Isolation and Purification of Potent Growth Inhibitors from Piper methysticum Root.

Piper methysticum (kava) root is known to possess promising weed suppressing activity. The present study was conducted to search for potent plant growth inhibitors from the root of this medicinal pepper plant. The ethyl acetate (EtOAc) extract exhibited the strongest reduction on growth of Raphanus sativus (radish) (IC50 shoot and root growth = 172.00 and 51.31 µg/mL respectively) among solvent extracts. From this active extract, nine potent growth inhibitors involved in the inhibitory activities of P. methysticum root were isolated, purified and characterized by column chromatography (CC), gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The six fractions purified by CC included two flavanones: 5-hydroxy-4',7-dimethoxyflavanone (C1) and 5,7-dihydroxy-4'-methoxy-6,8-dimethylflavanone (matteucinol, C2) and six kavalactones: 5,6-dehydro-kavain (C3), a mixture of kavain and yagonin (C4), yagonin (C5) and dihydro-5,6-dehydrokavain, 7,8-dihydrokavain, dihydromethysticin and methysticin (C6). The amounts of 5-hydroxy-4',7-dimethoxyflavanone, matteucinol, 5,6-dehydrokavain and yangonin were 0.76, 2.50, 2.75 and 2.09 mg/g dry weight (DW), respectively. The two flavanones C1 and C2 exhibited the strongest inhibition on shoot elongation (IC50 = 120.22 and 248.03 µg/mL, respectively), whilst the two kavalactone mixtures C4 and C6 showed the highest suppression on root growth of R. sativus (IC50 = 7.70 and 15.67 µg/mL, respectively). This study was the first to report the purification and inhibitory activities of the two flavanones 5-hydroxy-4',7-dimethoxyflavanone and matteucinol in P. methysticum root. The isolated constituents from P. methysticum root including the flavanones C1 and C2 and the mixtures C4 and C6 may possess distinct modes of action on plant growth. Findings of this study highlighted that the combinations of hexane-ethyl acetate by 9:1 and 8:2 ratios successfully purified flavanones and kavalactones in P. methysticum root.

Natural products, their derivatives, mimics and synthetic equivalents: role in agrochemical discovery.

Natural products (NPs) have a long history as a source of, and inspiration for, novel agrochemicals. Many of the existing herbicides, fungicides, and insecticides have their origins in a wide range of NPs from a variety of sources. Owing to the changing needs of agriculture, shifts in pest spectrum, development of resistance, and evolving regulatory requirements, the need for new agrochemical tools remains as critical as ever. As such, NPs continue to be an important source of models and templates for the development of new agrochemicals, demonstrated by the fact that NP models exist for many of the pest control agents that were discovered by other means. Interestingly, there appear to be distinct differences in the success of different NP sources for different pesticide uses. Although a few microbial NPs have been important starting points in recent discoveries of some insecticidal agrochemicals, historically plant sources have contributed the most to the discovery of new insecticides. In contrast, fungi have been the most important NP sources for new fungicides. Like insecticides, plant-sourced NPs have made the largest contribution to herbicide discovery. Available data on 2014 global sales and numbers of compounds in each class of pesticides indicate that the overall impact of NPs to the discovery of herbicides has been relatively modest compared to the impact observed for fungicides and insecticides. However, as new sourcing and approaches to NP discovery evolve, the impact of NPs in all agrochemical arenas will continue to expand. © 2016 Society of Chemical Industry.

Microbial degradation of herbicides.

Herbicides remain the most effective, efficient and economical way to control weeds; and its market continues to grow even with the plethora of generic products. With the development of herbicide-tolerant crops, use of herbicides is increasing around the world that has resulted in severe contamination of the environment. The strategies are now being developed to clean these substances in an economical and eco-friendly manner. In this review, an attempt has been made to pool all the available literature on the biodegradation of key herbicides, clodinafop propargyl, 2,4-dichlorophenoxyacetic acid, atrazine, metolachlor, diuron, glyphosate, imazapyr, pendimethalin and paraquat under the following objectives: (1) to highlight the general characteristic and mode of action, (2) to enlist toxicity in animals, (3) to pool microorganisms capable of degrading herbicides, (4) to discuss the assessment of herbicides degradation by efficient microbes, (5) to highlight biodegradation pathways, (6) to discuss the molecular basis of degradation, (7) to enlist the products of herbicides under degradation process, (8) to highlight the factors effecting biodegradation of herbicides and (9) to discuss the future aspects of herbicides degradation. This review may be useful in developing safer and economic microbiological methods for cleanup of soil and water contaminated with such compounds.

