Metabolic profiles and non-targeted LC-MS/MS approach as a complementary tool to targeted analysis in assessment of plant exposure to pesticides.

Liquid chromatography coupled with tandem mass spectrometry was employed for the detection of pesticides (thiamethoxam, lambda-cyhalothrin, deltamethrin, and metalaxyl) and their metabolites in Raphanus sativus var. longipinnatus exposed to these compounds under experimental conditions. Metalaxyl (0.008 mg/kg), metalaxyl acid (0.009 mg/kg), and (+)-trans-chrysanthemic acid (0.098 mg/kg) were identified in the plants exposed to the individual pesticides and their metabolites. Non-targeted analysis revealed the presence of thiamethoxam, lambda-cyhalothrin, and deltamethrin metabolites in plants exposed to these substances, despite the fact that the pesticide concentrations were below the analytical method's limit of quantification (0.005-0.006 mg/kg). Based on the non-targeted screening, non-specific (leucine and tyramine) and specific (epinephrine, dopamine, tryptamine, and serotonin) markers of plant exposure to the mentioned stress-inducing compounds were detected. These findings prove that non-targeted analysis is an indispensable tool for determining plants' exposure to pesticides, even when the parent compound has been completely metabolized.

Assessment of toxic interaction of nano zinc oxide and nano copper oxide on germination of Raphanus sativus seeds.

Multiple applications of nanoparticles (NPs) could result in their potential release into agricultural systems and raised concerns about food safety. The NPs once released in the environment may interact with numerous pollutants, including other NPs. Present study assessed the impact of a single (CuO and ZnO NPs) and binary mixture (CuO+ZnO NPs) on the germination of Raphanus sativus seeds with a wide range of exposure concentration (0-1000 mg/L). Both the NPs have shown a deleterious effect on seeds at exposure concentration greater than 10 mg/L. Antagonistic interaction between effects of CuO and ZnO NPs on seeds was noticed for all the exposed concentrations. CuO NPs showed higher absorption capacity on the seedling surface than ZnO NPs. Internal uptake of Zn in ZnO NP-exposed seedlings was found to be greater than that due to CuO NP-exposed seedlings. Three different types of exposure adversely affected seed germination (reduction in root length, shoot length, and fresh weight). Reduction in growth parameters (length and weight) with concentration was compared using log-logistic dose-response model of "DRC" package of the "R" software, and EC50 was determined. As per EC50 values, the toxicity of CuO NPs was found to be maximum followed by CuO+ZnO NPs and then minimum for ZnO NPs. Seedlings accumulated Cu and Zn metals, and higher uptake was recorded for Zn (reported as mg/g seedling dry weight). The order of toxicity was found as CuO NPs > binary mixture (CuO+ZnO) NPs > ZnO NPs. Exposure concentration greater than 10 mg/L resulted in significant toxicity and uptake in germinated seedlings. These findings indicated that exposure of the mixture of NPs during germination might give different effects and thus, further attempts could prove quite beneficial to the literature.

EDTA-Assisted Metal Uptake in Raphanus sativus L. and Brassica oleracea L.: Assessment of Toxicity and Food Safety.

A study was conducted to determine the effect of ethylenediaminetetraacetic acid (EDTA) on phytoextraction potential of radish and cabbage. Plant biomass, photosynthetic pigments, proline and phenolics were significantly affected by the accumulation of heavy metals (HM). The metal uptake potential was increased significantly by the application of EDTA. Target hazard quotient (THQ) associated with exposure of these contaminants to food chain was calculated. Agronomic interventions to increase mineral levels in crops often increases the leaf concentrations only, the mineral concentration in edible portions are not increased at desired level due to low mobility of Zn in phloem. Since the leaves of both these crops are edible and a component of staple vegetarian diet, biofortification through Zn present in soil and its solubilization and mobilization through chelators can be implemented. However in no such instance these crops should be consumed when grown on Pb contaminated soil due to associated hazards.

AHAS Trp574Leu substitution in Raphanus sativus L.: screening, enzyme activity and fitness cost.

