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Formulated glyphosate activates the DNA-response checkpoint of the cell cycle leading to the prevention of G2/M transition.

A glyphosate containing pesticide impedes at 10 mM glyphosate the G2/M transition as judged from analysis of the first cell cycle of sea urchin development. We show that formulated glyphosate prevented dephosphorylation of Tyr 15 of the cell cycle regulator CDK1/cyclin B in vivo, the end point target of the G2/M cell cycle checkpoint. Formulated glyphosate had no direct effect on the dual specific cdc25 phosphatase activity responsible for Tyr 15 dephosphorylation. At a concentration that efficiently impeded the cell cycle, formulated glyphosate inhibited the synthesis of DNA occurring in S phase of the cell cycle. The extent of the inhibition of DNA synthesis by formulated glyphosate was correlated with the effect on the cell cycle. We conclude that formulated glyphosate's effect on the cell cycle is exerted at the level of the DNA-response checkpoint of S phase. The resulting inhibition of CDK1/cyclin B Tyr 15 dephosphorylation leads to prevention of the G2/M transition and cell cycle progression.

Animals↗

Comparative action of glyphosate as a trigger of energy drain in eubacteria.

Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycine], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzymes, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of common-pathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry is an energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coli after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also profound effects upon glyphosate sensitivity.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

Cadmium adsorption in montmorillonite as affected by glyphosate.

Behaviors of soil heavy metals are often affected by coexisting herbicides due to their physical and chemical interaction. Effect of glyphosate, an herbicide containing -PO3(2-) and -COOH groups, on cadmium adsorption in montmorillonite was studied in detail. The results showed that the cadmium adsorption quantity in montmorillonite increased with increasing soil solution pH and cadmium concentration as usual, but decreased with glyphosate, which is due to the formation of a low affinity complex of Cd and glyphosate and decreasing solution pH induced by glyphosate addition. When the equilibrium solution pH was below 6.7, glyphosate has little effect on cadmium adsorption, but when the equilibrium solution pH was above 6.7, glyphosate significantly decreased cadmium adsorption quantity in montmorillonite. In addition, the adding order of Cd and glyphosate also influenced Cd adsorption quantity in montmorillonite.

Adsorption↗

Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.

Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

Enzymological basis for herbicidal action of glyphosate.

The effects of 1 millimolar glyphosate (N-[phosphonomethyl]glycine) upon the activities of enzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata [L.] Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by Mn(2+) (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co(2+) for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co(2+) is a V(max) activator with respect to both substrates, glyphosate cannot act simply by Co(2+) chelation because inhibition is competitive with respect to erythrose-4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimic acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal of growth inhibition in 7-day seedlings of mung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target.

Journal Article↗

Selection of Glyphosate-Tolerant Tobacco Calli and the Expression of this Tolerance in Regenerated Plants.

From nonmutagenized haploid suspensions of Nicotiana tabacum L. cv Wisconsin 38 cells, 51 cell lines capable of growth in the presence of 1 millimolar glyphosate (N-phosphonomethyl glycine) were initially isolated at a frequency of 2.3 x 10(-8). Eighteen cell lines retained tolerance when grown on selective medium for 3 years. Tolerance persisted for at least 14 months in six cell lines cultured in the absence of glyphosate. Some plants regenerated from four glyphosate-tolerant cell lines were tolerant. Glyphosate-tolerant tissue was isolated from some sensitive as well as some tolerant regenerated plants. Six of the tolerant cell lines were also tolerant to the herbicide amitrole (3-amino-1,2,4-triazole). Five cell lines selected for amitrole tolerance were glyphosate tolerant. Some plants regenerated from three of these five cell lines were glyphosate tolerant and glyphosate-tolerant tissue was obtained from several of these regenerated plants. Amitrole uptake in suspension cultures of several variants was assessed in terms of influx rate constants. This parameter was not sufficiently different indicating that altered membrane properties could not account for the herbicide tolerance.

Journal Article↗

Isolation of a Pseudomonas sp. Which Utilizes the Phosphonate Herbicide Glyphosate.

A strain of bacteria has been isolated which rapidly and efficiently utilizes the herbicide glyphosate (N-phosphonomethylglycine) as its sole phosphorus source in a synthetic medium. The strain (PG2982) was isolated by subculturing Pseudomonas aeruginosa ATCC 9027 in a synthetic broth medium containing glyphosate as the sole phosphorus source. Strain PG2982 differs from the culture of P. aeruginosa in that it is nonflagellated, does not produce pyocyanin, and has an absolute requirement for thiamine. Strain PG2982 has been tentatively identified as a Pseudomonas sp. strain by its biochemical activities and moles percent guanine plus cytosine. Measurements of glyphosate with an amino acid analyzer show that glyphosate rapidly disappears from the medium during exponential growth of strain PG2982. In batch culture at 30 degrees C, this isolate completely utilized 1.0 mM glyphosate in 96 h and yielded a cell density equal to that obtained with 1.0 mM phosphate as the phosphorus source. However, a longer lag phase and greater generation time were noted in the glyphosate-containing medium. Strain PG2982 can efficiently utilize glyphosate as an alternate phosphorus source.