[The method of multicomponent determination of herbicides of various chemical classes in water].

In the work there are considered results of the development of the multicomponent method of measurement of concentration of herbicides of various chemical nature under their joint presence in the water. There was justified the optimality of application of HPLC-DAD (the working wavelength of 240 nm) for the determination of levels of 10 active ingredients of herbicides of class of sulfonylurea (metsulfuron-methyl, nikosulfuron, sulfometuron-methyl, thifensulfuron-methyl, triflusulfuron-methyl), imidazolinone (imazapyr, imazethapyr), 2,6-Bis[(4,6-dimethoxy-2- pyrimidinyl)oxy]benzoic acid (bispyribac acid), triazol-pyrimidines (Penoxsulam), a benzoylpyrazole compound (Topramenzone). For the concentrating and cleaning of samples of water there were used cartridges for solid-phase extraction of Oasis HLB - the macro porous copolymer made on the basis of the balanced ratio of 2 monomers - lipophilic divinylbenzene and hydrophilic N-vinylpirrolidone. The range of the detected concentrations in water was volatile between 0.0005 and 0.005 mg/L, values of standard deviation vary in the range of 1.8-3.9%. Chlorine-containing acidic herbicides were analyzed by the method of GC-ECD and GC-MS (IE) after preliminary converting of compounds into flying derivatives with the use of diazomethane. Satisfactory extraction of substances from a water sample may be achieved by classical extraction in the system "liquid-liquid" with the application of Methyl tert-butyl ester. For cleaning of the derivatized sample there were used cartridges for solid-phase extraction on the basis of silica gel. The range of the determination of 9 active ingredients referring to classes of phenoxy-acetic acid (2,4- D, MCPA), pyridinecarboxylic (aminopyiralid, picloram, clopyralid), benzoic acids (dicamba), benzothiadiazinone (bentazone), biphenyl ester (acifluorfen) and a chloroacetamide (acetochlor) - 0.0001-0.001 mg/L, SD values vary in the range of 1.8-33%.

Performance of different herbicides in dry-seeded rice in Bangladesh.

A field study was conducted in the boro season of 2011-12 and aman season of 2012 at Jessore, Bangladesh, to evaluate the performance of sequential applications of preemergence herbicides (oxadiargyl 80 g ai ha(-1), pendimethalin 850 g ai ha(-1), acetachlor + bensulfuranmethyl 240 g ai ha(-1), and pyrazosulfuron 15 g ai ha(-1)) followed by a postemergence herbicide (ethoxysulfuron 18 g ai ha(-1)) in dry-seeded rice. All evaluated herbicides reduced weed density and biomass by a significant amount. Among herbicides, pendimethalin, oxadiargyl, and acetachlor + bensulfuranmethyl performed very well against grasses; pyrazosulfuron, on the other hand, was not effective. The best herbicide for broadleaf weed control was oxadiargyl (65-85% control); pendimethalin and acetachlor + bensulfuraonmethyl were not effective for this purpose. The best combination for weed control was oxadiargyl followed by ethoxysulfuron in the boro season and oxadiargyl followed by a one-time hand weeding in the aman season. Compared with the partial weedy plots (hand weeded once), oxadiargyl followed by ethoxysulfuron (4.13 t ha(-1)) provided a 62% higher yield in the boro season while oxadiargyl followed by a one-time hand weeding (4.08 t ha(-1)) provided a 37% higher yield in the aman season.

Genetic Programming as a tool for identification of analyte-specificity from complex response patterns using a non-specific whole-cell biosensor.

