Practical High-Throughput Method to Screen Compounds for Anthelmintic Activity against Caenorhabditis elegans.

In the present study, we established a practical and cost-effective high throughput screening assay, which relies on the measurement of the motility of Caenorhabditis elegans by infrared light-interference. Using this assay, we screened 14,400 small molecules from the "HitFinder" library (Maybridge), achieving a hit rate of 0.3%. We identified small molecules that reproducibly inhibited the motility of C. elegans (young adults) and assessed dose relationships for a subset of compounds. Future work will critically evaluate the potential of some of these hits as candidates for subsequent optimisation or repurposing as nematocides or nematostats. This high throughput screening assay has the advantage over many previous assays in that it is cost- and time-effective to carry out and achieves a markedly higher throughput (~10,000 compounds per week); therefore, it is suited to the screening of libraries of tens to hundreds of thousands of compounds for subsequent evaluation and development. The present phenotypic whole-worm assay should be readily adaptable to a range of socioeconomically important parasitic nematodes of humans and animals, depending on their dimensions and motility characteristics in vitro, for the discovery of new anthelmintic candidates. This focus is particularly important, given the widespread problems associated with drug resistance in many parasitic worms of livestock animals globally.

A fatal case after an intravenous injection of levamisole.

Levamisole is a drug originally prescribed as an antihelmintic. Because of the occurrence of severe cases of agranulocytosis and leukoencephalitis it was removed from the French market in 1998 for human use, while it remains available for veterinary use. Nowadays in France its only use in humans is regulated by authorization for temporary use for its immunomodulatory properties in the treatment of nephritic syndrome.A 52-year-old man was found dead at his farm. Injection points were observed on his arm and a syringe containing a dark orange-brown liquid was found near the body. At his home, the discovery of a letter highlighted suicidal intent. Analysis of the aforementioned liquid, peripheral blood and urine confirmed the unique presence of levamisole. The femoral blood concentration of levamisole was of 25 mg/L whereas the femoral blood concentrations reported in cases of fatalities after cocaine use do not exceed 0.0056 mg/L. In humans, levamisole can be detected in biological samples after cocaine use as this drug is also an adulterant and one of its metabolites (aminorex) seems to have amphetamine-like properties. In this case, the man consumed levamisole from time to time for its stimulant and strengthening effects.Cases of fatal poisoning using levamisole are very rare and poorly documented, which makes the interpretation of postmortem blood levamisole concentration difficult.

Nematicidal activity of 'major royal jelly protein'-containing glycoproteins from Acacia honey.

Parasitic nematodes infect more than two billion people worldwide particularly in developing countries. We previously reported nematicidal activity of natural honey using model nematode Caenorhabditis elegans. In this study, characterization of nematicidal effects of natural honey and its glycoproteins has been carried out. Chromatographically separated honey glycoproteins showed potent anti-C. elegans activity (LD50 = 100 ng proteins/µL). Honey glycoproteins with molecular masses of ∼260 kD and ∼160 kD comprised of 'major royal jelly protein-1'-containing complexes. In these complexes, MRJP1 was present in different glycosylation forms. Quantitative PCR based gene expression assays described molecular functions of C. elegans affected by honey and honey glycoproteins. Expression of 14 gene transcripts associated with key cellular and molecular functions including energy metabolism, cytoskeleton, cell division, transcription and translation was analyzed. Acacia honey exerted a concentration-dependent alteration of gene transcripts involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1, act-2, and arp6). Likewise, MRJP1-containing glycoproteins caused down-regulation of arp-6 and idhg-1; and up-regulation of act-1 and mdh-1 gene transcripts. Consistent down-regulation of isocitrate dehydrogenase encoding idhg-1 gene which is among the rate-controlling enzymes of the citric acid cycle was considered as main biochemical factor involved in the nematicidal activity of honey and MRJP-containing glycoproteins. Acacia honey suppressed the expression of gene transcripts encoding actin-2, while honey glycoproteins did not. Hence, honey partly exerted anti-C. elegans activity by decreasing the transcription of actin-2 gene transcripts, demonstrated by a defect in the movement and egg laying. Moreover, arp-6 gene transcripts encoding actin-related protein 6 was significantly and constantly down-regulated by honey and honey proteins.

Report: An ex vivo up-take of levamisole molecules by cestode (Monezia expensa) of goat (Capra hirsa) and its detection through RP-HPLC.

