Selectivity of metsulfuron applied to soybean before sowing in different intervals and soils.

This work aimed to evaluate the selectivity of the herbicide metsulfuron applied at different times on the development of soybeans grown in soils with different characteristics. The experiment was conducted in a randomized block design, in a factorial scheme (4 x 4), with four replicates. Factor A was application time (0, 15, 30, and 45 days before sowing, DBS) and factor B was soil type (Erechim, Itaqui, Piratini, and Santa Maria). Soybean plants cultivated in the Erechim soil showed moderate phytotoxicity, with greater damage to the leaf area and plant dry matter, mainly after application at 30 DBS. Those cultivated in Itaqui soil showed gradual phytotoxicity between 14 and 28 days after emergence (DAE). Soybean plants grown in the Piratini and Santa Maria soils showed the highest phytotoxicity and photosynthetic reduction, mainly at 15 and 0 DBS. Metsulfuron application at 45 DBS caused reduced plant growth by up to 40%, and reduced shoot development (30%) in soybean plants grown in Piratini and Santa Maria soils, respectively. There were gradual changes in phytotoxicity and the morphophysiological traits of soybean plants exposed to the residual effect of metsulfuron in different soils, which indicates that soybeans should be sown more than 45 days after the application of metsulfuron, regardless of soil characteristics.

Florasulam resistance status of flixweed (Descurainia sophia L.) and alternative herbicides for its chemical control in the North China plain.

Flixweed (Descurainia sophia L.) is widely distributed in winter wheat (Triticum aestivum L.) fields in the North China Plain and has evolved resistance to herbicides, including the acetolactate synthase (ALS) inhibitor florasulam. However, the florasulam resistance status of flixweed in the North China Plain is poorly understood, which hinders the integrated management of this weed in winter wheat production systems. Thus, 45 flixweed populations were collected in wheat fields in these areas, and their sensitivity to florasulam and ALS-inhibitor-resistant mutation diversity were assessed. Meanwhile, alternative herbicides/herbicide mixtures for the control of florasulam-resistant flixweed were screened and evaluated under greenhouse and field conditions. Of the populations, 30 showed florasulam resistance (RRR and RR), 9 had a high risk of evolving florasulam resistance (R?) and 6 were susceptible. These populations had 5.3 to 345.1-fold resistance to florasulam, and 4 ALS resistance mutations (P197H, P197S, P197T and W574L) were observed. The subsequent herbicide sensitivity assay showed that the SD-06 population (with ALS1 P197T and ALS2 W574L mutations) exhibited cross-resistance to all ALS inhibitors tested, but was sensitive to MCPA-Na, fluroxypyr, carfentrazone-ethyl and bipyrazone. Meanwhile, the other HN-07 population with non-target-site resistance (NTSR) also showed resistance to all tested ALS inhibitors, and it was "R?" to MCPA-Na while sensitive to fluroxypyr, carfentrazone-ethyl and bipyrazone. The field experiments were conducted at the research farm where the SD-06 population was collected, and the results suggested that florasulam at 3.75-4.5 g ai ha-1 had little efficacy (0.6-12.1%), whereas MCPA-Na + carfentrazone-ethyl (87.1-91.2%) and bipyrazone+fluroxypyr (90.1-97.8%) controlled the resistant flixweed.

A Trp-574-Leu mutation in the acetolactate synthase (ALS) gene of Lithospermum arvense L. confers broad-spectrum resistance to ALS inhibitors.

Lithospermum arvense is a troublesome dicotyledonous winter annual weed of wheat in China. A L. arvense population (HN01) suspected of being resistant to acetolactate synthase (ALS) inhibitors was found in Henan Province, China. This study aimed to testify the sensitivity of this HN01 population to eight herbicides from 3 different modes of action, and to explore the potential target-site-resistance mechanism to tribenuron-methyl. The whole-plant bioassays indicated that the population was highly resistant to tribenuron-methyl (SU, 350-fold), pyrithiobac sodium (PTB, 151-fold), pyroxsulam (TP, 62.7-fold), florasulam (TP, 80.6-fold), and imazethapyr (IMI, 136-fold), but was sensitive to carfentrazone-ethyl and fluroxypyr-meptyl. ALS gene sequencing revealed that the Trp (TGG) was substituted by Leu (TTG) at codon 574 in resistant plants. In in vitro ALS assays, the concentration of tribenuron-methyl required to inhibit 50% ALS activity (I50) for HN01 was 117-fold greater than that required to inhibit a susceptible population (HN05), indicating that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron-methyl. To the best of our knowledge, this is the first report of ALS gene Trp-574-Leu amino acid mutation confer resistance to tribenuron-methyl in L. arvense.

