different sediment depths. TOXSWA is used in the regulatory exposure assessment to simulate the behavior of pesticides in surface waters. Calibration of three driving input parameters by inverse modelling (diffusion-, adsorption coefficient and OM) revealed no potential for improvement. The core sampling technique used and the model may contribute to a more realistic determination of concentration to which the Chironomid larvae are exposed to. This applies to water sediment test systems where the test organisms do not evenly inhabit the sediment.

Comparison of the in vitro assays to investigate the hepatic metabolism of seven pesticides in Cyprinus carpio and Oncorhynchus mykiss.

Liver S9 fractions from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were incubated with seven pesticides (fenamidone, fenoxaprop-p-ethyl, penflufen, spirotetramat, tebuconazole, tembotrione and trifloxystrobin) and the metabolic pathways of the applied chemicals were determined by HPLC-high-resolution mass spectrometry. Five of the seven pesticides (fenamidone, penflufen, spirotetramat, trifloxystrobin and fenoxaprop-p-ethyl) revealed a higher metabolic capacity of rainbow trout liver fractions compared to carp liver fractions. The other two pesticides (tebuconazole and tembotrione) showed a similar and marginal biotransformation for liver S9 fractions of both species. Furthermore, four compounds (penflufen, spirotetramat, tembotrione and tebuconazole) were incubated with cryo-preserved hepatocytes of rainbow trout showing additional conjugated metabolites compared to liver S9 fractions. The incubations were performed with concentrations of 1 and 10 µM for experiments with liver S9 fractions and 5 µM with hepatocytes for up to 120 (liver S9 fractions) or 240 min (hepatocytes). A set of positive controls was used to confirm the metabolic capability of the in vitro systems. The comparison of the in vitro results from hepatocyte assays of penflufen and tebuconazole with the data from corresponding in vivo studies performed according to OECD (Organisation for Economic Co-operation and Development) guideline 305 exhibited a similar metabolic behavior for these pesticides and emphasizes the reliability of the in vitro assays. Besides investigation of the metabolism of plant protection products for research purposes, inter-species comparison by in vitro assays and the use of PBTK modelling approaches will allow improved environmental and dietary risk assessments.

In vitro metabolism of tebuconazole, flurtamone, fenhexamid, metalaxyl-M and spirodiclofen in Cannabis sativa L. (hemp) callus cultures.

BACKGROUND: Cannabis sativa L. (hemp) is a medicinal plant producing various cannabinoids. Its consumption is legalized for medical use due to the alleged positive health effects of these cannabinoids. To satisfy the demand, C. sativa plants are propagated in contained growth chambers. During indoor propagation, pesticides usually are used to ensure efficient production. However, pesticide registration and safe application in C. sativa has not been investigated in detail. RESULTS: With this study the metabolic degradation of pesticides in recently established C. sativa callus cultures was examined. Tebuconazole, metalaxyl-M fenhexamid, flurtamone and spirodiclofen were applied at 10 µm for 21 days. Results were compared with metabolism data obtained from Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Tritium aestivum L. callus cultures as well as in metabolism guideline studies. The successfully established C. sativa callus cultures were able to degrade pesticides by oxidation, demethylation, and cleavage of ester bonds in phase I, as well as glycosylation and conjugation with malonic acid in phase II and III. Initial metabolites were detected after Day (D)7 and were traced at D21. CONCLUSION: The resulting pathways demonstrate the same main degradation strategies as crop plants. Because metabolites could be the main residue, the exposure of consumers to these residues will be of high importance. We present here an in vitro assay for a first estimation of pesticide metabolism in C. sativa. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

What is the actual exposure of organic compounds on Chironomus riparius? - A novel methodology enabling the depth-related analysis in sediment microcosms.

