Cyromazine Residues Research Articles

Biodegradation of Cyromazine by Mycobacterium sp. M15: Performance, Degradation Pathways, and Key Enzymes.

Cyromazine, a triazine insecticide, raises food safety concerns due to residues in vegetables like cowpeas. Microbial metabolism is key for pesticide elimination, but bacteria efficient in cyromazine degradation are limited, with uncharacterized enzymes. This study isolated a highly efficient cyromazine-degrading bacterium, Mycobacterium sp. M15, from a cowpea field. M15 utilized cyromazine as the sole carbon source for its growth and completely degraded 0.5 mM cyromazine within 24 h. The degradation pathway involved hydrolyzing cyromazine to N-cyclopropylammeline and further to N-cyclopropylammelide, with amino groups removed sequentially. The cyclopropylamine group in N-cyclopropionamide continued to hydrolyze to cyanuric acid. A protein, CriA, identified as an aminohydrolase in M15, degraded cyromazine to N-cyclopropylammeline. Using CriA reduced cyromazine residues on cowpea surfaces and completely degraded them in immersion solutions. These findings offer insights into cyromazine's microbial degradation mechanism and highlight the potential of cyromazine-degrading enzymes in enhancing food safety.

Technology for Rapid Detection of Cyromazine Residues in Fruits and Vegetables: Molecularly Imprinted Electrochemical Sensors.

Cyromazine is an insect growth regulator insecticide with high selectivity and is widely used in the production and cultivation of fruits and vegetables. In recent years, incidents of excessive cyromazine residues in food have occurred frequently, and it is urgent to establish an accurate, fast, and convenient method for the detection of cyromazine residues to ensure the safety of edible agricultural products. To achieve rapid detection of cyromazine residues, we prepared a molecularly imprinted electrochemical sensor for the detection of cyromazine residues in agricultural products. Samples of tomato (Lycopersicon esculentum Miller), cowpea (Vigna unguiculata), and water were tested for the recovery rate of cyromazine. The results showed that the concentration of cyromazine showed a good linear relationship with the peak response current of the sensor developed in this study. The lower limit of detection for cyromazine was 0.5 µmol/L, and the sensor also had good reproducibility and interference resistance. This paper can be used as a basis for the study of methods for the detection of cyromazine residues in edible agricultural products.

Evaluation of cyromazine transferred from feed to chicken products and subsequent assessment of dietary risks to Chinese consumers.

In this work, levels of cyromazine and its metabolite melamine in chicken products and its transfer factors (TFs) and dietary risks assessment for Chinese consumers are presented. Cyromazine was added to chicken feed at doses of 5, 25, and 50mg/kg for 42 days. Cyromazine residues were found in frequently consumed chicken products (meat, heart, liver, gizzard, and eggs), with liver and eggs displaying the highest concentrations (0.935 and 1.281mg/kg, respectively). Low levels of melamine residues (<0.029mg/kg) were detected in chicken products from chickens treated with 25 and 50mg/kg cyromazine, but not in eggs from chickens treated with 5mg/kg cyromazine. The TFs for the investigated chicken products varied from 0.0074 to 0.0229 across the dosage levels. The chronic exposure assessment showed that 0.001 to 0.190% of the acceptable daily intake (0.06mg/kg body weight [b.w.]/day) of cyromazine was consumed through chicken products among the various age and gender groups of Chinese consumers. The acute exposure assessments of different age and gender groups were 0.0004% to 0.178% of the acute reference dose (0.1mg/kg b.w.). Although the results suggest that the risk associated with cyromazine residues in chicken products was low in China, the possible risk associated with cyromazine residues in chicken products should not be ignored while cyromazine remains a legal feed additive. PRACTICAL APPLICATION: The present analytical method could be used for cyromazine and melamine detection in different chicken products, and dietary risk assessments of cyromazine provided a support for the work of regulatory bodies to conduct surveillance programs regarding food safety evaluation of cyromazine.