Effects of Sublethal Doses of Herbicides on the Competitive Interactions Between 2 Nontarget Plants, Centaurea cyanus L. and Silene noctiflora L.

Plant competitive interactions influence the effect of herbicides, and the effect of competitive interactions on plant responses may be important to include in the ecological risk assessment of herbicides. In the present study the effect of competitive interactions and sublethal doses of 2 herbicides on plant species was investigated in competition experiments and fitted to empirical competition models. Two nontarget species commonly found in agroecosystems (Centaurea cyanus L. and Silene noctiflora L.) and 2 herbicides (glyphosate and metsulfuron methyl) were used in separate experiments. Plants were sprayed at the 6- to 8-leaf stage. Effects of herbicide treatments and plant density were modeled by generalization of a discrete hyperbolic competition model. The 10% effective dose (ED10) was calculated for C. cyanus. All experiments showed that as density increased, plants were negatively affected. Furthermore, in all cases, C. cyanus remained a better competitor than S. noctiflora. Nevertheless, the density of S. noctiflora (competitor) was an influential element in determining the ED10 of C. cyanus measured at the mature stage. With herbicide exposure, the competitive interactions were further altered; C. cyanus was less affected by glyphosate when S. noctiflora increased to high density. In contrast, at the young stage, conspecific density was important in determining the sensitivity of C. cyanus to metsulfuron methyl, whereas the density of the competitor S. noctiflora had a limited influence. Overall, the results demonstrate the importance of integrating the effect of herbicide and species interactions measured at the reproductive stage into the ecological risk assessments of pesticides. Environ Toxicol Chem 2019;38:2053-2064. © 2019 SETAC.

Laboratory and field tests for risk assessment of metsulfuron-methyl-based herbicides for soil fauna.

Metsulfuron-methyl is one of the most used sulfonylurea herbicides, being applied alone in pre-emergence and with a mineral oil (as adjuvant) in post-emergence. In risk assessment of pesticides, ecotoxicity tests have been applied to assess the effects of products and mixtures under laboratory conditions, but they are limited in their ecological relevance when compared to field assessments. Considering the differences between laboratory and field exposure, and the lack of data on the effects of metsulfuron-methyl in natural soils, this study consisted in a set of tests to assess the ecotoxicity of this herbicide applied alone, combined with an adjuvant (mineral oil) and the adjuvant applied alone, both under laboratory and field exposure, with artificial and natural soil respectively. Reproduction tests with four non-target soil invertebrates species were performed in laboratory, while two experiments were performed in field evaluating avoidance behaviour, feeding activity, mesofauna abundance and pesticide residual. Laboratory results showed that metsulfuron-methyl alone is not a threat to soil fauna on the recommended doses. However, the presence of mineral oil as adjuvant showed ecotoxicity to Eisenia andrei, Enchytraeus crypticus and Proisotoma minuta on laboratory tests. Field evaluations indicated that metsulfuron-methyl and the adjuvant do not impaired the feeding activity of the soil fauna. The low abundance of native communities could be related to soil management. Results showed that laboratory and field evaluations are necessary to better understanding of the effects of pesticides to soil fauna and adjuvant addition should be considered on pesticides risk assessment.

Resistance of Rapistrum rugosum to tribenuron and imazamox due to Trp574 or Pro197 substitution in the acetolactate synthase.

Ten putative resistant and two susceptible Rapistrum rugosum populations originating from Greece were studied for resistance to acetolactate synthase (ALS)-inhibiting herbicides, using dose-response assays, sequencing of als gene and in vitro ALS activity assays. The dose-response assays showed that one (P1) out of ten putative resistant populations was cross-resistant to tribenuron and imazamox, while another population (P4) was resistant to tribenuron only. All populations were susceptible to MCPA at the recommended rate. Gene sequencing of als revealed that the P4 population had a point mutation at Pro197 by His providing resistance to tribenuron, whereas the P1 had a Trp574 by Leu point mutation conferring cross-resistance to tribenuron and imazamox. The in vitro activity of the ALS enzyme indicated I50 values (tribenuron concentration required for 50% reduction of the ALS activity) ranging from 66.68 to 137.01 µM, whereas the respective value for the S populations ranged from 0.29 to 0.54 µM. These results strongly support that two R. rugosum populations evolved resistance to ALS-inhibiting herbicides due different point mutations in the als gene.