Detection of various molecules of drugs remained a prime issue especially in tissues of animals, humans and in their target parasites. The cestode/tapeworms pose a dilemma because of their weird body composition and uptake pattern of nutrients and medicines especially through absorption by tegument. We selected levamisole; thought to be potent antiparasitic/ani-cestodal drug. The uptake of levamisole (LEV) through cestodeal tissues is studied through HPCL in this paper. High performance liquid chromatography technique has been utilized to know the uptake of levamisole in tissues of cestodes of Goat (Monezia expensa) in small ruminants. The drug was exposed to M. expensa by in vitro till its death or a parasite ceases its movement. The tissue/ part of proglattids of the M. expensa were homogenized with some modifications and levamisole extraction was performed with liquid phase extraction method. The evaporation of solvent was done and the residual cestodal tissues were cleaned by solid phase. After the solid phase extraction method, the recovery of drug, detection and quantification of levamisole from cestodal tissues was determined through Reverse Phase Column High Performance Liquid Chromatography (RP-HPLC). Levamisole (LEV) molecules assay was obtained on a C18 reverse-phase (20um, 6mm x 150mm) column at flow rate of 1ml/min using acetonitrile and ammonium acetate as mobile phase and UV detection was done at 254nm. The development of method of Levamisole (LEV) detection from cestodal tissues by HPLC in vitro samples has been demonstrated first time in Pakistan, which can provide the solution of parasitic control and provide in sight in to the uptake of anti cestodal drugs either against human or livestock parasites.

Plasma pharmacokinetics and muscle residue dynamics of mebendazole in Carassius auratus.

Mebendazole is approved for use in aquatic animals and is widely used in Chinese aquaculture. We developed a pharmacokinetic and residue analysis for mebendazole levels in the goldfish (Carassius auratus). Plasma and muscle samples of C. auratus were taken after oral administration of 10 mg/kg mebendazole. The maximal drug plasma concentration of 0.55 mg/L was achieved at 48 hr and then declined with the elimination half-life (T1/2ß ) of 7.99 hr. Administration of 10 mg/kg by oral gavage for 5 successive days resulted in a peak mebendazole concentration of 0.70 mg/kg in muscle at 96 hr after the last dose. The drug was then eliminated at a relatively slow rate from muscle with T1/2ß of 68.41 hr. There was no detectable mebendazole in any muscle samples at 24 days postadministration. The AUClast in plasma and muscle was 19.42 and 105.33 mg hr/L, respectively. These data provide information for dosage recommendations and withdrawal time determinations for mebendazole use in aquariums.

Closantel plasma and milk disposition in dairy goats: assessment of drug residues in cheese and ricotta.

Closantel (CLS) is currently used in programs for the strategic control of gastrointestinal nematodes. CLS is extralabel used in different dairy goat production systems. From available data in dairy cows, it can be concluded that residues of CLS persist in milk. The current work evaluated the concentration profiles of CLS in plasma and milk from lactating orally treated dairy goats to assess the residues pattern in dairy products such as cheese and ricotta. Six (6) female Saanen dairy goats were treated orally with CLS administered at 10 mg/kg. Blood and milk samples were collected between 0 and 36 days post-treatment. The whole milk production was collected at 1, 4, 7, and 10 days post-treatment to produce soft cheese and ricotta. CLS concentrations in plasma, milk, cheese, whey, and ricotta were determined by HPLC. The concentrations of CLS measured in plasma were higher than those measured in milk at all sampling times. However, the calculated withdrawal time for CLS in milk was between 39 and 43 days postadministration to dairy goats. CLS residual concentrations in cheese (between 0.93 and 1.8 µg/g) were higher than those measured in the milk used for its production. CLS concentrations in ricotta were sixfold higher than those in the milk and 20-fold higher than those in the whey used for its production. The persistent and high residual concentrations of CLS in the milk and in the cheese and ricotta should be seriously considered before issuing any recommendation on the extralabel use of CLS in dairy goat farms.

Residue and dissipation kinetics of novel nematicide fluopyram in rice assessed by QuEChERS-liquid chromatography-tandem mass spectrometry.