Whole-cell biosensors are mostly non-specific with respect to their detection capabilities for toxicants, and therefore offering an interesting perspective in environmental monitoring. However, to fully employ this feature, a robust classification method needs to be implemented into these sensor systems to allow further identification of detected substances. Substance-specific information can be extracted from signals derived from biosensors harbouring one or multiple biological components. Here, a major task is the identification of substance-specific information among considerable amounts of biosensor data. For this purpose, several approaches make use of statistical methods or machine learning algorithms. Genetic Programming (GP), a heuristic machine learning technique offers several advantages compared to other machine learning approaches and consequently may be a promising tool for biosensor data classification. In the present study, we have evaluated the use of GP for the classification of herbicides and herbicide classes (chemical classes) by analysis of substance-specific patterns derived from a whole-cell multi-species biosensor. We re-analysed data from a previously described array-based biosensor system employing diverse microalgae (Podola and Melkonian, 2005), aiming on the identification of five individual herbicides as well as two herbicide classes. GP analyses were performed using the commercially available GP software 'Discipulus', resulting in classifiers (computer programs) for the binary classification of each individual herbicide or herbicide class. GP-generated classifiers both for individual herbicides and herbicide classes were able to perform a statistically significant identification of herbicides or herbicide classes, respectively. The majority of classifiers were able to perform correct classifications (sensitivity) of about 80-95% of test data sets, whereas the false positive rate (specificity) was lower than 20% for most classifiers. Results suggest that a higher number of data sets may lead to a better classification performance. In the present paper, GP-based classification was combined with a biosensor for the first time. Our results demonstrate GP was able to identify substance-specific information within complex biosensor response patterns and furthermore use this information for successful toxicant classification in unknown samples. This suggests further research to assess perspectives and limitations of this approach in the field of biosensors.

Solvent (ionic liquid) impregnated resin-based extraction coupled with dynamic ultrasonic desorption for separation and concentration of four herbicides in environmental water.

A new method was developed for the determination of monolinuron, propazine, linuron, and prebane in environmental water samples. The solvent (ionic liquid) impregnated resin (IL-SIR)-based extraction coupled with dynamic ultrasonic desorption (DUSD) was applied to the separation and concentration of the analytes. The high performance liquid chromatography (HPLC) was applied to the determination of the analytes. The ionic liquid [C(6)MIM][PF(6)] was immobilized on Diaion HP20 resin by immersing the resin in ethanol solution containing [C(6)MIM][PF(6)]. The effect of extraction parameters, including pH value of sample solution, salt concentration in sample and extraction time, and elution conditions, including the concentration of ethanol in elution solvent, the flow rate of elution solvent and the ultrasonic power, were examined and optimized. The limits of detection and quantification for the analytes were in the range of 0.15-0.29 µg L(-1) and 0.51-0.98 µg L(-1), respectively. Some environmental water samples were analyzed and the analytical results were satisfactory.

The action of herbicides on fatty acid biosynthesis and elongation in barley and cucumber.

BACKGROUND: Herbicides that affect lipid metabolism have been used commercially for many years. Here, napropamide, diphenamid, dimethachlor and cafenstrole are compared; these have all been classified by the Herbicide Resistance Action Committee (HRAC) as K(3) herbicides and inhibitors of cell division and/or synthesis of very-long-chain fatty acids (VLCFAs). In addition, spiro-decanedione A and pinoxaden dione are compared as inhibitors of lipid synthesis through inhibition of acetyl-CoA carboxylase (ACCase). RESULTS: Whereas the chloracetamide dimethachlor and the carboxyamide cafenstrole potently inhibited VLCFA synthesis in both barley and cucumber, the acetamides napropamide and diphenamid which are also classified as K(3) herbicides and likewise the unclassified herbicide cinmethylin did not. The graminicide pinoxaden dione inhibited de novo fatty acid synthesis in barley, but not in cucumber, and correspondingly inhibited the plastid form of maize ACCase much more than the cytosolic form (IC(50) values of 0.1 and 17 microM). By contrast, spiro-decanedione A exhibited herbicidal effects not only on grasses but also on broad leaves, strongly inhibited maize cytosolic ACCase and inhibited synthesis of VLCFAs in cucumber. CONCLUSIONS: The acetamides napropamide and diphenamid, which do not inhibit VLCFA synthesis, should be classified separately from K(3) herbicides that do. Pinoxaden dione and spiro-decanedione A represent new classes of chemicals acting on plant lipid synthesis.

