Combination of photoinduced fluorescence and GC-MS for elucidating the photodegradation mechanisms of diflubenzuron and fenuron pesticides.

Diflubenzuron (DFB) and fenuron (FEN) are benzoylurea and phenylurea pesticides, widely used in Senegal, that do not exhibit any natural fluorescence, but can be determined by means of photoinduced fluorescence (PIF) methods. Photodegradation of DFB and FEN yielded a number of fluorescent and non-fluorescent photoproducts. For both pesticides, at least 10 photoproducts were detected and identified by gas chromatography-mass spectrometry (GC/MS). To identify the formed fluorescent DFB and FEN photoproducts, their fluorescence spectra were compared with those of standard compounds, including phenol and p-hydroxyaniline.

Metabolism of Diflubenzuron in Lizard ( Eremias argus) and Comparative Toxicity of Diflubenzuron and Its Metabolite.

The metabolic process of diflubenzuron in rat or fish has been well studied, but little is known about its elimination pathway in lizard. The current study predicted the metabolic route of diflubenzuron in lizard feces and compared the toxicity of diflubenzuron and 4-chloroaniline on lizard thyroid system. The amido bond cleavage was the major route for diflubenzuron elimination in lizard feces. 4-Chloroaniline as the most toxic diflubenzuron metabolite was also abundant in feces. According to liver slices, 4-chloroaniline exposure induced significant changes of nuclear shape, while diflubenzuron exposure caused significant hepatocytes clustering. On the basis of thyroid hormone and thyroid-related gene levels, triiodothyronine (T3) level in lizard liver was regulated by thyroid hormone receptors, while thyroxine (T4) concentration was modulated by dio2 and udp genes after diflubenzuron or 4-chloroaniline exposure. These results showed that both diflubenzuron and 4-chloroaniline could disrupt lizard thyroid system, which could provide evidence for lizard population decline.

Different residue behaviors of four pesticides in mushroom using two different application methods.

Pesticide residue in mushrooms is less known. In this study, the risks of beta-cypermethrin, pyriproxyfen, avermectin, and diflubenzuron in oyster and shiitake mushrooms were evaluated using two different treatments: substrate mixture and surface spraying. Almost all the concentrations of these pesticides at day 90 were higher than 80% of the initial concentrations, while it was less than 45% for all cases within 35 days by spraying. For surface spraying, the residues of beta-cypermethrin were 0.0843-1.22 mg kg-1 in shiitake mushrooms and below 0.005 mg kg-1 in oyster mushrooms; the residues of pyriproxyfen, avermectin, and diflubenzuron were 0.122-4.84, 0.00501-0.111, and 0.0681-1.91 mg kg-1, respectively. The residues of beta-cypermethrin, pyriproxyfen, and diflubenzuron in oyster mushrooms (in shiitake mushrooms) at interval of 0, 3, 5 days (1, 5, 7 days) were below their MRLs in China or Japan. The residue of avermectin in both mushrooms was lower than its limit of detection. These results provide information to safe and proper use of the pesticides in oyster and shiitake mushrooms.

Persistence and Stability of Teflubenzuron and Diflubenzuron When Associated to Organic Particles in Marine Sediment.

The persistence and stability of the oral administered anti salmon-lice drugs teflubenzuron and diflubenzuron were tested when associated to organic material as faecal particles from Atlantic salmon and medicated food pellets. This laboratory study was performed in seawater under aerobic conditions, at 7°C in the dark and showed that both compounds were remarkably persistent and stable since no significant reduction in the concentrations of flubenzurons in sediment were seen after 24 weeks. Therefore neither chemical or microbial degradation nor outwashing seems to be important pathways for these drugs to disappear from sediment under fish farms. Thus, it is more likely that the decrease of flubenzurons from marine sediments described in field investigations is caused by either bioturbation, resuspension of organic particles or a combination of these.

Toxicological assessment of the anti-salmon lice drug diflubenzuron on Atlantic cod Gadus morhua.

Increasing use of the chitin synthesis inhibitor diflubenzuron against the ectoparasitic salmon louse Lepeophtheirus salmonis in marine aquaculture has raised concerns over its environmental impacts. This study evaluated how diflubenzuron affects Atlantic cod Gadus morhua, a fish species often found near Atlantic salmon Salmo salar farms, focusing on uptake kinetics and hepatic transcriptional responses. Two experiments were conducted, one time-series trial in which the fish were given a daily dose (3 mg kg-1 fish) of diflubenzuron for 14 d followed by a 3 wk depuration period, and one dose-response trial with increasing concentrations (3, 10 and 50 mg kg-1 fish). The highest diflubenzuron concentrations were found in the liver at Day 15. No detectable levels of diflubenzuron were found in liver or muscle 3 wk after the end of the treatment. At the molecular level, small effects of diflubenzuron treatment on gene transcription were observed. In the time-series experiment, the strongest effects were seen at Day 8, with 2 transcripts being upregulated (bclx2 and cpt1a) and 8 transcripts being downregulated (gstp1, gstm1, gstt1, ugt1a, nat2, cat, p53 and slc16a9a). Five transcripts (cyp3a, cpt1a, ptgs2, elovl5 and mapk1) responded significantly to diflubenzuron exposure in the dose-response experiment. This study shows that diflubenzuron can be taken up by Atlantic cod, that it is rapidly cleared from the body and that when present this pharmaceutical causes only small effects on the expression of genes involved in detoxification pathways. Taken together, our data suggest that accumulated diflubenzuron at the levels studied would have a relatively small effect on wild Atlantic cod.