Supervised field trial was conducted to study the dissipation pattern of fluopyram in rice plant after application of fluopyram 400 g/L SC(Velum Prime) as soil drenching at the time of sowing in nursery bed at X (500 g a.i. ha-1) and 1.25X (625.2 g a.i. ha-1) doses. Samples of rice plant were collected on 0, 1, 3, 7, 10, 15, and 20 days after transplanting. QuEChERS-based extraction method was validated and adopted to determine the residues of fluopyram in rice seedlings, whole rice grains (with husk), polished rice grain, husk, straw, and soil using LC-MS/MS (liquid chromatography-tandem mass spectrometry).The initial deposit of fluopyram in rice plant recorded were 0.27 and 0.41 mg kg-1in X and 1.25X doses, respectively. Fluopyram residues dissipated following first-order kinetics with half-life of 2.53 and 2.57 days at X and 1.25X doses, respectively. Residues were detected in seedlings up to 15 days after transplanting and were at below LOQ in whole rice grains (with husk), polished rice grain, husk, straw, and soil collected at harvest. Monitoring study revealed that application of novel nematicide fluopyram for the management of nematodes in rice does not pose any risks to consumers.

[Biologically active substances of preparation avercom].

The preparation avercom created on the basis of ethanol extracts from the biomass of Streptomyces avermitilis UCM Ac-2179, contains an antiparasitic antibiotic avermectin, as well as a complex of biologically active substances: amino acids, lipids, including nonsaturated fatty acids, and phytohormones, particularly: auxins, cytokinins, hybberellins. The above mentioned complex is characterized by nematocidical, phytostimulating and elicitor effect upon plants which has been confirmed with the results of production experiments on the cucumber variety Angelina.

Nematocidal Effect of Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) against Haemonchus contortus.

The nematocidal effect of Pleurotus ostreatus (white variety of oyster mushroom) aqueous extract (AE) was evaluated against Haemonchus contortus eggs and infective larvae (L3) in vitro and in artificially infected gerbils (Meriones unguiculatus). The chemical analyses indicated that constituents of AE are tridecanoic, tetradecanoic, linolelaidic, 9,15-octadecadienoic, and oxalic acids. P. ostreatus extract inhibited larval hatching by 100% at the concentration of 2.24 mg/mL and (50% effective concentration) EC50 of 0.73 mg/mL. In the larval development test, AE induced a larvicidal effect at the concentration of 50 mg/mL and EC50 of 17.24 mg/mL. The larval migration test revealed a reduction of 94.7% at a concentration of as low as 4 mg/mL and EC50 of 1.25 mg/mL. No significant effects of treatment with P. ostreatus AE were seen on H. contortus in the gerbil model. Thus, our results demonstrate an important nematocidal in vitro effect of P. ostreatus AE against the parasite H. contortus. However, further investigations are necessary to confirm the anthelmintic potential of P. ostreatus extract in small ruminants.

Transport of a nematicide in surface and groundwaters in a tropical volcanic catchment.

The aim of this article is to determine how the nematicide cadusafos [S,S-di-sec-butyl O-ethyl phosphorodithioate] contaminates water and soils at two scales, subcatchment and catchment. The study site was a small banana (Musa spp.)-growing catchment on the tropical volcanic island of Guadeloupe in the Caribbean. Two application campaigns were conducted, one in 2003 on 40% of the catchment and one in 2006 on 12%. The study involved monitoring for 100 d the surface water and groundwater flows and the cadusafos concentrations in the soil and in surface and groundwaters in a 2400 m(2) subcatchment and a 17.8 ha catchment. The results show that at the subcatchment scale the high retention in the A horizon of the soil limits the transport of cadusafos by runoff, whereas the lower retention of the molecule in the B horizon favors percolation toward the shallow groundwater. Comparing the losses of cadusafos at the subcatchment and at the catchment scales revealed that the nematicide re-infiltrated in the hydrographic network. Two successive phases of stream water contamination were observed, corresponding to two distinct contamination mechanisms: an event-dominated contamination phase (of <30 d) when transport was linked to overland flow during precipitation shortly after application, and a stabilized contamination phase when transport originated mainly from the drainage of the shallow aquifer. Lastly, comparing the losses of the two phases during 2003 and 2006 showed that shallow groundwater, which is promoted in such permeable soils under abundant tropical rainfalls, seems to be the main contributor to stream contamination.

Influence of Bi 58 Nowy (38% dimethoate) on pyrantel embonate concentration in the liver of rats.