High performance liquid chromatographic determination of cyromazine and its derivative melamine in poultry meats and eggs

A high performance liquid chromatography (HPLC) with NH2 column and 75% acetonitrile eluate to determine the insecticide cyromazine residues in poultry meats and eggs was developed. Samples were treated with NaOH, extracted with acetonitrile containing 20% concentrated NH4OH, and cleaned up through C18 Sep-Pak cartridge. The peaks of cyromazine and its metabolite melamine were successfully separated within 12 min and cleanly differentiated from interfering peaks of the samples. The detection limit of both compounds was 0.02 ppm. Recoveries of cyromazine and melamine ranged from 92.8-97.3% and 91.0-96.1% at fortified levels of 0.2-0.7 ppm, respectively. Each reaction coefficient of linear standard curve of cyromazine and melamine was better than 0.999. Using this HPLC determination, only one beef sample from 46 tested samples was detected to contain 0.04 ppm cyromazine. This was further confirmed by gas chromatography-mass spectroscopy.

Plasma pharmacokinetics and tissue depletion of cyromazine and its metabolite melamine following oral administration in laying chickens.

The study was designed to characterize the plasma pharmacokinetics and tissue depletion profiles (including eggs) of cyromazine (CYR) in chickens following oral administration alone or in combination with melamine (MEL). In order to assess the pharmacokinetic profile of CYR, chickens were administered 1 or 10mg/kg (single oral doses), whereas residue studies were conducted in chickens fed CYR alone (5 or 10mg/kg) or CYR (5mg/kg) and MEL (5mg/kg) for a period of 14days. Estimates for the apparent volume of distribution (1.66L/kg), clearance (7.17mL/kg/min), and elimination half-life (2.82h) were derived by noncompartmental analyses. The highest concentration of CYR occurred in liver but fell below detectable limits within 3days following drug withdrawal from feed. Combined feeding of MEL with CYR did not significantly alter CYR tissue levels. CYR residues were detected only in egg white and were undetectable at the 2nd day postadministration. No MEL was found in eggs unless it had been added to the feed, and when present, it almost exclusively restricted to the egg white. Based upon the results of this initial study of CYR pharmacokinetics and residue depletion, it appears that use of CYR as a feed additive either alone (5 or 10mg/kg) or in combination with MEL (both agents at 5mg/kg) does not produce unsafe residue levels in edible products as long as appropriate withdrawal periods are followed for tissues (3days) and eggs (2days). However, our results indicate that adoption of a zero-day withdrawal period should be reconsidered in light of these results.

Sample Preparation of Eggs From Laying Hens Using QuEChERS Dispersive Extraction for the Simultaneous Determination of Melamine and Cyromazine Residues by HPLC-DAD.

A quick, easy, cheap, effective, rugged, and safe (QuEChERS) dispersive extraction method is proposed herein for the isolation and cleanup of melamine and cyromazine from chicken egg yolk. Analytes are determined by high-performance liquid chromatography using photodiode array detector after separation on a LiChroCART® (250 × 4 mm)—LiChrospher® RP-8e, 5 μm analytical column using a mobile phase of 0.1% trifluoracetic acid and methanol (80:20 v/v) delivered isocratically at a flow rate of 1 mL/minute. Extraction of isolated compounds was achieved by methanol and acetonitrile mixture (1:1 v/v). Recovery rates ranged between 74.5% and 115.8%. The method was validated in terms of 657/2002/EC decision. The within-laboratory reproducibility, expressed as a relative standard deviation, was <11%. Decision limits (CCalfa) were 2.56 mg/kg for melamine and 0.22 mg/kg−1 for cyromazine, and the corresponding results for detection capability (CCbeta) were 2.8 mg/kg for melamine and 0.24 mg/kg for cyromazine. Ruggedness was estimated according to the Youden approach studying egg yolk mass, sorbent mass, centrifugation time, organic solvents volume, evaporation temperature, and vortex time.