Biomonitoring of genotoxic risk in agricultural workers from five colombian regions: association to occupational exposure to glyphosate.

In order to assess possible human effects associated with glyphosate formulations used in the Colombian aerial spray program for control of illicit crops, a cytogenetic biomonitoring study was carried out in subjects from five Colombian regions, characterized by different exposure to glyphosate and other pesticides. Women of reproductive age (137 persons 15-49 yr old) and their spouses (137 persons) were interviewed to obtain data on current health status, history, lifestyle, including past and current occupational exposure to pesticides, and factors including those known to be associated with increased frequency of micronuclei (MN). In regions where glyphosate was being sprayed, blood samples were taken prior to spraying (indicative of baseline exposure), 5 d after spraying, and 4 mo after spraying. Lymphocytes were cultured and a cytokinesis-block micronucleus cytome assay was applied to evaluate chromosomal damage and cytotoxicity. Compared with Santa Marta, where organic coffee is grown without pesticides, the baseline frequency of binucleated cells with micronuclei (BNMN) was significantly greater in subjects from the other four regions. The highest frequency of BNMN was in Boyaca, where no aerial eradication spraying of glyphosate was conducted, and in Valle del Cauca, where glyphosate was used for maturation of sugar cane. Region, gender, and older age (> or =35 yr) were the only variables associated with the frequency of BNMN measured before spraying. A significant increase in frequency of BNMN between first and second sampling was observed in Narino, Putumayo, and Valle immediately (<5 d) after spraying. In the post-spray sample, those who reported direct contact with the eradication spray showed a higher quantitative frequency of BNMN compared to those without glyphosate exposure. The increase in frequency of BNMN observed immediately after the glyphosate spraying was not consistent with the rates of application used in the regions and there was no association between self-reported direct contact with eradication sprays and frequency of BNMN. Four months after spraying, a statistically significant decrease in the mean frequency of BNMN compared with the second sampling was observed in Narino, but not in Putumayo and Valle del Cauca. Overall, data suggest that genotoxic damage associated with glyphosate spraying for control of illicit crops as evidenced by MN test is small and appears to be transient. Evidence indicates that the genotoxic risk potentially associated with exposure to glyphosate in the areas where the herbicide is applied for coca and poppy eradication is low.

Natural products that have been used commercially as crop protection agents.

Many compounds derived from living organisms have found a use in crop protection. These compounds have formed the basis of chemical synthesis programmes to derive new chemical products; they have been used to identify new biochemical modes of action that can be exploited by industry-led discovery programmes; some have been used as starting materials for semi-synthetic derivatives; and many have been used or continue to be used directly as crop protection agents. This review examines only those compounds derived from living organisms that are currently used as pesticides. Plant growth regulators and semiochemicals have been excluded from the review, as have living organisms that exert their effects by the production of biologically active secondary metabolites.

Pesticides selectivity list to beneficial arthropods in four field vegetable crops.