The aim of the study was to determine the concentration of pyrantel residues in the liver of rats in different time points after oral administration of pyrantel embonate as well as combined administration of the Bi 58 Nowy preparation (38% of dimethoate) and pyrantel embonate. The experiment was conducted in two stages involving different doses of compounds and modes of exposure. At the first stage, the animals were administered pyrantel embonate with a stomach tube at a dose of 1000 mg/kg b.w. twice in a two-week interval, i.e. on day 14 and 28, and the Bi 58 Nowy preparation with drinking water at a dose of 15.48 mg/kg b.w. for 28 days. At the second stage, the rats received pyrantel embonate at a dose of 400 mg/kg b.w. with a stomach tube for 3 consecutive days, whereas the Bi 58 Nowy preparation was administered at a dose of 38.7 mg/kg b.w. also with a stomach tube for 5 consecutive days. In the rats doubly administered with pyrantel embonate, its residues were present until day 14, whereas when the drug was administered for 3 consecutive days they were present until day 7 of the experiment. The maximum concentration of pyrantel embonate was found in the liver after the 3rd hour, whereas a considerable decrease occurred between the 3rd and the 12th hour. The combined administration of pyrantel embonate and the Bi 58 Nowy preparation caused a significant decrease in the concentration of pyrantel residues in the liver 3 and 6 hours after exposure, as compared to the rats receiving the drug alone.

Discovery of potential ovicidal natural products using metabolomics.

Plant extracts are a potential source of new compounds for nematode control and may be an excellent alternative for the control gastrointestinal nematodes that are resistant to conventional anthelmintics. However, research involving natural products is a complex process. The main challenge is the identification of bioactive compounds. Online analytical techniques with universal detectors, such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), together with metabolomics could enable the fast, accurate evaluation of a massive amount of data, constituting a viable option for the identification of active compounds in plant extracts. This study focused on the evaluation of the ovicidal activity of ethanol extracts from 17 plants collected from the Pantanal wetland in the state of Mato Grosso do Sul, Brazil, against eggs of Haemonchus placei using the egg hatchability test. The ethanol extracts were obtained using accelerated solvent extraction. The data on ovicidal activity, mass spectrometry and metabolomics were evaluated using HPLC-DAD-MS, partial least squares regression analysis (PLS-DA) and a correlation map (univariate correlation analyses) to detect compounds that have a positive correlation with biological activity. Among the ten metabolites with the best correlation coefficients, six were phenylpropanoids, two were triterpene saponins, one was a brevipolide, and one was a flavonoid. Combinations of metabolites with high ovicidal action were also identified, such as phenylpropanoids combined with the triterpene saponins and the flavonoid, flavonoids combined with iridoid and phenylpropanoids, and saponins combined with phenylpropanoid. The positive correlation between classes of compounds in plants belonging to different genera and biological activity (as previously identified in the literature) reinforces the robustness of the statistical data and demonstrates the efficacy of this method for the selection of bioactive compounds without the need for isolation and reevaluation. The proposed method also enables the determination of synergism among the classes, which would be impracticable using traditional methods. The present investigation demonstrates that the metabolomic technique was efficient at detecting secondary metabolites with ovicidal activity against H. placei. Thus, the use of metabolomics can be a tool to accelerate and simplify bioprospecting research with plant extracts in veterinary parasitology.

In vitro assessment of plant lectins with anti-pinwood nematode activity.

Two lectin proteins were purified from the corms of Pinellia ternata and Lycoris radiata. Both P. ternata agglutinin (PTA) protein and L. radiata agglutinin (LRA) protein formed polymers and coagulated both rabbit red blood cells and yeast cells. The two proteins were each diluted to different concentration and then mixed with pinewood nematodes, and nematode survival was measured. Results showed that the two lectin proteins showed significant levels of resistance against nematodes and the nematode population was significantly reduced, compared to PBS buffer without protein control group. The mean number of nematodes of two lectin proteins group was significantly lower than that of control group constantly throughout the assay period with differences being very significant at P<0.01 after 24 h. After 96 h, when 500 microg/ml proteins were used, nematode number significantly declined to an average of 26 (approximately 43% of the controls) and 32.2 (approximately 53.3% of the controls) nematodes at LRA and PTA protein, respectively, compared to the control group. Results also indicated that higher concentrations of protein were more toxic to the pinewood nematode. Even when the concentration was as low as 30 microg/ml, the toxic proteins retained their anti-nematode activity. Furthermore, pinewood nematode was exposed to the proteins for longer, more pinewood nematodes were killed. Our results indicated the two lectin proteins both apparently have a toxic effect on the pinewood nematode that affects its survival in vitro.

Sulfamethazine and flubendazole in seepage water after the sprinkling of manured areas.