A novel dispersive micro solid phase extraction using PCX as the sorbent for the determination of melamine and cyromazine in milk and milk powder by UHPLC-HRMS/MS

A novel dispersive micro solid phase extraction (DMSPE) cleanup method based on the PCX sorbent (a kind of cation exchange polymer material) was applied to the analysis of melamine and cyromazine residues in milk and milk powder, and ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC–HRMS) was used as instrument detection. Milk powder samples were first extracted with 1% formic acid in acetonitrile/water (1:1v/v), and milk samples were cleaned up directly without any pre-extraction. Then, melamine and cyromazine in the extracts or milk were adsorbed to the PCX powder. Subsequently, the analytes in PCX sorbent were eluted with ammonium hydroxide/acetonitrile (2.5:97.5v/v) through a simple unit device equipped with 1mL syringe and 0.22μm nylon syringe filter. All the samples were analyzed by UHPLC–HRMS/MS on a Waters Acquity BEH HILIC column with 0.1% formic acid and 4mM ammonium formate in water/acetonitrile as the mobile phase with gradient elution. The matrix effect, recovery, and repeatability, within laboratory reproducibility, CCα and CCβ of the DMSPE cleanup method were investigated. The proposed method provided a significant improvement for the determination of melamine and cyromazine in milk and milk powder in terms of efficient, rapid, economical, and miniaturized sample preparation methods, which yielded fewer matrix effects compared with SPE method. The established cleanup method is expected to be widely applied for the sample preparation of alkaline contaminants at trace levels in the future.

A Sensitive and Validated HPLC Method for the Determination of Cyromazine and Melamine in Herbal and Edible Plants Using Accelerated Solvent Extraction and Cleanup with SPE

A highly sensitive method was developed for the determination of the residues of cyromazine (CYR) and its metabolite, melamine (MEL), in herbal and edible plant samples by using reversed phase high-performance liquid chromatography-diode-array detection (RP-HPLC-DAD) with accelerated solvent extraction and solid phase extraction cleanup. The conditions of separation and detection were investigated and optimized. A Waters C18 column (250 × 4.6 mm i.d., 5 µm) was used for the RP-HPLC, with a mobile phase composed of 0.1% trifluoroacetic acid solution and methanol (85:15, v/v, pH 2.6). Under the optimized conditions, good linearity was achieved with a correlation coefficient of 0.9998. The limits of quantification of the method were 2.15 µg/kg for CYR and 2.51 µg/kg for MEL, which are maximum residue limits as low as three orders of magnitude. The recovery values at three spiked concentrations were in the range of 96.2-107.1% with relative standard deviations (RSDs) of 1.1-5.7% for CYR, and 92.7-104.9% with RSDs of 1.7-6.1% for MEL. The proposed method allows detection at levels as low as µg/kg levels for CYR and MEL. The method was validated by liquid chromatography-tandem mass spectrometry, and can be used for the routine determination of CYR and MEL in herbal and edible plant samples with the characteristics of speed, high sensitivity and accuracy, and low consumption of reagents.

Simultaneous determination of cyromazine and dicyclanil in animal edible tissues using UPLC-MS/MS

Cyromazine and dicyclanil are both used as insect growth regulators. This paper describes an easy and innovative simultaneous extraction method for residues of cyromazine and dicyclanil in food of animal origin, and a confirmation procedure using UHPLC-MS/MS. The sample was extracted, deproteinised by 1% trichloracetic acid in water-acetonitrile, followed by selective defatting using hexane based on the degree of matrix complexity; cleaned-up on an mixed-mode cation exchange (MCX) cartridge; and quantified by using matrix-matched calibration. The mean recoveries were all between 62.0% and 99.2%, and the RSDs were all below 9.94%. The present method was rapid, sensitive and reliable, which was applied to the quantitative analysis of these residues in animal tissues.

Degradation of diflubenzuron, ivermectin, cyromazine and temephos in soil following surface disposal of sheep dipping and jetting solutions

Dissipation of diflubenzuron, ivermectin and cyromazine residues in soil was measured following the disposal of 250-L batches of dipping or jetting solutions by spraying onto 64-m2 pasture plots. Half-lives for the three insecticides in soil were estimated to be 80, 39 and 17 days, respectively. Temephos residues in soil were monitored for 224 days after disposal of 4800 L of dip solution (365 mg/L) onto a 450-m2 bunded plot. A half-life in soil of 62 days was estimated. This is significantly longer than that for diazinon, which it has largely replaced as the most commonly applied sheep dip insecticide. In consideration of the volume of dip solutions likely to be discharged on-farm and the half-lives of these commonly used insecticides the following recommendations should apply: permanent exclusion of grazing stock from land treated with diflubenzuron, and temporary exclusion of grazing stock for 3–6 months from land treated with cyromazine or temephos. No additional precautions over those already listed on ivermectin jetting product labels are considered to be warranted by these data.