Selectivity of pesticides to beneficial arthropods is a key data for the implementation of IPM program. In the context of field vegetables crop, a set of 16 fungicides, 17 herbicides and 14 insecticides commonly used in Belgium were tested on 5 indicator species: the parasitic hymenoptera Aphidius rhopalosiphi (De Stefani-Perez) (Hym., Aphidiidae), the aphid foliage dwelling predators Adalia bipunctata (L.) (Col., Coccinellidae) and Episyrphus balteatus (Dipt., Syrphidae) and the ground-dwelling predators Aleochara bilineata (Col., Staphyllinidae) and Bembidion lampros (Col., Carabidae). Pesticides were tested according a testing scheme including a first assessment on inert substrate (glass plates for adults of A. rhopalosiphi, larvae of A. bipunctata and E. balteatus, sand on adults of A. bilineata and B. lampros) and, for product that were toxic, a second assessment on natural substrate (barley seedlings for A. rhopalosiphi, french bean plants for A. bipunctato and E. balteatus and two type of soil for 8. lampros and A. bilineato). The effects of the product were assessed on basis on mortality, except for A. bilineata (Onion fly pupae parasitism). According to the final results obtained at the end of this testing scheme, the product were listed in toxicity class: green list if effect < or =30%, yellow list 30% < effect < 60% and orange list 60% < effect < or =80%. Products with toxicity higher than 80% on plants or on soils, or that reduce parasitism more than 80% on soil were put in red list and are not recommended for IPM. Results showed that all fungicides and herbicides were included in the green list except tebuconazole and boscalid + pyraclostrobin that were labeled as yellow for A. bipunctata. In opposite, no foliar insecticide was totally selective for all beneficial tested. However some products are in green list for one or several species. Soil insecticides were all are very toxic for ground dwelling arthropods and classed in red list. All results obtained during this study and further upgrade will be available on www.cra.wallonie.be/selectivite. In conclusions, fungicides and herbicides tested are compatible with IPM programs. For foliar insecticides, some treatments can be used carefully according to the selectivity. But for soil insecticide treatments, their toxicity raise the question of their use in IPM programs in vegetables and the need of new compounds or development of alternative pest control programs.

Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides.

Development of hybrid rice has greatly contributed to increased yields during the past three decades. Two bentazon-lethal mutants 8077S and Norin8m are being utilized in developing new hybrid rice systems. When the male sterile lines are developed in such a mutant background, the problem of F1 seed contamination by self-seeds from the sterile lines can be solved by spraying bentazon at seedling stage. We first determined the sensitivity of the mutant plants to bentazon. Both mutants showed symptoms to bentazon starting from 100 mg/l, which was about 60-fold, lower than the sensitivity threshold of their wild-type controls. In addition, both mutants were sensitive to sulfonylurea-type herbicides. The locus for the mutant phenotype is bel for 8077S and bsl for Norin8m. Tests showed that the two loci are allelic to each other. The two genes were cloned by map-based cloning. Interestingly, both mutant alleles had a single-base deletion, which was confirmed by PCR-RFLP. The two loci are renamed bel ( a ) (for bel) and bel ( b ) (for bsl). The wild-type Bel gene encodes a novel cytochrome P450 monooxgenase, named CYP81A6. Analysis of the mutant protein sequence also revealed the reason for bel ( a ) being slightly tolerant than bel ( b ). Introduction of the wild-type Bel gene rescued the bentazon- and sulfonylurea-sensitive phenotype of bel ( a ) mutant. On the other hand, expression of antisense Bel in W6154S induced a mutant phenotype. Based on these results we conclude that the novel cytochrome P450 monooxygenase CYP81A6 encoded by Bel confers resistance to two different classes of herbicides.

Toxicity assessment of metabolites of fungal biocontrol agents using two different (Artemia salina and Daphnia magna) invertebrate bioassays.

Fungal biocontrol agents (BCAs) have been marketed for control of crop pests, weeds, and diseases. However, BCAs may produce toxic metabolites, whose presence in the formulated products, in the crops and in the environment should be considered along with the associated risk. Two invertebrate models, viz. Artemia salina and Daphnia magna were used to assess the acute toxicity of seven BCA metabolites, characterized by different chemical nature and mode of action, namely alamethicin (ALA), paracelsin (PCS), antiamoebin (AAM), gliotoxin (GTX), destruxin A (DA), oosporein (OOS), and elsinochrome A (EA). The two invertebrates were very sensitive to all the metabolites examined, except OOS. The LC50s after 24 and 36 h exposures showed the following toxicity ranks: A. salina, DA > ALA > EA > GTX > AAM > PCS (LC50s ranging from 9.78 to 40.84 microg/ml at 24 h and from 2.92 to 18.56 microg/ml at 36 h); D. magna, DA > GTX = EA > ALA > PCS > AAM (LC50s ranging from 0.20 to 24.41 microg/ml at 24h and from 0.16 to 11.98 microg/ml at 36 h). LC50 of OOS to D. magna increased dramatically in 36 h exposure, compared to 24 h exposures (5.84 and 68.40 microg/ml, respectively). A. salina and D. magna proved to be suitable models for rapid and inexpensive screening of toxicity of BCAs at an early stage of product development.