Pharmaceuticals are widely used in modern livestock production and can reach the environment via the application of manure containing excreted drugs. Limited information is available on the transport and fate of veterinary medicines applied to soils. Therefore, we assessed the potential for the sulphonamide antibiotic sulfamethazine (SMT) and the antiparasitic drug flubendazole (FLUB) including their metabolites to move from agricultural manure to drainage waters at 1m depth. A comparison was made of losses from sites under different land use (grassland versus arable cropping) as well as losses from neighbouring plots under the same land use. Liquid manure from pigs treated with SMT and FLUB was spread on 10 x 30 m2 plots (750l per plot). SMT concentration in slurry ranged from 600 to 1700 microg l(-1) (metabolite acetyl-SMT 280-1700 microg l(-1)) and FLUB concentration ranging from 25 to 56 microg l(-1) (metabolite amino-FLUB 32-110 microg l(-1), hydroxy-FLUB 19-38 microg l(-1)). About 1h after application heavy rainfall (50mm in 2.5h) was simulated by sprinkler irrigation. Drainage flow started within 1h after the commencement of sprinkling. SMT and FLUB concentrations in leachate reached values of up to 16 microg l(-1) and 0.3 microg l(-1), respectively. Loss rates (relative to the applied amounts) from the neighbouring sites under arable cropping ranged from 2.8% to 5.4% for SMT and 0.8% to 3.1% for FLUB (including metabolites). On the permanent grassland plot, due to its multitude of macropores, loss rates reached values up to 10% for SMT and 16% for FLUB (including metabolites). These results demonstrate that the variability in leaching of veterinary drugs may be high even between large neighbouring plots, depending on site heterogeneity and land use.

Toxicity and anthelmintic efficacy of crude aqueous of extract of the bark of Sacoglottis gabonensis.

The water extract of the stem bark of Sacoglottis gabonensis was evaluated for its preliminary acute toxicity and anthelmintic efficacy against gastro-intestinal nematodes of small ruminants and mice in both in vitro and in vivo studies. Intra-peritoneal administration of doses ranging from 400 to 3200 mg/kg of the aqueous stem bark extract produced varying degrees of toxicity manifested as depression, drowsiness and unsteady gait, paralysis of the hind limbs, dyspnoea, coma and death. The pathological lesions noted at necropsy were mainly congestion and edema of the lungs, bronchi and bronchioles and hepatomegally with focal necrosis of liver cells. The severity of the clinical symptoms and pathological lesions were dose-related. In the in vitro study, the extract significantly (P<0.05) reduced the hatching of strongyline nematode eggs from naturally infected small ruminants. The 100 mg/ml concentration of the extract produced the highest (94.4%) inhibition on nematode egg hatch and the result was comparable to similar effect produced by either levamisole (100% at 15 mg/ml) or albendazole (99.7% at 6.25 mg/ml). In rats experimentally infected with Heligmosomoides polygyrus, treatment with the S. gabonensis stem bark aqueous extract significantly (P<0.05) reduced adult worm burden and completely inhibited faecal egg output 5 days post treatment.

Kainic acid in the seaweed Palmaria palmata (dulse).

Twenty samples of the seaweed Palmaria palmata (dulse) purchased mainly from commercial Internet shops on the European market were analysed by a liquid chromatograph coupled with a tandem mass spectrometer method for the content of kainic acid, a naturally occurring neurotoxic compound in P. palmata. Kainic acid levels in the samples ranged widely from trace levels to approximately 560 µg g-1 dry weight.

Effects of biochar amendment on sorption, dissipation, and uptake of fenamiphos and cadusafos nematicides in sandy soil.

BACKGROUND: The application of biochar to soil is supposed to alter its adsorption/desorption potential toward pesticides, thereby affecting their bioavailability and efficacy. This is particularly relevant in the case of nematicides because these pesticides are directly applied to soil. RESULTS: Biochar was produced from date palm (PB) and eucalyptus (EB) waste at 450 °C and added at a rate of 1% to a sandy soil. The half-life (t½ ) of fenamiphos was increased from 2.7 to 18.3 and 18.6 days in PB- and EB-amended soils, respectively. By contrast, the half-life of cadusafos was unaffected. Freundlich Kf values increased from 1.22 and 0.39 (µg1-Nf g-1 mLNf ) to 4.49 and 6.84 in 1% PB-amended soil, and to 3.49 and 4.62 in 1% EB-amended soil for cadusafos and fenamiphos, respectively. Plant uptake of both nematicides in tomato seedlings was reduced by approximately 97% (cadusafos) and 85% (fenamiphos). Although nematicide efficacy against Meloidogyne incognita was not altered at the recommended dosage, it was negatively affected at a half-dose rate. Under these conditions, it decreased from 43.1% in unamended sandy soil to only 18.3% in 1% PB-amended soil. CONCLUSIONS: Biochar addition increased the sorption capacity of soil. This resulted in a decrease of nematicide bioavailability, together with a reduction of both the dissipation rate and uptake by tomato plants. © 2018 Society of Chemical Industry.