Screening effects of metsulfuron-methyl to collembolans and earthworms: the role of adjuvant addition on ecotoxicity.

Metsulfuron-methyl is a common active ingredient recommended for use in pre- and post-emergence control of annual grasses and broadleaf weeds in crops, usually applied with mineral oil as adjuvant to enhance its efficiency. Despite the increasing use of this herbicide, there are no information on its ecotoxicity effects to soil fauna. Avoidance and lethality tests were performed with earthworms and collembolans using tropical artificial soil contaminated with formulated products Ally® (600 g L-1 metsulfuron-methyl) and Assist® (756 g L-1 mineral oil) as adjuvant. Lethality test with earthworms showed no difference when tested with or without adjuvant. When Ally® was tested alone, it caused avoidance behavior only at high concentrations (5000 and 10,000 times field predicted dose). However, Assist® addition changed the response of soil invertebrates increasing the avoidance even at field predicted doses. The toxicity of the adjuvant was confirmed in tests exposing collembolans and earthworms to Assist® alone resulting in avoidance behavior. The results clearly show that the addition of mineral oil enhanced the ecotoxicity of metsulfuron-methyl. This study provides an important contribution to the knowledge on the toxicity of metsulfuron-methyl and indicates that adjuvants should be considered in risk assessment of pesticides, considering that under field conditions, these products are applied together.

Individual and combined effects of herbicide tribenuron-methyl and fungicide tebuconazole on soil earthworm Eisenia fetida.

Earthworms are soil engineers that alter the soil bio-physical properties to favor plant growth whereas pesticides represent a significant threat to their abundance and soil health. Thus, we investigated the toxic effects of tribenuron-methyl (TBM) and tebuconazole (TEB) on the soil earthworm, Eisenia fetida. The TBM demonstrated low toxicity to E. fetida in the contact filter paper and artificial soil tests, with median lethal concentrations (LC50) of 135.6 µg cm-2 at 48 h and 511 mg kg-1 on day 14, respectively. Similarly, TEB also showed low toxicity to E. fetida in the artificial soil test with LC50 of 287 mg kg-1 on day 14. However, TEB was highly toxic to earthworm in the contact filter paper test with LC50 of 5.7 µg cm-2 at 48 h. The mixture of two pesticides had an antagonistic effect on the earthworm. Under 0.1 LC50 of TBM and TEB, either single or combined application of pesticides induced oxidative stress and inhibited cellulase activity in early days of the earthworm exposure. However, both pesticides did not damage the earthworm DNA. Our results suggest that pesticides can negatively affect soil earthworms and provide valuable information regarding the responses of soil biological engineers to the lethal agrochemicals.

Identification of N-arylsulfonylpyrimidones as anticancer agents.

For confirming the role of five membered ring of imidazolidinone moiety of N-arylsulfonylimidazolidinones (7) previously reported with highly potent anticancer agent, a series of N-arylsulfonylpyrimidones (10a-g) and N-arylsulfonyltetrahydropyrimidones (11a-e) were prepared and their anti-proliferating activity was measured against human cancer cell lines (renal ACHN, colon HCT-15, breast MDA-MB-231, lung NCI-H23, stomach NUGC-3, and prostate PC-3) using XTT assay. Among them, 1-(1-acetylindolin-5-ylsulfonyl)-4-phenyltetrahydropyrimidin-2(1H)-one (11d, mean GI50 = 3.50 µM) and ethyl 5-(2-oxo-4-phenyltetrahydropyrimidin-1(2H)-ylsulfonyl)-indoline-1-carboxylate (11e, mean GI50 = 0.26 µM) showed best growth inhibitory activity against human cancer cell lines. Considering the activity results, N-arylsulfonyltetrahydropyrimidones (11) exhibited more potent activity compared to N-arylsulfonylpyrimidones (10) and comparable activity to N-arylsulfonylimidazolidinones (7). Especially, tetrahydropyrimidin-2(1H)-one analogs containing acylindolin-5-ylsulfonyl moiety at position 1 demonstrated their strong growth inhibitory activity against human cancer cell lines.