Determination of diflubenzuron residues in milk and cattle tissues / Determinação de resíduos de diflubenzuron em leite e tecidos bovinos

Diflubenzuron (DFB) is used to control ectoparasitic infestation by inhibiting larvae development in the manure and feces of treated animals. It is also currently been used to control tick infestations. In this study, milk and tissues from cattle treated orally with DFB for a 77-120 day period with a commercial product containing the compound were analyzed for the presence of residues. DFB residues were determined by using extraction with acetonitrile, cleanup with C18 SPE and chromatographic analysis by HPLC with UV detection (254nm). DFB was not detected in any of the analysed samples (<0.006mg kg-1 for fat, <0.014mg kg-1 for muscle, <0.015mg kg-1 for kidney, <0.016mg kg-1 for liver and <0.0006mg kg-1 for milk). In this manner, the use of this compound, according to the manufacturer's suggested doses may result in cattle milk, liver, kidneys, fat and muscles being considered safe regarding the presence of DFB residues.

O diflubenzuron (DFB) é um inibidor de desenvolvimento de insetos que inibe a síntese de quitina com atividade ovicida e larvicida e está sendo utilizado na pecuária para o controle do carrapato. Leite e tecidos provenientes de bovinos tratados por um período de 77 a 120 dias com um produto comercial contendo DFB foram analisados quanto à presença de resíduos. Os resíduos de DFB foram determinados utilizando-se extração com acetonitrila, limpeza por SPE C18 e cromatografia líquida de alta eficiência com detecção por UV (254nm). DFB não foi detectado em nenhuma das amostras analisadas (<0.006mg kg-1 para gordura, <0,014mg kg-1 para músculo, <0,015mg kg-1 para rim, <0,016mg kg-1 para fígado e <0.0006mg kg-1 para leite). Dessa forma, a utilização do princípio ativo conforme recomendado pelo fabricante e em níveis suficientes para se obter o efeito larvicida desejado deve resultar em leite, fígado, rins, gordura e músculos que podem ser considerados seguros para o consumo em termos da presença DFB.

Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods.

Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a population-level bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase (CHS1) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods.

Determination of the insecticide diflubenzuron in mushrooms by kinetic method and high-performance liquid chromatographic method.

In the present study, a new sensitive and simple kinetic-spectrophotometric method for the determination of the insecticide diflubenzuron [1-(4-chlorophenyl)-3-(2,6-diflubenzoil)urea] is proposed. The method is based on the inhibited effect of diflubenzuron on the oxidation of sulphanilic acid (SA) by hydrogen peroxide in phosphate buffer in presence Cu(II) ion. Diflubenzuron was determined with linear calibration graph in the interval from 0.31 to 3.1 µg mL⁻¹ and from 3.1 to 31.0 µg mL⁻¹. The optimized conditions yielded a theoretical detection limit of 0.18 µg mL⁻¹ corresponding to 0.036 mg kg(-1)mushroom sample based on the 3S(b) criterion. The RSD is 5.03-1.83 % and 2.81-0.71 % for the concentration interval of diflubenzuron 0.31-3.1 µg mL⁻¹ and 3.1-31.0 µg mL⁻¹, respectively. The reaction was followed spectrophotometrically at 370 nm. The kinetic parameters of the reaction are reported, and the rate equations are suggested. The developed procedure was successfully applied to the rapid determination of diflubenzuron in spiked mushroom samples of different mushroom species. The HPLC method was used like a comparative method to verify results.

Effects of benzoylphenylurea on chitin synthesis and orientation in the cuticle of the Drosophila larva.

Chitin is an essential constituent of the insect exoskeleton, the cuticle, which is an extracellular matrix (ECM) covering the animal. It is produced by the glycosyltransferase chitin synthase at the apical plasma membrane of epidermal and tracheal cells. To fulfil its role in cuticle elasticity and stiffness it associates with proteins, thereby adopting a stereotypic arrangement of helicoidally stacked sheets, which run parallel to the surface of the animal. One approach to understand the mechanisms of chitin synthesis and organisation is to dissect these processes genetically. However, since only a few genes coding for factors involved in chitin synthesis and organisation have been identified to date using the model arthropod Drosophila melanogaster insight arising from mutant analysis is rather limited. To collect new data on the role of chitin during insect cuticle differentiation, we have analysed the effects of chitin synthesis inhibitors on Drosophila embryogenesis. For this purpose, we have chosen the benzoylphenylurea diflubenzuron and lufenuron that are widely used as insect growth regulators. Our data allow mainly two important conclusions. First, correct organisation of chitin seems to directly depend on the amount of chitin synthesised. Second, chitin synthesis and organisation are cell-autonomous processes as insecticide-treated larvae display a mosaic of cuticle defects. As benzoylphenylurea are used not only as insecticides but also as anti-diabetic drugs, the study of their impact on Drosophila cuticle differentiation may be fruitful for understanding their mode of action on a cellular pathway that is seemingly conserved between vertebrates and invertebrates.