Pesticide residues in heterogeneous plant populations, a model-based approach applied to nematicides in banana (Musa spp.).

Nematicides are widely used to control plant-parasitic nematodes in intensive export banana (Musa spp.) cropping systems. Data show that the concentration of fosthiazate in banana fruits varies from zero to 0.035 g kg-1, under the maximal residue limit (MRL=0.05 mg kg-1). The fosthiazate concentration in fruit is described by a Gaussian envelope curve function of the interval between pesticide application and fruit harvest (preharvest interval). The heterogeneity of phenological stages in a banana population increases over time, and thus the preharvest interval of fruits harvested after a pesticide application varies over time. A phenological model was used to simulate the long-term harvest dynamics of banana at field scale. Simulations show that the mean fosthiazate concentration in fruits varies according to nematicide application program, climate (temperature), and planting date of the banana field. This method is used to assess the percentage of harvested bunches that exceed a residue threshold and to help farmers minimize fosthiazate residues in bananas.

Leaching of the organophosphorus nematicide fosthiazate.

Fosthiazate is an organophosphorus nematicide which was recently included in Annex I of the Directive 91/414/EEC under the clause that it should be used with special care in soils vulnerable to leaching. Thus, the leaching of fosthiazate was investigated in columns packed with three different soils which represented situations of high (site 2), intermediate (site 1) and low (site 3) leaching potential. The recommended dose of fosthiazate was applied at the surface of the soil columns and fosthiazate fate and transport was investigated for the next two months. Fosthiazate concentrations in the leachate collected from the bottom of the columns packed with soil from site 2 exceeded 0.1 microgl(-1) in most cases. This soil was characterized as acidic, indicating longer fosthiazate persistence, with low organic matter content, indicating weak adsorption, thus representing a situation vulnerable to leaching. In contrast, the lowest concentrations of fosthiazate in the leachate were evident in the columns packed with soil from site 3. This soil was characterized as alkaline, indicating faster degradation, with higher organic matter content, indicating stronger adsorption, thus representing a situation not favoring leaching of fosthiazate. The highest concentration of fosthiazate in the leachate from the columns packed with soil from site 2 was 3.44 microgl(-1) compared to 1.17 and 0.16 microgl(-1), which were the corresponding maximum values measured in columns packed with soil from sites 1 and 3, respectively. The results of the current study further suggest that fosthiazate is mobile in soil and can leach under conducive soil conditions like acidic soils with low organic matter content.

Stereoisomeric separation and toxicity of the nematicide fosthiazate.

Considerable attention has been paid to the enantiomeric resolution and toxicity of some chiral organophosphorous pesticides (OPs) with one asymmetric center, but research concerning chiral OPs with two asymmetric centers is still limited. In the present study, the stereoisomeric separation and toxicity of fosthiazate, a chiral OP with two asymmetric centers on phosphorus and carbon atoms, was investigated. All four stereoisomers of fosthiazate were separated successfully with a Chiralpak(R) AD [amylase tris(3,5-dimethyl-phenyl carbamate)] column on high-performance liquid chromatography. The resolved isomers and the pairs of enantiomers were further distinguished using a circular dichroism detector and an optical rotation detector, designating the first (pk1) and third (pk3) eluted peaks as one pair of enantiomers and the second (pk2) and fourth (pk4) peaks as the other pair. The developed method was used to prepare microquantities of individual stereoisomers that were used for in vitro and in vivo bioassays. The inhibition potencies of the stereoisomers against acetylcholinesterase of Electrophorus electricus were slightly stereoselective, with a maximum difference of 1.4-fold among the isomers. A 3.1-fold difference, however, was observed in the acute toxicity of isomers to Daphnia magna. The 48-h toxicity of isomers to D. magna followed the order pk1 > pk2 > pk4 > racemate approximately pk3. The stereoselective toxicity to D. magna found in fosthiazate suggests that the environmental safety of fosthiazate should be evaluated on the basis of its individual isomers.