Analysis of reproductive toxicity and classification of glufosinate-ammonium.

CONCLUSION REGARDING CLASSIFICATION OF GLUFOSINATE-AMMONIUM: Science Partners' Evaluation Group (Evaluation Group) has conducted an independent analysis of the herbicide glufosinate-ammonium (GA) relative to its potential to cause reproductive toxicity in humans. Further, the Evaluation Group has evaluated the implementation of Annex 6 of Commission Directive 2001/59/EC (28th ATP of Council Directive 67/548/EEC) and Council Directive 91/414/EEC, with respect to classification of chemicals posing potential reproductive hazards. After consideration of all information available to us relevant to the potential of glufosinate-ammonium (GA) to cause reproductive toxicity, the Science Partners Evaluation Group concludes that no classification of GA is justified. The following form the basis of this conclusion. There are no human data to suggest that GA causes reproductive toxicity in women or in their conceptus. The issue concerning possible reproductive hazard to humans is raised solely on the basis of positive animal test results that show GA to cause preimplantation or implantation losses in rats. SPECIFICALLY: a. Daily treatment with GA had no detectable effect on the earliest stages of the reproductive sequence including gametogenesis, ovulation, mating and conception; b. Treatment with GA interfered with rat gestation before and at the stage when the conceptus implants into the uterus. This effect occurred at doses of 360 ppm in the feed (corresponding to daily doses of 27.8 mg/kg bw) and above; and c. After implantation, no further effect of GA on prenatal and post-natal development was recognized. Previous concerns that GA might be toxic to embryonic stages after implantation were not supported by the data. Abortions and stillbirth seen were associated with, and regarded as secondary to, maternal toxicity. There was no evidence suggesting the induction of malformations in the offspring. The mechanism underlying this adverse effect in experimental laboratory animals is identified-inhibition of glutamine synthetase. Glutamine is essential to the viability of the embryo. The embryo is dependent on a maternal source of the amino acid. For embryo lethality to occur, a significant reduction of maternal glutamine is required. Such reduction in maternal glutamine depends on a significant inhibition of glutamine synthetase by GA. This can only occur when the mother is exposed to very high levels of GA. SPECIFICALLY: a. The reproductive toxicity of GA is confined to very short, early stages of reproduction, during which the conceptus is dependent on maternal glutamine; and b. In order for the effect to occur, significant reduction in maternal blood glutamine level is required, which in turn depends on a significant inhibition of glutamine synthetase, induced by high levels of GA in the maternal system. There is no evidence for accumulation of GA in the mammalian organism beyond a factor of two and no evidence for its metabolic toxification. To raise a concern in humans, women would have to be exposed to GA during the very limited time frame of preimplantation or implantation and the exposure would have to be to the exceedingly high levels necessary to alter the maternal metabolism and, correspondingly, result in glutamine levels in maternal tissue and blood plasma being drastically reduced. There is no basis to suggest that such exposures would occur under conditions of normal handling and use. SPECIFICALLY: a. Under conditions of normal handling and use, operators would never be exposed to GA levels that could potentially inhibit glutamine synthetase to the extent that this inhibition could impair preimplantation or implantation. b. All acceptable exposure measurements and predictive calculations confirm this conclusion, and in fact demonstrate that reasonably foreseeable exposure of workers would be to levels significantly below the AOEL. c. The evidence is also clear that there is no reproductive toxicity hazard to workers upon reentry tosprayed fields, bystanders, consumers or toddlers. The safety margin compared to the NOAEL in animal studies is sufficiently large to assure protection of the health of workers using GA as well as bystanders, consumers, and toddlers. Pursuant to Annex 6 of Commission Directive 2001/59/EC (28th ATP of Council Directive 67/548/EEC), to justify a classification of category 2 there must be sufficient evidence to produce a strong presumption that human exposure to the substance may result in impaired fertility in humans. It is the conclusion of the Science Partners Evaluation Group that there is no reasonable evidence to suggest a strong presumption of impairment. To the contrary, there is clear evidence demonstrating a strong presumption that exposure to GA would not cause the adverse effect demonstrated in rats. Pursuant to Annex 6 of Commission Directive 2001/59/EC (28th ATP of Council Directive 67/548/EEC), to justify a classification of category 3, there must be sufficient evidence to provide a strong suspicion of impaired fertility in humans. There is no basis to conclude that the animal data demonstrating impaired preimplantation or implantation has any relevance to humans in that the effect found in rats only occurs at levels which would never be experienced by workers under conditions of normal handling and use or by bystanders, consumers, or toddlers.