A novel active sampling method enabled determination of sediment depth profiles revealing the spatial distribution of model compounds N,N-dimethylsulfamide, fluopyram and bixafen (low, medium, high adsorption affinity) in sediment microcosms according to OECD Test 218/219 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment/Spiked Water). After the overlying water was removed, plastic tubes were inserted into the sediment and the microcosms were frozen. For depth-related analysis, each "sediment core" was mounted in a cutting device and sawed into three 5-mm-slices, respectively (top, middle, bottom). Each slice was centrifuged for sediment and pore water separation. By various sampling dates within 28 days, we could follow the behavior of model compounds depending on sorption affinities and display specific distribution patterns within the sediment. N,N-dimethylsulfamide showing no sediment adsorption, migrated unhindered in (OECD 219) and out (OECD 218) of the sediment via pore water, resulting in homogenous distributions in both test designs. Fluopyram with moderate adsorption affinity revealed a concentration gradient with declining amounts from top to bottom layer (OECD 219) and higher amounts in the middle and bottom layer as compared to the top layer (OECD 218). Bixafen providing a strong adsorption affinity accumulated in the top layer in OECD 219, while no concentration gradients became visible in OECD 218. For establishing a Toxic Substances in Surface Waters (TOXSWA) model, we compared our measurements with simulated results revealing good agreements. The presented methodology is a useful tool to determine more realistic sediment and pore water concentrations, which the Chironomid larvae are exposed to.

Lipophilicity matters - A new look at experimental plant uptake data from literature.

Peer-reviewed Transpiration Stream Concentration Factor (TSCF) values were analysed to elucidate whether pH-induced changes in lipophilicity can explain some of the variability in reported TSCF and whether a potential relationship between lipophilicity and TSCF can be described by a simple mathematical model. The data set for this investigation combined TSCF values of 42 non-ionisable and ionisable compounds from hydroponic tests with intact plants and publicly available lipophilicity data for the tested compounds. The data set was not homogenous in terms of molecular weight of the tested compounds, plant species used for testing and experimental conditions, but a strong effect of one of these factors on variation in reported TSCF was not detected. Variation in TSCF was high for the same or similar predicted octanol/water partitioning coefficient (log P) but could be reduced by considering octanol/water distribution coefficients (log D) instead. The TSCF data set was split into a training and a test data set in order to identify and test a best-fit model describing the relationship between log D and TSCF. Comparing different types of models (linear, sigmoidal, Gaussian), the Gaussian model fitted to the training data set after removal of two outliers was identified as best-fit model based on visual assessment and fit statistics (RMSE = 0.20, NSE = 0.57, R = 0.75 (p < 0.001)). The 95% confidence interval around the best-fit model contained about 70% of data points in the training set and the test set, respectively. In conclusion, compound lipophilicity expressed as log D is a more appropriate descriptor of uptake by plant roots and subsequent translocation than log P when ionisable compounds are considered. Furthermore, findings in this study suggest that a relationship exists between log D and TSCF for uptake tests with intact plants which can be described by a simple bell-shaped Gaussian model.

Evaluation of Callus Cultures to Elucidate the Metabolism of Tebuconazole, Flurtamone, Fenhexamid, and Metalaxyl-M in Brassica napus L., Glycine max (L.) Merr., Zea mays L., and Triticum aestivum L.

Plant cell cultures can be used to identify the metabolic degradation of pesticides in crops. Therefore, Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Triticum aestivum L. were used to elucidate the metabolic degradation of the following pesticides: tebuconazole, flurtamone, fenhexamid, and metalaxyl-M. Callus cultures were treated with 10 µM of the named pesticides by passive diffusion out of the nutrition agar while young plants were hydroponically exposed to it. After 14 days, the comparison of in planta and in vitro experiments showed that the metabolic degradation is well described by in vitro callus cultures. The intracellular uptake of all pesticides and a broad spectrum of exemplarily hydroxylated and conjugated metabolites were detectable. Overall, the comparability of the nature of residues out of both experiments with the regulatory guideline metabolism studies could be demonstrated. Therefore, we recommend it as a potential screening tool to elucidate the metabolism of pesticides in crops.

Anxiolytic and antidepressant effects of Ziziphus mucronata hydromethanolic extract in male rats exposed to unpredictable chronic mild stress: Possible mechanisms of actions.