Journal Article↗

Degradation of the Herbicide Glyphosate by Members of the Family Rhizobiaceae.

Several strains of the family Rhizobiaceae were tested for their ability to degrade the phosphonate herbicide glyphosate (isopropylamine salt of N-phosphonomethylglycine). All organisms tested (seven Rhizobium meliloti strains, Rhizobium leguminosarum, Rhizobium galega, Rhizobium trifolii, Agrobacterium rhizogenes, and Agrobacterium tumefaciens) were able to grow on glyphosate as the sole source of phosphorus in the presence of the aromatic amino acids, although growth on glyphosate was not as fast as on P(i). These results suggest that glyphosate degradation ability is widespread in the family Rhizobiaceae. Uptake and metabolism of glyphosate were studied by using R. meliloti 1021. Sarcosine was found to be the immediate breakdown product, indicating that the initial cleavage of glyphosate was at the C-P bond. Therefore, glyphosate breakdown in R. meliloti 1021 is achieved by a C-P lyase activity.

Journal Article↗

Studies on a new group of biodegradable surfactants for glyphosate.

The effectiveness of a homologous series of biodegradable rapeseed oil derivatives (triglyceride ethoxylates; Agnique RSO series containing an average of 5, 10, 30 and 60 units of ethylene oxide (EO) as adjuvants for foliage-applied, water-soluble, systemic active ingredients was evaluated employing glyphosate as an example. Previous experiments had revealed that the surfactants used are not phytotoxic at concentrations ranging from 1 to 10 g litre-1. The experiments were performed using Phaseolus vulgaris L and nine selected weed species, grown in a growth chamber at 25/20 (+/- 2) degrees C day/night temperature and 40/70 (+/- 10)% relative humidity. The surfactants were evaluated for enhancement of spray retention, and foliar penetration biological efficacy of glyphosate. Glyphosate was applied at a concentration of 43 mM. The surfactants were added at concentrations of 1 g litre-1. The commercial glyphosate 360 g AE litre-1 SL Roundup Ultra and unformulated glyphosate served as references. The surfactants used improved spray retention, foliar penetration and biological efficacy. Some of the formulations were comparable to the performance of Roundup Ultra in the aspects evaluated; some were even more effective in enhancing spray liquid retention and promoting glyphosate phytotoxicity in several plant species. In these studies Agnique RSO 60 generally was most effective.

Glycine↗

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells.

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli.

The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19) is essential for the biosynthesis of aromatic compounds in plants and microbes and is the unique target of the herbicide glyphosate. One of the first glyphosate-insensitive enzymes reported was a Gly96Ala mutant of EPSP synthase from Klebsiella pneumoniae. We have introduced this single-site mutation into the highly homologous EPSP synthase from Escherichia coli. The mutant enzyme is insensitive to glyphosate with unaltered affinity for its first substrate, shikimate-3-phosphate (S3P), but displays a 30-fold lower affinity for its second substrate, phosphoenolpyruvate (PEP). Using X-ray crystallography, we solved the structure of Gly96Ala-EPSP synthase liganded with S3P to 0.17 nm resolution. The crystal structure shows that the additional methyl group from Ala96 protrudes into the active site of the enzyme. While the interactions between enzyme and S3P remain unaffected, the accessible volume for glyphosate binding is substantially reduced. Exploiting the crystallographic results for molecular modeling, we demonstrate that PEP but not glyphosate can be docked in the Gly96Ala-modified binding site. The predicted PEP binding site satisfies the earlier proposed interaction pattern for PEP with EPSP synthase and corroborates the assumption that glyphosate and PEP target the same binding site.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

Transfer of hexazinone and glyphosate through undisturbed soil columns in soils under Christmas tree cultivation.