Determination of Residues of Cyromazine and Its Metabolite, Melamine, in Animal-Derived Food by Gas Chromatography−Mass Spectrometry with Derivatization

A gas chromatography-mass spectrometric (GC-MS) method was established for the determination of cyromazine and its metabolite, melamine, in animal-derived food. Chicken and tilapia muscle samples were spiked with (15)N(3)-melamine, extracted with an acidic acetonitrile/water solution, and defatted with dichloromethane. Egg and milk samples were directly extracted with 3% trichloroacetic acid. The extracts were purified using mixed cation-exchange cartridges, derived with N,O-bis(trimethylsilyl)trifluoroacetamide, and detected by GC-MS. Cyromazine and melamine were quantified by external standard methods except for the determination of melamine in animal muscle, which used an internal standard method. Recoveries ranged from 75.0 to 110.0%, and relative standard deviations were <15.0%. In animal muscle the limits of quantification (LOQs) were 20 microg/kg and the limits of detection (LODs) were 10 microg/kg for cyromazine and melamine. In milk and eggs the LOQs were 10 microg/kg and the LODs were 5 microg/kg for both analytes.

Application and Fate of Cyromazine in a Closed-Cycle Hydroponic Cultivation of Bean (Phaseolus vulgaris L.)

The fate of cyromazine applied via the nutrient solution (20, 40, and 60 mg of active ingredient per plant) in a closed-cycle soilless cultivation of bean with zero discharge of effluents was traced in both the recycled drainage solution and the plant tissues for 99 days. The insecticide was applied once, 15 days after planting (16 days prior to the first harvest). In addition to cyromazine, the residues of melamine, its metabolite, in the drainage solution and plant tissues were also regularly determined during the 99 days. The two higher application doses induced toxicity symptoms on the leaves of the bean plant. The maximum cyromazine levels were measured 8 days after application in the drainage solution (17-46 mg l(-1)), 16 days in the roots (1.1-2.4 mg kg(-1) fresh weight [f. wt.]) and the vegetative shoot (4.5-9.5 mg kg(-1) f. wt.), and 24 days after application in the pods (2.6-4.1 mg kg(-1) f. wt.). However, the cyromazine residues in pods were clearly below the maximum acceptable levels for bean. The half-life of cyromazine in the drainage solution ranged from 16 to 19 days for the three doses. The melamine residues in the drainage solution and in the roots reached a concentration peak 16 days after cyromazine application, whereas in the vegetative shoot and the pods they were constantly increasing over the 99 days after application. Nevertheless, the melamine residues were constantly much lower than those of cyromazine, although on the last sampling day (99) they tended toward convergence. Cyromazine proved to be highly persistent, as indicated by the remarkably high residues measured in both the drainage solution and the plant tissues, even 99 days after application. Nevertheless, the application of cyromazine via the nutrient solution to beans grown in closed-cycle hydroponic systems at doses not exceeding 20 mg per plant seems to be safe with respect to both phytotoxicity and residue levels in the edible pods.

Fate of cyromazine applied in nutrient solution to a gerbera ( Gerbera jamesonii) crop grown in a closed hydroponic system