Tribenuron-methyl resistance and mutation diversity of the AHAS gene in shepherd's purse (Capsella bursa-pastoris (L.) Medik.) in Henan Province, China.

Shepherd's purse is a troublesome dicot weed that occurs in the major wheat-producing areas in China. Twenty-eight shepherd's purse populations were collected from winter wheat-planting areas in Henan Province and used to evaluate tribenuron-methyl resistance and acetohydroxyacid synthase (AHAS) gene-mutation diversity. The results indicate that all 28 shepherd's purse populations were resistant to tribenuron-methyl at different levels compared with the susceptible population. Mutation of the 197 codon (CCT) changed proline (Pro) into tyrosine (Tyr), histidine (His), leucine (Leu), serine (Ser), arginine (Arg), alanine (Ala) and threonine (Thr), whereas mutation of the 574 codon (TGG) changed tryptophan (Trp) into leucine (Leu). Among these amino acid changes, a co-concurrence of Pro197Leu and Trp574Leu substitutions was identified for the first time in resistant weed species. Furthermore, Pro197Tyr, Pro197Arg and Pro197Ala substitutions have not been previously reported in shepherd's purse. The results of the in vitro AHAS assay suggest that an insensitive AHAS is likely involved in the resistance to tribenuron-methyl in the R populations with AHAS gene mutations, and the non-target-site based resistance might exist in some populations.

Exposure to trace amounts of sulfonylurea herbicide tribenuron-methyl causes male sterility in 17 species or subspecies of cruciferous plants.

BACKGROUND: For most cruciferous plants, which are known as important crops and a number of weeds, hybrid breeding is hampered by the unavailability of a pollination control system. Male sterility induced by a gametocide can be useful for the utilization of plant heterosis. RESULTS: The gametocidal effect of sulfonylurea herbicide tribenuron-methyl was tested across seventeen cruciferous species or subspecies including Brassica juncea, B. carinata, B. oleracea ssp. capitata, B. oleracea ssp. acephala, B. rapa ssp. pekinensis, B. rapa ssp. chinensis, B. rapa ssp. parachinensis, B. nigra, Orychophragmus violaceus, Matthiola incana, Raphanus sativa, Sisymbrium altissimum, Eruca sativa, Sinapis alba, Sinapis arvensis, Capsella bursa-pastoris and Camelina sativa. The plants of 23 cultivars in these species or subspecies were foliar sprayed with 10 ml of 0.2 or 0.4 mg/L of tribenuron-methyl before the vacuolated microspore formed in the largest flower buds; the application was repeated ten to twelve days afterwards. Tribenuron-methyl exposure significantly changed the flowering phenology and reproductive function. The treated plants demonstrated a one to four day delay in flowering time and a shortened duration of flowering, as well as other slight phytotoxic effects including a reduction in plant height and floral organ size. Approximately 80% to 100% male sterility, which was estimated by both pollen staining and selfing seed-set rate, was induced in the plants. As a result, plants were rendered functionally able to out-cross, with an average 87% and 54% manually pollinated seed-set rate compared to the corresponding controls at the 0.2 mg/L and 0.4 mg/L doses, respectively. CONCLUSIONS: The results suggested that male reproductive function was much more sensitive to tribenuron-methyl exposure than female function. This sulfonylurea herbicide has a promising use as the gametocide for hybrid production in cruciferous plants.

Tribenuron-Methyl Induces Male Sterility through Anther-Specific Inhibition of Acetolactate Synthase Leading to Autophagic Cell Death.