Field studies monitoring pesticide pollution in the Morvan region (France) have revealed surface water contamination by some herbicides. The purpose of this study was to investigate in greater detail the transport of two herbicides, used in Christmas tree production in the Morvan, under controlled laboratory conditions. Thus, the leaching of hexazinone (3-cyclohexyl-6-dimethyl-amino-1-methyl-1,3,5-triazine-2,4 (1H,3H) dione) and glyphosate (N-(phosphono-methyl-glycine)) through structured soil columns was studied using one loamy sand and two sandy loams from sites currently under Christmas tree cultivation in the Morvan. The three soils were cultivated sandy brunisol [Sound reference base for soils, D. Baize, M.C. Girard (Coord.), INRA, Versailles, 1998, 322 p] or, according to the FAO [FAO, World reference base for soil resources, ISSS-ISRIC-FAO, FAO, Rome, Italy, 1998], the La Garenne was an arenosol and the two other soils were cambisols. The clay contents of the soils ranged from 86 to 156 g kg(-1) and the organic carbon ranged from 98 to 347 g kg(-1). After 160 mm of simulated rainfall applied over 12 days, 2-11% of the applied hexazinone was recovered in the leachate. The recovery was much higher than that of glyphosate, which was less than 0.01%. The greater mobility of hexazinone might be related to its much lower adsorption coefficient, K(oc), 19-300 l kg(-1), compared with 8.5-10231 l kg(-1) for glyphosate (literature values). Another factor that may explain the higher amounts of hexazinone recovered in the leachates of the three soil columns is its greater persistence (19.7-91 days) relative to that of glyphosate (7.9-14.4 days). The mobility of both herbicides was greater in the soils with higher gravel contents, coarser textures, and lower organic carbon contents. Moreover, glyphosate migration seems negatively correlated not only to soil organic carbon, but also to aluminium and iron contents of soils. This soil column study suggests that at the watershed scale, surface water contamination by hexazinone could occur via the horizontal subsurface flow in upper centimeters of soil. In contrast, the surface water contamination with glyphosate by this mechanism appears unlikely.

Carbon↗

Study on the photocatalytic degradation of glyphosate by TiO(2) photocatalyst.

In this paper, the photocatalytic degradation of glyphosate selected as the deputy of organic pollutant in aqueous solution with TiO(2) powder as a photocatalyst has been studied. The effects of various parameters, such as the amount of the photocatalyst, illumination time, initial pH value, electron acceptors, metal ions, and anions on the photocatalytic degradation of glyphosate were investigated. From the studies, the best condition for the effect of the parameters on the photocatalytic degradation of glyphosate was obtained. The results show that the optimum amount of the photocatalyst used is 6.0 g l(-1) for the photocatalytic reactions. The photodegradation efficiency of glyphosate increases with the increase of the illumination time. With the addition of Fe(3+), Cu(2+), H(2)O(2), K(2)S(2)O(8) or KBrO(3), the photocatalytic degradation of glyphosate is accelerated. However, with the addition of Na(+), K(+), Mg(2+), Ca(2+), Zn(2+), Co(2+) and Ni(2+), or with the addition of trace amounts of Cl(-), Br(-), SO(4)(2-), there are no obvious effects on the reactions. Acidic or alkaline mediums are favorable for the photocatalytic degradation of glyphosate. The possible roles of the additives on the reactions and the possible mechanisms of effect were discussed.

Adsorption↗

Improved coupled-column liquid chromatographic method for the determination of glyphosate and aminomethylphosphonic acid residues in environmental waters.

An existing method for the determination of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) in water has been improved. It is based on precolumn derivatization with the fluorescent reagent 9-fluorenylmethylcloroformate (FMOC) followed by large-volume injection in a coupled-column LC system using fluorescence detection (LC-LC-FD). The derivatization step was slightly modified by changing parameters such as volume and/or concentration of sample and reagents to decrease the limits of quantification (LOQ) of glyphosate and AMPA to 0.1 microg/l. Additionally, the use of Amberlite IRA-900 for preconcentration of glyphosate, prior to the derivatization step, was investigated; the LOQ of glyphosate was lowered to 0.02 microg/l. Drinking, surface and ground water spiked with glyphosate and AMPA at 0.1-10 microg/l concentrations were analysed by the improved LC-LC-FD method. Recoveries were 87-106% with relative standard deviations lower than 8%. Drinking and ground water spiked with glyphosate at 0.02 and 0.1 microg/l were analysed after preconcentration on the anion-exchange resin with satisfactory recoveries (94-105%) and precision (better than 8%).

Glycine↗

Re-evaluation of glyphosate determination in water by liquid chromatography coupled to electrospray tandem mass spectrometry.

An analytical method based on on-line solid-phase extraction-liquid chromatography coupled to electrospray tandem mass spectrometry (SPE-LC-ESI-MS/MS) for the determination of glyphosate and aminomethylphosphonic acid (AMPA) residues has been applied to the analysis of water samples within a Round Robin Study. The method had been previously validated in a variety of water samples and it fulfilled all the parameters of precision, accuracy, sensitivity and unequivocal confirmation. The results within the study that we participated were highly satisfactory in all cases with the only exception of glyphosate in groundwater samples, where surprisingly recoveries around 15% were obtained despite the use of isotope-labeled glyphosate as internal standard (I.S.). A slight modification has been introduced in the method, simply consisting of the acidification of sample with hydrochloric acid (HCl) to pH 1. Then, the sample is neutralized and immediately derivatized with 9-fluorenylmethylchloroformate (FMOC) before LC-MS/MS determination. Round Robin Study samples were reanalyzed using this approach, and the recoveries increased up to 98%. A possible explanation might be the slow kinetic interaction between glyphosate and some components of the matrix. These components might act as chelating agents, making glyphosate unavailable for derivatization and therefore for analysis. Several water samples collected at the Mediterranean area of Spain, and previously analyzed and being found to contain glyphosate, were also reanalyzed using this approach, obtaining higher concentrations (between 2 and 14 times) in most of cases.