Cyromazine was applied at two doses (375 and 750 mg l −1) via the nutrient solution in a winter crop of gerbera ( Gerbera jamesonii) grown on pumice in a closed-loop system. The persistence of cyromazine in the system and its uptake and distribution within the plant were studied for a period of 121 days by regularly collecting and analysing samples of nutrient solution, drainage solution, substrate, roots, leaves, and flowers. The residues of cyromazine in leaf were further studied in a summer experiment which lasted 50 days. The concentration of cyromazine fluctuated within a relatively high range in both the drainage solution and the nutrient solution supplied to the plants during the first day after its application but declined progressively thereafter. Nevertheless, 37 days after application, mean concentrations of 6.2 and 10.9 mg l −1 cyromazine were found in the drainage solution at low and high doses, respectively. The corresponding cyromazine concentrations 121 days after its application were 0.02 and 0.03 mg l −1. Similar values were measured also in the nutrient solution. The adsorption of cyromazine by the pumice was lower than 0.02 mg kg −1. The half-life time of cyromazine in the closed system was 9.65 days. In the winter experiment, the highest cyromazine concentrations were determined 17 days after application in root and 56 days in leaf and flower, regardless of the dose, while on day 121 the leaf concentrations were 52 and 62 mg kg −1 fresh weight (f. wt) at the low and the high dose, respectively. In the summer experiment, the leaf concentration of cyromazine reached a maximum (232 mg kg −1 f. wt) 14 days after its application and decreased only slightly on day 24, but dropped rapidly to 66 mg kg −1 f. wt on day 50. The above results indicate that cyromazine applied via the nutrient solution may persist for more than 120 days in closed hydroponic systems, while its maximum concentration in the plant tissues occurs several days after application.

Effects of cyromazine and abamectin on the pea leafminer Liriomyza huidobrensis (Diptera: Agromyzidae) and its parasitoid Diglyphus isaea (Hymenoptera: Eulophidae) in potatoes

The pea leafminer, Liriomyza huidobrensis (Blanchard), arrived in Israel almost a decade ago. Although adults are resistant to insecticides, growers treat fields with translaminar insecticides (abamectin and cyromazine) after they see large numbers of adults. Field trials were conducted for 2 years in potatoes ( Solanum tuberosum L.) to evaluate the effects of a single, properly timed, application of these two insecticides on L. huidobrensis and its eulophid parasitoid, Diglyphus isaea Walker. Abamectin and cyromazine were applied at recommended field rates once, early in the growing season. Yellow sticky traps were used to monitor adult population levels, and leaf samples were used to monitor larval population levels. Cyromazine residues were monitored within the potato plants by a gas chromatograph–mass spectrometer. Both insecticides significantly reduced leafminers as compared to non-treated control; however, cyromazine was significantly more effective than abamectin. Similarly, parasitoid populations from both insecticide treatments were significantly reduced as compared to the non-treated control; however, parasitoid populations from abamectin treated plots recovered sooner than in cyromazine treated plots. Cyromazine residues in the plant declined over time, with a half-life of ≈1 wk. Implications of these results on management practices are discussed.

Effects of cyromazine and abamectin on the leafminer, Liriomyza huidobrensis and its parasitoid, Diglyphus isaea in celery

SUMMARYField trials were conducted for 2 yr on celery (Apium graveolens L.) to determine the effects of two translaminar insecticides (abamectin and cyromazine) on the agromyzid leafminer, Liriomyza huidobrensis (Blanchard), and the eulophid parasitoid, Diglyphus isaea Walker. Abamectin and cyromazine were applied once early in the growing season by spray or drip application. Yellow sticky traps and leaf samples were used to monitor population levels; a gas chromatograph‐mass spectrometer was used to monitor cyromazine residues within the celery plants during 1 yr. Spray applications of abamectin and cyromazine significantly reduced larval leafminers and D. isaea. Contrary to grower practices, application of cyromazine by drip irrigation systems had minor effects on larval leafminer and D. isaea as compared to spray application. Cyromazine residues in the plants treated by spray application declined sharply after 1 wk, and no residues were detected 2 wk after application; less than 0.05 ppm cyromazine residues were detected in plants treated by drip application. Implications of these results on management practices are discussed.

Studies on the Absorption, Residues and Metabolism of Cyromazine in Tomatoes

The residues and metabolism of radio-labeled cyromazine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine) formulated as a 750 g kg -1 WP ('Trigard' 75WP)® was studied in greenhouse tomatoes. Residues of cyromazine and its metabolites were remarkably persistent, with only a slight loss occurring within a 30-day period. Findings with tomatoes were similar to those with glass plates when exposed to cyromazine. Little volatilization occurred from either tomato or glass surfaces kept under greenhouse conditions. The only metabolite identified was melamine on both glass plates and tomatoes. Two other metabolites were found but were not identified.