Effects of five rice herbicides on the growth of two threatened aquatic ferns.

The effects of five rice herbicides bensulfuron methyl, mefenacet, quinoclamine, simetryn, and thiobencarb on the growth of two threatened aquatic ferns Azolla japonica and Salvinia natans were tested using 12-day exposure experiments at 0.1-100 nM which are expected to be present in drainages and rivers in Japan. As a reference species, Lenma minor was also used to examine the toxicity of bensulfuron methyl. Bensulfuron methyl had the most pronounced effect on the relative growth rate (RGR) of A. japonica, S. natans, and L. minor with an EC50 of 5.0, 0.54, and 10 nM, respectively. The other herbicides reduced the RGR of the aquatic ferns only at the highest concentration (100 nM) or not at all. S. natans showed the highest susceptibility to bensulfuron methyl among the three species, and the EC50 for this species was comparable to or below the maximum concentration (0.49-5.6 nM) that had been previously detected in 7 of 11 rivers in Japan. These results suggest that bensulfuron methyl runoff in drainages and rivers in Japan is expected to have adverse effects on the growth of threatened aquatic ferns in some cases, and that no or small effects occur for the other four herbicides tested.

Toxicity assessment of 40 herbicides to the green alga Raphidocelis subcapitata.

The effects of 40 herbicides with nine modes of action on the green alga Raphidocelis subcapitata were studied by 96-h acute toxicity tests. Results showed that the EC50 of the herbicides with respect to the photosynthetic processes of R. subcapitata ranged from 0.0007 to 4.2286 mgL(-1). Photosynthesis was the process of the green alga most sensitive to the tested herbicides. The most toxic herbicides were atrazine, ametryme, simazine, prometryne, cyanazine, isoproturon, chlorotoluron, diuron, methabenzthiazuron, and paraquat. The EC50 of the protoporphyrinogen oxidase inhibitor oxadiargyl, which was the parameter least sensitive to the herbicides tested in this study, was 42.5 mgL(-1). The descending order of the average acute toxicity to R. subcapitata of herbicides with regard to the nine modes of action was as follows: photosynthetic process>cell division>lipid synthesis, acetyl-coenzyme A carboxylase>acetolactate synthase> 5-enolpyruvyl-shikimate-3-phosphate synthase, glutamine synthase, hormone synthesis>protoporphyrinogen oxidase.

Detection of thyroid system-disrupting chemicals using in vitro and in vivo screening assays in Xenopus laevis.

We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T(3)) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T(3)), > L-thyroxine (T(4)) > 3,3',5'-L-triiodothyronine (rT(3)). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T(3)- antagonist activity at concentrations ranging from 10(-6) to 10(-5) M. These chemicals also inhibited the expression of the endogenous primary T(3)-response TH nuclear receptor beta (TRbeta) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T(3)-dependent activation of TRbeta gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T(3)-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo.

The ecotoxicological effects of Photosystem II herbicides on corals.