BACKGROUND: Feral radish (Raphanus sativus L.) is a problematic weed that has become resistant to acetohydroxyacid synthase (AHAS) inhibitor herbicides due to the Trp574Leu mutation. An AHAS gene mutation that causes herbicide resistance may have negative pleiotropic effects on plant fitness. This study reports the effects of the Trp574Leu mutation on AHAS activity and reproductive traits of R. sativus. RESULTS: Eight of 17 feral radish accessions presented individuals resistant to metsulfuron-methyl at 0.5% to >90.0% and all the resistant individuals analyzed showed the Trp574Leu mutation. Without herbicide selection, the AHAS activity was 3.2-fold higher in the susceptible accession than in the resistant one. The resistant accession was >9000-fold more resistant to metsulfuron-methyl and imazethapyr than the susceptible accession. Under low intraspecific competition during two growing seasons, AHAS-resistant feral radish accessions showed 22-38% and 21-47% lower seed numbers and yield per plant than the susceptible accession. CONCLUSION: This is the first report of fitness cost associated with the AHAS Trp574Leu mutation in R. sativus populations. This fitness cost could reduce frequency of the resistant allele without herbicide selection. © 2018 Society of Chemical Industry.

Scenario-targeted toxicity assessment through multiple endpoint bioassays in a soil posing unacceptable environmental risk according to regulatory screening values.

Lanestosa is a chronically polluted site (derelict mine) where the soil (Lanestosa (LA) soil) exceeds screening values (SVs) of regulatory policies in force (Basque Country; Europe) for Zn, Pb and Cd. A scenario-targeted toxicity assessment was carried out on the basis of a multi-endpoint bioassay approach. Acute and chronic toxicity bioassays were conducted with selected test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates and with bioaccumulation studies in earthworms. Besides, the toxicity profile was compared with that of the mine runoff (RO) soil and of a fresh artificially polluted soil (LAAPS) resembling LA soil pollutant profile. Extractability studies in LA soil revealed that Pb, Zn and Cd were highly available for exchange and/or release into the environment. Indeed, Pb and Zn were accumulated in earthworms and LA soil resulted to be toxic. Soil respiration, V. fischeri, vegetative and developmental cycles of D. discoideum and survival and juvenile production of E. fetida were severely affected. These results confirmed that LA soil had unacceptable environmental risk and demanded intervention. In contrast, although Pb and Zn concentrations in RO soil revealed also unacceptable risk, both metal extractability and toxicity were much lower than in LA soil. Thus, within the polluted site, the need for intervention varied between areas that posed dissimilar risk. Besides, since LAAPS, with a high exchangeable metal fraction, was the most toxic, ageing under in situ natural conditions seemingly contributed to attenuate LA soil risk. As a whole, combining multi-endpoint bioassays with scenario-targeted analysis (including leaching and ageing) provides reliable risk assessment in soils posing unacceptable environmental risk according to SVs, which is useful to optimise the required intervention measures.

Evaluation of biological endpoints in crop plants after exposure to non-steroidal anti-inflammatory drugs (NSAIDs): implications for phytotoxicological assessment of novel contaminants.

Human pharmaceuticals have been detected in the terrestrial environment at µg to mg kg(-1) concentrations. Repeated application of sewage sludge (biosolids) and increasing reclaimed wastewater use for irrigation could lead to accumulation of these novel contaminants in soil systems. Despite this, potential phytotoxicological effects on higher plants have rarely been evaluated. These studies aimed to test effects upon germination, development, growth and physiology of two crop plants, namely radish (Raphanus sativus Spakler 3) and lettuce (Lactuca sativa All Year Around), after exposure to different, but structurally related non-steroidal anti-inflammatory drugs (NSAIDs) at environmentally relevant concentrations. A range of biological endpoints comprising biomass, length, water content, specific root and shoot length, root to shoot ratio, daily progress of stages of cell elongation and organ emergence (primary root, hypocotyl elongation, cotyledon emergence, cotyledon opening, and no change), as well as photosynthetic measurements were evaluated. Compounds from the fenamic acid class were found to affect R. sativus root endpoints (root length and water content), while ibuprofen affected early root development of L. sativa. In general, phytotoxicological effects on root endpoints demonstrated that impacts upon higher plants are not only compound specific, but also differ between plant species. It was found that the usage of a wide range of biological endpoints (all simple, cost-effective and ecologically relevant) were beneficial in detecting differences in plant responses to NSAID exposure. Due to paucity and discrepancy within the few previously available phytotoxicological studies with pharmaceuticals, it is now essential to allocate time and resources to consider development of suitable chronic toxicity tests, and some suggestions regarding this are presented.