Comparative studies on effects of three chitin synthesis inhibitors on common malaria mosquito (Diptera: Culicidae).

Toxicities of three chitin synthesis inhibitors (diflubenzuron, nikkomycin Z and polyoxin D) were evaluated using second instars of the common malaria mosquito, Anopheles quadrimaculatus Say (Diptera: Culicidae). Neither nikkomycin Z nor polyoxin D at 50 microg/liter caused significant larval mortality, although they reduced the body weight of the survivors by 20.5 and 33.8%, respectively, in 48 h. In contrast, exposures of the larvae to diflubenzuron at 12.5 microg/liter for 48 h resulted in 86.7% larval mortality and reduced the body weight of the survivors by 29.1%. Exposure of the pupae (<12 h old) to diflubenzuron at 100 microg/liter for 48 h caused 18.9% pupal mortality and consequently reduced the adult emergence by 24.7% from the surviving pupae. Furthermore, exposure of third instars to diflubenzuron at 4, 20, 100, and 500 microg/liter for 24 h resulted in the reduction of larval chitin contents by 4.25, 33.2, 35.2, and 57.7%, respectively. Such an effect seemed to be associated with only cuticular chitin synthesis because the same exposures did not significantly affect chitin contents in the guts. Our results indicated that diflubenzuron was highly toxic to second instars by not only causing high larval mortality but also by affecting their growth. Diflubenzuron was also fairly toxic to pupae by not only causing pupal mortality but also affecting the adult emergence. Our results suggest that diflubenzuron might affect only chitin synthesis in the cuticle but not in the peritrophic matrix, which is probably due to diflubenzuron's direct contact to mosquito larvae in water, slow distribution in insect body, rapid degradation in the insect gut, or a combination.

Determination of benzoylurea insecticide residues in tomatoes by high-performance liquid chromatography with ultraviolet-diode array and atmospheric pressure chemical ionization-mass spectrometry detection.

A simple and sensitive method using high-performance liquid chromatography/ mass spectrometry (LC/MS) was developed and validated for simultaneous determination of 5 benzoylurea insecticides-diflubenzuron, triflumuron, teflubenzuron, lufenuron, and flufenoxuron-in tomatoes. Residues were successfully separated on a C18 column by methanol-water isocratic elution. Detection was carried out by an ultraviolet diode array detector (UV-DAD) coupled with a quadrupole mass spectrometer, using atmospheric pressure chemical ionization (APCI) in negative-ion mode. The main ions were the deprotonated molecules [M-H]- for triflumuron, and the anions formed by elimination of hydrofluoric acid [M-H-HF]- for diflubenzuron and flufenoxuron, and [M-2H-HF] for lufenuron and teflubenzuron. The calibration plots were linear for both detectors over the range 0.05 to 10 microg/mL, and the method presented good quality parameters. The limits of detection for standard solutions were 0.008-0.01 mg/L (equivalent to 0.08-0.1 ng injected) for both detectors, and the limits of quantification (LOQs) were approximately 10 times lower than national maximum residue levels (MRLs). Depending on the compound and the detector, the LOQ values ranged from 0.2 to 0.4 ng injected. The optimum LC-UV-DAD/APCI-MS conditions were applied to the analysis of benzoylureas in tomatoes. The obtained recoveries from fortified tomato samples (50 g), extracted with ethyl acetate and purified by solid-phase extraction on silica sorbent, were 88-100 and 92.9-105% for the UV-DAD and MS detectors, respectively, with precision values (relative standard deviations) of 2.9-11 and 3.7-14%, respectively. The method was applied to 12 tomato samples from local markets, and diflubenzuron and lufenuron were detected in only one sample at concentrations lower than the MRLs. The results indicate that the developed LC/MS method is accurate, precise, and sensitive for quantitative and qualitative analysis at low levels of benzoylureas required by legislation.

Pharmaceutical development of a parenteral formulation of the investigational anticancer drug clanfenur.

A stable parenteral dosage form for the investigational cytotoxic drug clanfenur was designed, and the bulk drug was characterized by its nuclear magnetic resonance, mass spectrometry, infrared, and ultraviolet spectra. The 1H and 13C spectra show clanfenur to be a mixture of two stereoisomers. Because of poor solubility in aqueous solution and precipitation in co-solvent, surfactant, or emulsion systems, a two-pump infusion system was developed for intravenous administration. Clanfenur, solubilized in a Cremophor EL/ethanol (1:1, w/v) solution (concentration, 15 mg/mL), can be simultaneously infused with 5% dextrose infusion fluid. Total doses of up to 1,680 mg of clanfenur (and 56 g of Cremophor EL) theoretically can be administered to patients over a 6-hour period. From accelerated stability testing of clanfenur in the Cremophor EL/ethanol (1:1, w/v) formulation, a shelf life of 3.5 years at 4 degrees C and of 4 months at 25 degrees C is calculated.