The recent discovery of contamination of the tropical marine environment by Photosystem II (PSII) herbicides used in agriculture and antifouling paints has led to concerns regarding the effects on corals and their symbiotic dinoflagellate algae. In reviewing the ecotoxicological studies conducted so far, PSII herbicides appear able to readily penetrate the coral tissues and rapidly (within minutes) reduce the photochemical efficiency of the intracellular algal symbionts. The dinoflagellates appear at least as sensitive to PSII herbicides as other phototrophs tested so far, with photosynthesis being affected at exceptionally low concentrations (i.e. in the ngl(-1) range). At these levels and over short exposure periods, the effects can be fully reversible (i.e. when corals are returned to clean seawater) and vary according to type of herbicide; however, when exposed to higher concentrations in the light or over longer exposure periods, it results in a long-term sustained reduction of the photochemical efficiency of the algae (symptomatic of chronic photoinhibition). This can result in the dissociation of the symbiosis (bleaching) which is a common but nevertheless significant sub lethal stress response requiring many months to recover from. It is argued that the reliance of corals on an endosymbiotic photoautotrophic energy source, together with predilection for the symbiosis to dissociate when photosynthesis of the algae is affected, renders coral particularly susceptible to changes in environmental conditions-and especially phytotoxins such as PSII herbicides.

Selectivity lists of pesticides to beneficial arthropods for IPM programs in carrot--first results.

In order to improve IPM programs in carrot, 7 fungicides, 12 herbicides and 9 insecticides commonly used in Belgium were tested for their toxicity towards five beneficial arthropods representative of most important natural enemies encountered in carrot: parasitic wasps - Aphidius rhopalosiphi (De Stefani-Perez) (Hym., Aphidiidae), ladybirds - Adalia bipunctata (L.) (Col., Coccinellidae), hoverfly - Episyrphus balteatus (Dipt.. Syrphidae), rove beetle - Aleochara bilineata (Col., Staphylinidae) and carabid beetle - Bembidion lampros (Col., Carabidae). Initialy, all plant protection products were tested on inert substrate glass plates or sand according to the insect. Products with a corrected mortality (CM) or a parasitism reduction (PR) lower than 30% were kept for the constitution of positive list (green list). The other compounds were further tested on plant for A. rhopalosiphi, A. bipunctata, E. balteatus and soil for B. lampros and A. bilineata. With these extended laboratory tests results, products were listed in toxicity class: green category [CM or PR < or = 30%], yellow category [30% < CM or PR < or = 60%] and orange category [60% < CM or PR < or = 80%]. Products with toxicity higher than 80% on plants or that reduce parasitism more than 80% on soil were put in red category and are not recommended to Integrated Pest Management programs in carrot. Results showed that all fungicides tested were harmless to beneficials except Tebuconazole, which was slightly harmful for A. bipunctata. Herbicides were also harmless for soil beneficials, except Chlorpropham. This product was very toxic on sand towards A. bilineata and must be tested on soil. All soil insecticides tested were very toxic for ground beneficials and considered as non-selective. Their use in IPM is subject to questioning in view of negative impacts on beneficials. Among foliar insecticides, Dimethoate and Deltamethrin are not recommended for IPM because their high toxicity for all beneficials. The other foliar insecticides were more selective; any of them were harmless for all species tested.

Analytical mass spectrometry of herbicides.

Herbicides are chemical substances that are applied to agricultural soils, gardens, lawns, or plants to destroy or to prevent the growth of undesirable vegetation. The herbicides included in this review are generally synthetic organic compounds that are ingredients in commercial herbicide products that were designated active during late 2002 in the U.S. Environmental Protection Agency's database of registered and canceled pesticide products. The compounds are organized into 21 categories according to their general chemical structures or a common structural group. The herbicides in each category are discussed in terms of their structures, their database electron ionization mass spectra, and their amenability to separation and measurement with gas chromatography, reversed-phase liquid chromatography, and capillary electrophoresis combined with mass spectrometry. Ionization techniques that are considered here are mainly electron ionization, electrospray, and atmospheric pressure chemical ionization. Sixty-six references are provided to herbicide reviews, and to the recent herbicide analytical chemistry and mass spectrometry research literature.