ETHNOPHARMACOLOGICAL RELEVANCE: Ziziphus mucronata (ZM) is used traditionally in the treatment of mood and depression. However, no existing scientific data is confirming this traditional claim. AIM OF THE STUDY: The present study was planned to investigate the anxiolytic and antidepressant-like effects of this plant in a stressed-induced depression model in rats. MATERIALS AND METHODS: Depressive-like behaviors were induced by exposing rats to different stress paradigms daily for 30 days. A sucrose preference test was performed to assess anhedonia in rats. Anxiety and depression-related behavior were assessed. The oxidative parameters (lipid peroxidation, SOD and catalase activities) were evaluated. Pindolol and Flumazenil were also used to assess the mechanism of action of ZM extract. RESULTS: The results showed that chronic administration of ZM (150, 300, and 600 mg/kg, p.o., 30 days) and imipramine treatment (20 mg/kg, p.o, 30 days) remarkably (P < 0.05) reversed the UCMS-induced behavioral changes observed in stress vehicle treated rats by reducing sucrose preference, decreased the immobility period in the FST and latency in NSF. Besides, ZM (300 and 600 mg/kg, p.o., 30 days) raised the percentages of time spent and number of open arms entries as well as the number of transitions. Also, ZM (300 mg/kg, (P < 0.05) decreased lipid peroxidation and increased both SOD and catalase activities (300 and 600 mg/kg, (P < 0.05)). These aforementioned behavioral indices were also completely nullified by pindolol a ß-adrenoceptors blocker and 5-HT 1A/1B receptor antagonist but not by flumazenil, a benzodiazepine receptors antagonist. CONCLUSION: ZM improved symptoms of anxiety and depression in behavioral despair paradigm in chronically stressed rats. The observed effects could be due to its capacities to restore the antioxidant status, and probably the modulation of monoamines transmissions.

Evaluation of a novel test design to determine uptake of chemicals by plant roots.

A new hydroponic study design to determine uptake of chemicals by plant roots was tested by (i) investigating uptake of [14C]-1,2,4-triazole by wheat plants in a ring test with ten laboratory organizations and (ii) studying uptake of ten other radiolabelled chemicals by potato, tomato or wheat plants in two laboratories. Replicate data from the ring test were used to calculate plant uptake factor (PUF) values (uptake into roots and shoots) and transpiration stream concentration factor (TSCF) values (uptake into shoots). Average PUF for 1,2,4-triazole was 0.73 (n=39, 95% confidence interval (CI): 0.64, 0.82) and the corresponding TSCF value was 1.03 (n=49, 95% CI: 0.76, 1.3). Boxplots and subsequent classification tree analysis of PUF and TSCF values showed that potential outlier values were >1.38 and were observed for PUF replicates with low biomass increase (ratio of final to initial biomass ≤1.739) and small initial biomass (≤1.55g) and for TSCF replicates with an increase in biomass of <0.67g over a period of eight days. Considering only valid replicate data, average values of PUF and TSCF were 0.65 (n=33, 95% CI: 0.57, 0.73) and 0.64 (n=39, 95% CI: 0.58, 0.70). The additional experiments with ten chemicals and three plant species showed that uptake was low for polar substances of high molecular weight (≥394g/mol) and that TSCF values increased with log Kow values of the tested chemicals ranging from -1.54 to 1.88 (polynomial equation with R2=0.64). A cluster analysis for three of the compounds that were tested on wheat and tomato indicated that the plant uptake was mainly determined by the substance. Overall, the findings show that the hydroponic study design allows for reliable quantification of plant uptake over a range of compound/crop combinations.

Enzymatic oxidation of rutin by horseradish peroxidase: kinetic mechanism and identification of a dimeric product by LC-Orbitrap mass spectrometry.

Flavonoid oxidation is important issue in food processing and quality. The kinetic mechanism of enzymatic oxidation of rutin by horseradish peroxidase (HRP) was studied. Rutin oxidation reaction was followed by recording of spectral changes over the time at 360 nm. The studied oxidation is mostly enzymatic and less part non-enzymatic. The reaction with HRP has a higher rate compared with the reaction without of HRP, whereby is part of non-enzymatic reaction about 10% of the total reaction. Kinetic parameters were determined from graphics of linear Michaelis-Menten equation, and it was found that investigated reactions of rutin oxidation by HRP take place in a ping-pong kinetic mechanism. High resolution HPLC-MS analysis of the mixture of oxidized products of rutin revealed the presence of rutin dimer. Because of widely distribution of rutin as well as presence of peroxidases and hydrogen peroxide in fresh foods identification of this enzymatic modification product can be beneficial for foods quality and safety.