Toxicity assessment through multiple endpoint bioassays in soils posing environmental risk according to regulatory screening values.

Toxicity profiles of two soils (a brownfield in Legazpi and an abandoned iron mine in Zugaztieta; Basque Country) contaminated with several metals (As, Zn, Pb and Cu in Legazpi; Zn, Pb, Cd and Cu in Zugaztieta) and petroleum hydrocarbons (in Legazpi) were determined using a multi-endpoint bioassay approach. Investigated soils exceeded screening values (SVs) of regulatory policies in force (Basque Country; Europe). Acute and chronic toxicity bioassays were conducted with a selected set of test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates, as well as with bioaccumulation studies in earthworms. The sensitivity of the test species and the toxicity endpoints varied depending on the soil. It was concluded that whilst Zugaztieta soil showed very little or no toxicity, Legazpi soil was toxic according to almost all the toxicity tests (solid phase Microtox, D. discoideum inhibition of fruiting body formation and developmental cycle solid phase assays, lettuce seed germination and root elongation test, earthworm acute toxicity and reproduction tests, D. discoideum cell viability and replication elutriate assays). Thus, albeit both soils had similar SVs, their ecotoxicological risk, and therefore the need for intervening, was different for each soil as unveiled after toxicity profiling based on multiple endpoint bioassays. Such a toxicity profiling approach is suitable to be applied for scenario-targeted soil risk assessment in those cases where applicable national/regional soil legislation based on SVs demands further toxicity assessment.

Plants as models for chromium and nickel risk assessment.

The adverse effects of Cr(III), Cr(VI), and Ni(II) expressed as root and shoot growth inhibition, metal accumulation and translocation throughout plants, and genotoxicity study were examined. To examine phytoxicity and metal accumulation, Vicia sativa, Raphanus sativus, Zea mays and Sinapis alba plants were used. Except for S. alba root growth inhibition, Ni had the strongest inhibitory effect on root and shoot growth. The inhibitory rank order based on IC50 values was Ni(II) > Cr(VI) > Cr(III). Z. mays was the least sensitive to all metals. While the accumulation of Cr was higher in the roots than the upper plant parts, Ni transport to shoots was at least two times higher than that of Cr. The highest accumulation of Cr was found in Z. mays and that of Ni in V. sativa and Z. mays roots. For all plants, the translocation factor was higher for Cr(VI) than for Cr(III). The translocation factor for Ni was several times higher than those of Cr. For mutagenicity assay, root tips of V. sativa, R. sativus and Z. mays were used. All metals exerted a significant increase of chromosomal aberrations and the rank order of aberrations was: Cr(VI) > Ni(II) > Cr(III). Genotoxic effects of metals were also determined by analysis of micronuclei frequency in the pollen tetrads of Tradescantia plants. None of metals significantly stimulated micronuclei frequency and the genotoxic effect decreased in the following order: Cr(VI) ≥ Ni(II) > Cr(III).

Assessment of the variability in response of radish and brinjal at biochemical and physiological levels under similar ozone exposure conditions.