Chromatography, Liquid↗

Influence of glyphosate and its formulation (Roundup) on the toxicity and bioavailability of metals to Ceriodaphnia dubia.

This study examined the toxicological interaction between glyphosate (or its formulation, Roundup) and several heavy metals to a freshwater cladoceran, Ceriodaphnia dubia. We demonstrated that all binary combinations of Roundup and metals (Cd, Cu, Cr, Ni, Pb, Se and Zn) exhibited "less than additive" mixture toxicity, with 48-h LC50 toxic unit > 1. Addition of glyphosate alone could significantly reduce the acute toxicity of Ag, Cd, Cr, Cu, Ni, Pb and Zn (but not Hg and Se). The ratio between glyphosate and metal ions was important in determining the mitigation of metal toxicity by glyphosate. A bioaccumulation study showed that in the presence of glyphosate the uptake of some metals (e.g. Ag) was halted but that of others (e.g. Hg) was increased significantly. Therefore, our study strongly suggests that glyphosate and its commercial formulations can control the toxicity as well as the bioavailability of heavy metals in aquatic ecosystems where both groups of chemicals can co-occur.

Adsorption↗

A glyphosate-based pesticide impinges on transcription.

Widely spread chemicals used for human benefits may exert adverse effects on health or the environment, the identification of which are a major challenge. The early development of the sea urchin constitutes an appropriate model for the identification of undesirable cellular and molecular targets of pollutants. The widespread glyphosate-based pesticide affected sea urchin development by impeding the hatching process at millimolar range concentration of glyphosate. Glyphosate, the active herbicide ingredient of Roundup, by itself delayed hatching as judged from the comparable effect of different commercial glyphosate-based pesticides and from the effect of pure glyphosate addition to a threshold concentration of Roundup. The surfactant polyoxyethylene amine (POEA), the major component of commercial Roundup, was found to be highly toxic to the embryos when tested alone and therefore could contribute to the inhibition of hatching. Hatching, a landmark of early development, is a transcription-dependent process. Correlatively, the herbicide inhibited the global transcription, which follows fertilization at the 16-cell stage. Transcription inhibition was dose-dependent in the millimolar glyphosate range concentration. A 1257-bp fragment of the hatching enzyme transcript from Sphaerechinus granularis was cloned and sequenced; its transcription was delayed by 2 h in the pesticide-treated embryos. Because transcription is a fundamental basic biological process, the pesticide may be of health concern by inhalation near herbicide spraying at a concentration 25 times the adverse transcription concentration in the sprayed microdroplets.

Animals↗

Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors.

Glyphosate-based herbicides (e.g. Roundup) are extensively used in the aquatic environment, but there is a paucity of data on the toxicity of the formulated products and the influences by environmental factors. In this study, the acute toxicity of technical-grade glyphosate acid, isopropylamine (IPA) salt of glyphosate, Roundup and its surfactant polyoxyethylene amine (POEA) to Microtox bacterium (Vibrio fischeri), microalgae (Selenastrum capricornutum and Skeletonema costatum), protozoa (Tetrahymena pyriformis and Euplotes vannus) and crustaceans (Ceriodaphnia dubia and Acartia tonsa) was examined and the relative toxicity contributions of POEA to Roundup were calculated. The effects of four environmental factors (temperature, pH, suspended sediment and algal food concentrations) on the acute toxicity of Roundup to C. dubia were also examined. Generally, the toxicity order of the chemicals was: POEA>Roundup>glyphosate acid>IPA salt of glyphosate, while the toxicity of glyphosate acid was mainly due to its high acidity. Microtox bacterium and protozoa had similar sensitivities towards Roundup toxicity (i.e. IC50 from 23.5 to 29.5 mg AE/l). In contrast, microalgae and crustaceans were 4-5 folds more sensitive to Roundup toxicity than bacteria and protozoa. Except photosynthetic microalgae, POEA accounted for more than 86% of Roundup toxicity and the toxicity contribution of POEA was shown to be species-dependent. Increase in pH (6-9) and increase of suspended sediment concentration (0-200 mg/l) significantly increased the toxicity of Roundup to C. dubia, but there were no significant effects due to temperature change and food addition.

Animals↗