Tribenuron-methyl (TM) is a powerful sulfonylurea herbicide that inhibits branched-chain amino acid (BCAA) biosynthesis by targeting the catalytic subunit (CSR1) of acetolactate synthase (ALS). Selective induction of male sterility by foliar spraying of TM at low doses has been widely used for hybrid seed production in rapeseed (Brassica napus); however, the underlying mechanism remains unknown. Here, we report greater TM accumulation and subsequent stronger ALS inhibition and BCAA starvation in anthers than in leaves and stems after TM application. Constitutive or anther-specific expression of csr1-1D (a CSR1 mutant) eliminated anther-selective ALS inhibition and reversed the TM-induced male sterile phenotype in both rapeseed and Arabidopsis. The results of TM daub-stem experiments, combined with the observations of little TM accumulation in anthers and reversion of TM-induced male sterility by targeted expression of the TM metabolism gene Bel in either the mesophyll or phloem, suggested that foliar-sprayed TM was polar-transported to anthers mainly through the mesophyll and phloem. Microscopy and immunoblotting revealed that autophagy, a bulk degradation process induced during cell death, was elevated in TM-induced male sterile anthers and by anther-specific knockdown of ALS. Moreover, TM-induced pollen abortion was significantly inhibited by the autophagy inhibitor 3-MA. These data suggested that TM was polar-transported to anthers, resulting in BCAA starvation via anther-specific ALS inhibition and, ultimately, autophagic cell death in anthers.

Histopathological and ultramicroscopical changes in gill, liver and kidney of Anabas testudineus (Bloch) after chronic intoxication of almix (metsulfuron methyl 10.1%+chlorimuron ethyl 10.1%) herbicide.

Present study aimed to investigate the effects of almix herbicide on histopathological and ultrastructural changes in freshwater teleostean fish, Anabas testudineus (Bloch) under field and laboratory conditions with a dose of 8 g/acre and 66.7 mg/L respectively for 30 days. In field experiment fish species were reared in special type of cage submerged in pond. Cellular alterations of the concerned organs namely gills, liver and kidney were observed through light, scanning and transmission electron microscopy (SEM and TEM). Gill showed hypertrophy and oedema in secondary gill epithelium, and epithelial lifting under laboratory condition but in field condition hyperplasia was prominent. SEM study revealed necrosis, hyperplasia, excessive secretion of mucus and loss of microridges; while TEM study depicted degenerative changes in mitochondria and chloride cells and severe cytoplsmic vacuolation but the changes were comparatively less under field study. In liver, damage in acinar cells of hepatopancreas, degenerative changes in zymogen granules and detachment of hepatopancreatic acinar cells under laboratory condition were the serious changes, but in field condition fatty deposition is prominent. TEM study depicted dilated, degranulated and vesiculated rough endoplasmic reticulum, dilation and swelling of mitochondria and vacuolation in hepatocytes, but under field condition dilation of mitochondria and appearance of vacuolation were well marked. Kidney showed shrinkage of glomerulus, damaged and hypertrophied proximal convoluted tubule, loss of haematopoietic tissues under light microscopy; while ultrastructural changes like degenerative changes in mitochondria, deformed nucleus, dilation, fragmentation and vesiculation of rough endoplasmic reticulum, severe vacuolation in cytoplasm and necrosis were of very serious concern under laboratory condition, but in field condition epithelial cells showed less damage. Responses depicted that symptoms of lesions were more pronounced under laboratory than field condition and the degree of responses in different tissues were different under these two conditions. Therefore, evaluation of these histopathological lesions in concerned tissues may be established as symptomatic indicators for toxicity study in aquatic ecosystem.

Tribenuron-methyl resistance and mutation diversity of Pro197 in flixweed (Descurainia Sophia L.) accessions from China.

Flixweed (Descurainia Sophia L.) is a problematic weed in winter wheat fields in China, which causes great loss of wheat yield. A total of 46 flixweed accessions from winter wheat-planting areas were collected and used for the survey of resistance to tribenuron-methyl and Pro197 mutation diversity. According to the "R" resistance rating system, 16 flixweed accessions have evolved resistance to tribenuron-methyl, 13 accessions have high risk of developing resistance to this herbicide and 17 accessions are susceptible. The mutation of Pro197 codon (CCT) changed proline (Pro) into leucine (Leu) (homozygous, RR), serine (Ser, RR), histidine (His, RR), threonine (Thr, RR), Pro/Leu (heterozygous, RS), Pro/Ser (RS), Pro/His, Pro/Thr (RS) and Pro/Tyr (RS). Among these amino acid changes, a Pro197-Pro/Tyr (heterozygous, RS) substitution caused by the mutation of two successive nucleotides was identified for the first time in resistant weed species. In addition, the Pro197-His and Pro197-Pro/His mutations have not been reported previously in flixweed. Finally, a CPAS marker was developed to identify flixweed plants with or without Pro197 mutation.