The present investigation was done to evaluate the effects of ambient air pollutants on physiological and biochemical characteristics of radish (Raphnus sativa L. var. Pusa Reshmi) and brinjal (Solanum melongena L. var. Pusa hybrid-6) plants grown in open-top chambers with filtered (FCs) and non-filtered (NFCs) treatments at a suburban site in Varanasi, India. Eight hourly mean concentrations of 11.8, 20.8, and 40.8 ppb for SO2, NO2, and O3, respectively, were recorded. O3 was the most significant pollutant affecting the plant performance. Photosynthetic rate and stomatal conductance declined in both the test plants in NFCs as compared to FCs. Lipid peroxidation was higher in NFCs, but the increase was more in radish compared to brinjal. The constitutive levels of the antioxidants as well as their increments upon O3 exposure were of higher magnitude in brinjal as compared to radish. Reduction in Fv/Fm ratio of the plants in NFCs was a regulatory mechanism to cope with the inefficiency of Calvin cycle. The data indicate that O3 triggered the protective mechanisms in plants which resulted in increments in enzymatic and non-enzymatic antioxidants of O3-exposed plants. The variability of the magnitude of responses in radish and brinjal due to O3 stress suggests that radish is more susceptible to ambient O3 injury compared to brinjal.

The effect of calcinated calcium and chlorine treatments on Escherichia coli O157:H7 87-23 population reduction in radish sprouts.

UNLABELLED: The effect of calcinated calcium spray on Escherichia coli O157:H7 87-23 population reduction during radish sprout production was studied. Artificially inoculated radish seeds were soaked in sodium hypochlorite (NaOCl) solutions (200 and 20000 ppm), rinsed in distilled water, and sprayed with water or a calcinated calcium solution during sprouting. Microbial plate count was obtained at each step of the process and germination rate was determined after 72 h of sprouting. Scanning electron microscopy (SEM) was done on treated seeds and sprouts to locate which parts were populated by the E. coli cells. The results showed that the active compound in the calcinated calcium was calcium oxide. The treatment of 200 ppm NaOCl soaking followed by 0.04% calcinated calcium spray resulted in no microbial growth after a 72-h sprouting, while maintaining a high germination rate. The 0.4% calcinated calcium spray significantly reduced the germination rate and is therefore not recommended. Soaking the seeds in a 20000 ppm chlorine solution achieved the highest E. coli count reduction (1.65 log CFU/g). However, the E. coli cells that survived the 20000 ppm chlorine soak grew to 6 log CFU/g sprouts after a 72-h sprouting, significantly higher than the initial count on the seeds. The SEM microimages showed that the bacteria were mostly located in the roots of the radish sprouts and all across the seed surface. The E. coli O157:H7 87-23 cells appeared to be located in biofilms or embedded into the radish sprout tissues during sprouting. PRACTICAL APPLICATION: The seed sanitation treatment with 20000 ppm chlorine solution that is currently used by the sprout industry was once again found to be ineffective in eliminating inoculated pathogenic cells. More importantly, the remaining cells that have survived the chlorine wash would grow during sprouting to reach an alarmingly high cell concentration. The new observation of E. coli cells and sprout tissue interaction manifested as embedding of the cells in sprout tissues, if confirmed, will have a significant impact on the microbial safety intervention strategies used in the sprout industry. This research demonstrated the importance of eliminating all pathogens on the seeds before germination and sprouting.

Assessment of heavy metals phytotoxicity using seed germination and root elongation tests: a comparison of two growth substrates.

Seed germination and root elongation test is used to evaluate hazardous waste sites and to assess toxicity of organic and inorganic compounds. Paper substrate, especially circular filter paper placed inside a Petri dish has long been used for this test. Same reports indicate that filter paper might interfere with the toxicity of inorganic substances, especially metal cations. This study evaluate toxicity of Cd, Pb, Ni, Cu on lettuce, broccoli, tomato and radish seed using two bed material: agar and filter paper. The results show that percent germination is not affected by substrates; vice versa, as for root elongation, the test in agar showed to be more sensible than that the one on filter paper. The radical growth inhibition depends on the metal, on the tested concentration and on the species; among the tested metals, cadmium was the one determining the highest toxic effects on different species and lettuce was the plant that suffered more. From the comparison, it is clearly evident the greater sensibility of the test in agar; on the other hand, the lower sensibility of the test on the filter paper might be caused by the partial and not homogeneous exposition of the root to metal cations.