Effect of fly ash on persistence, mobility and bio-efficacy of metribuzin and metsulfuron-methyl in crop fields.

Field evaluation of two fly ashes (40t/ha) on persistence, mobility and bioactivity of metribuzin and metsulfuron-methyl was studied in soybean and wheat crops, respectively. Metribuzin was applied as pre-emergence at 0.5kg/ha, while metsulfuron-methyl was applied post-emergence at 8g/ha. Results suggested that metribuzin in surface (0-15cm) soil of fly ash unamended plots reached below detectable limit in 60 days, while herbicide persisted till 112 days in surface soil of fly ash amended plots. No metribuzin leached down to subsurface (15-30cm) soil in fly ash amended plots, while traces of metribuzin (0.6-1.2µg/kg) were recovered in subsurface soil of fly ash unamended plot. Metsulfuron-methyl in surface soil persisted till 15 days in control and 20 days in fly ash amended plots and no metsulfuron-methyl leached down to subsurface soil. Fly ash amendment had no adverse effect on the bioactivity of herbicides and yield of soybean and wheat. The study suggested that fly ash amendment to soil can be exploited to retain applied herbicides in surface soil.

Joint ecotoxicology of cadmium and metsulfuron-methyl in wheat (Triticum aestivum).

Herbicide is indispensable for crop production. However, substantial usage of herbicide has led to its increasing accumulation in soils and crops. In addition, cadmium has become one of the widely occurring contaminants in soils due to its significant release into environment via anthropogenic activities. In this study, ecotoxicological investigations were made by exposing the food crop wheat to joint contaminations of Cd and metsulfuron-methyl, a sulfonylurea herbicide. We analyzed growth and physiological and molecular responses in wheat exposed to 0.5 mg kg(-1) Cd and 0.02 mg kg(-1) metsulfuron-methyl (MSM). Soils contaminated with Cd and MSM complex caused significantly detrimental effect on wheat growth and physiological process. Combinative treatments with Cd and MSM damage more severely the plant cells as compared with Cd or MSM treatment alone. Compared with the growth parameter, the biochemical and molecular responses of wheat appeared more pronounced to Cd and MSM complex. Furthermore, compared with control, wheat plants exposed to Cd + MSM generated more O(2-.)and H2O2, both of which were shown to be the cause of enhanced activity of several antioxidant enzymes. Native polyacrylamide gel eletrophoresis and molecular response analyses were performed to validate the results indicated above. Our results indicated that joint contamination with Cd and MSM was more toxic to wheat than a single contamination. These sensitive biological parameters can be used as biomarkers monitoring the ecotoxicological process in plants.

Effects of repeated pulsed herbicide exposures on the growth of aquatic macrophytes.

Many contaminants are released into aquatic systems intermittently in a series of pulses. Pulse timing and magnitude can vary according to usage, compound-specific physicochemical properties, and use area characteristics. Standard laboratory ecotoxicity tests typically employ continuous exposure concentrations over defined durations and thus may not accurately and realistically reflect the effects of certain compounds on aquatic organisms, resulting in potential over- or underestimation. Consequently, the relative effects of pulsed (2 and 4 d) and continuous exposures of the duckweed Lemna minor to isoproturon, metsulfuron-methyl, and pentachlorophenol over a period of 42 d were explored in the present study. At the highest test concentrations, exposure of L. minor to pulses of metsulfuron-methyl resulted in effects on growth similar to those of an equivalent continuous exposure. For isoproturon, pulsed exposures had a lower impact than a corresponding continuous exposure, whereas the effect of pentachlorophenol delivered in pulses was greater. These differences may be explained by compound-specific uptake and degradation or dissipation rates in plants and the recovery potential that occurs following pulses for different pesticides. Given these results, use of a simple time-weighted average approach to estimate effects of intermittent exposures from short-term standard toxicity studies may not provide an accurate prediction that reflects realistic exposure scenarios. Development of mechanistic modeling approaches may facilitate better estimates of effects from intermittent exposures.

Designing allosteric regulators of thrombin. Monosulfated benzofuran dimers selectively interact with Arg173 of exosite 2 to induce inhibition.

Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. J. Med. Chem.201154 5522-5531). To identify the site of binding of SBDs, we studied thrombin inhibition in the presence of exosite 1 and 2 ligands. Whereas hirudin peptide and heparin octasaccharide did not affect the IC(50) of thrombin inhibition by a high affinity SBD, the presence of full-length heparin reduced inhibition potency by 4-fold. The presence of γ' fibrinogen peptide, which recognizes Arg93, Arg97, Arg173, Arg175, and other residues, resulted in a loss of affinity that correlated with the ideal Dixon-Webb competitive profile. Replacement of several arginines and lysines of exosite 2 with alanine did not affect thrombin inhibition potency, except for Arg173, which displayed a 22-fold reduction in IC(50). Docking studies suggested a hydrophobic patch around Arg173 as a plausible site of SBD binding to thrombin. The absence of the Arg173-like residue in factor Xa supported the observed selectivity of inhibition by SBDs. Cellular toxicity studies indicated that SBDs are essentially nontoxic to cells at concentrations as high as 250 mg/kg. Overall, the work presents the localization of the SBD binding site, which could lead to allosteric modulators of thrombin that are completely different from all clinically used anticoagulants.

Use of duckweed (Lemna disperma) to assess the phytotoxicity of the products of Fenton oxidation of metsulfuron methyl.

Because of pressure on water supplies world-wide, there is increasing interest in methods of remediating contaminated ground waters. However, with some remediation processes, the breakdown products are more toxic than the original contaminant. Organic matter and salinity may also influence degradation efficiency. This study tested the efficiency of Fenton oxidation in degrading the sulfonylurea herbicide metsulfuron methyl (MeS), and tested the reaction products for phytotoxicity with the Lemna (duckweed) bioassay. The efficiency of degradation by Fenton's reagent (Fe(2+)=0.09 mM; H(2)O(2)=1.76 mM, 4h) decreased with increasing initial MeS concentration, from 98% with 5 mg/L MeS, to 63% with 70 mg/L MeS. Addition of NaCl (10mM) and organic matter (humic acid at 0.2 and 2.0mg C/L as Total Organic Carbon) reduced the efficiency of degradation at low initial MeS concentrations (5 and 10 mg/L), but had no effect at high concentrations. The residual Fenton's reagent after Fenton's oxidation was toxic to Lemna. After removal of residual iron and H(2)O(2), the measured toxicity to Lemna in the treated samples could be explained by the concentrations of MeS as measured by HPLC/UV detection, so there was no evidence of additional toxicity or amelioration due to the by-products or formulation materials.

Can time-weighted average concentrations be used to assess the risks of metsulfuron-methyl to Myriophyllum spicatum under different time-variable exposure regimes?

We tested the effects of the herbicide metsulfuron-methyl on growth of the submerged macrophyte Myriophyllum spicatum under laboratory conditions using different exposure scenarios. The exposures of each scenario were comparable in the concentration × time factor, viz., the same 21-d time-weighted average (TWA) concentrations but variable in peak exposure concentrations (ranging from 0.1 to 21000 ng ai L⁻¹) and exposure periods (1, 3, 7, 14 or 21 d). To study recovery potential of the exposed M. spicatum plants we continued the observation on shoot and root growth for another 21 d in herbicide-free medium so that the total observation period was 42 d. Non-destructive endpoints, length and number of new shoots and roots, were determined weekly from day 14 onwards. Destructive endpoints, dry-weight (DW) of main shoots, new shoots and new roots, were measured at the end of the experiment (t=42 d). Metsulfuron-methyl exposure in particular inhibited new tissue formation but was not lethal to main shoots. On days 21 and 42 after start exposure, EC10/EC50 values for new tissues expressed in terms of peak concentration (=measured concentration during exposure periods of different length) showed large differences between exposure scenarios in contrast to EC10/EC50 values for days 21 and 42 expressed in terms of 21-d and 42-d TWA concentrations, respectively. At the end of the experiment (day 42), 42-d TWA EC(x) values were remarkably similar between exposure scenarios, while a similar trend could already be observed on day 21 for 21-d TWA EC(x) values. For the macrophyte M. spicatum and exposure to the herbicide metsulfuron-methyl the TWA approach seems to be appropriate to use in the risk assessment. However, the data from the toxicity experiment suggest that on day 21 also the absolute height of the pulse exposure played a (minor) role in the exposure - response relationships observed.