Validation of QuEChERS Method for Estimation of Imidacloprid and its Metabolites in Cotton Flower, Pollen, Nectariferous Tissue, and Honey.

BACKGROUND: Exposure of Apis mellifera to neonicotinoid insecticides is one of the factors attributed to the recent decline in A. mellifera populations resulting in economic and ecological losses due to loss of pollination services. Honeybees can get exposed to neonicotinoids like imidacloprid directly in the field at the time of application as well as during consumption of pollen and nectar from treated plants. In addition, some metabolites of imidacloprid are more toxic than the parent compound. So, the fate of imidacloprid and its metabolites in commodities to which honeybees get exposed needs to be overhauled. OBJECTIVE: To validate QuEChERS method for estimation of imidacloprid and its metabolites in cotton flower, pollen, nectariferous tissue, and honey using HPLC. METHODS: The QuEChERS method was validated in terms of selectivity, linearity, LOD, LOQ, matrix match, accuracy, and precision. The estimation of residues was done by HPLC. RESULTS: Recoveries of imidacloprid and its metabolites for cotton flowers, nectariferous tissue, pollen and honey samples were in the range of 80.42-99. 83%. LOQ for imidacloprid and its metabolites was 0.01 µg/g. Acceptable precision (RSD < 20%) was obtained. CONCLUSION: The method allows simple and fast extraction of imidacloprid and its metabolites from cotton flower, pollen, nectariferous tissue, and honey. HIGHLIGHTS: An accurate, simple, and sensitive analytical method was validated for imidacloprid and its metabolites. The method was validated according to the SANTE/12682/2019 guidelines.

Dissipation kinetics of imidacloprid in cotton flower, nectariferous tissue, pollen and Apis mellifera products using QuEChERS method.

BACKGROUND: Exposure of Apis mellifera to neonicotinoid insecticides is one of the factors attributed to the recent decline in A. mellifera populations resulting in economic and ecological losses due to loss of pollination services. Honey bees can get exposed to neonicotinoids like imidacloprid directly in the field at the time of application as well as during consumption of pollen and nectar from treated plants. So, the fate of imidacloprid in commodities to which honey bees get exposed needs to be overhauled. RESULTS: Residue of imidacloprid was investigated following imidacloprid application as seed treatment (2.4 and 4.8 g a.i. kg-1 seed) at the time of sowing and as foliar spray (17.8 and 35.6 g a.i. ha-1 ) at 70 days after sowing when the crop was at full bloom stage. The imidacloprid residue was below limit of quantification (LOQ) in flowers, necatariferous tissue, pollen from seed-treated cotton plants and honey collected from hives kept in plots with seed-treated cotton plants. However, average initial imidacloprid residue (2 h after spray) was 1.84 and 1.95 mg kg-1 in flowers; 0.22 and 0.24 mg kg-1 in nectariferous tissue; 0.88 and 0.96 mg kg-1 in pollen collected from plants sprayed with imidacloprid @ 17.8 g a.i. ha-1 at locations Faridkot and Bathinda, respectively. The average initial imidacloprid residue (21 days after spray) in honey collected from hives was 0.01 mg kg-1 . CONCLUSION: The residue in different substrates sampled from seed treated cotton plants was below LOQ. However, its foliar spray at bloom time resulted in imidacloprid residue in flower, nectariferous tissue, pollen and honey sampled from hives placed in plots. © 2021 Society of Chemical Industry.

Effectiveness of community-based treatment programs for treatment of uncomplicated severe acute malnourished children aged 6-59 months using locally produced nutrient dense foods: protocol for a multicentric longitudinal quasi-experimental study.

BACKGROUND: Severe acute malnutrition (SAM) is a major underlying cause of mortality among children. Around one third of the world's acutely malnourished children live in India. The WHO recommends community-based management of acute malnutrition (CMAM) for managing children with SAM. In India, different states are implementing community-based SAM treatment programme, hereinafter called CSAM, using varieties of locally produced nutrient dense food items with different nutrient compositions. The study will assess the effectiveness of these state specific CSAM interventions. METHODS: The longitudinal quasi-experimental study will be undertaken in two purposively selected blocks of one district each in the four intervention states and one comparison state. From each state, 200 SAM children identified using weight-for-length/height z-score (WHZ) < - 3 criteria will be enrolled in the study. Their anthropometric data and skinfold thickness will be taken on admission, at sixth week and at discharge by trained field investigators. Other child details, incidence of morbidity and socio-economic details will be collected on admission. To assess food consumption pattern including consumption of locally produced nutrient dense food supplements, dietary assessment, using 24-h dietary recall will be conducted on admission, at sixth week and at discharge. In addition, body composition parameters will be assessed for a sub-set of children using bio-electrical impedance analysis on admission and at discharge to analyse changes in total body water, fat-free mass, and fat mass. Post discharge, all study participants will be followed up monthly until 6 months. Atleast 10% of the sample will be checked for quality assessment. The study's primary outcome is cure rate defined as children attaining WHZ ≥ -2. Secondary outcomes include mean weight gain, mean length of stay, body composition parameters, relapse and mortality rates. Additionally, process evaluation and cost effectiveness analysis will be conducted. DISCUSSION: There is a shortage of robust evidence regarding the effectiveness of locally produced nutrient dense food supplements provided as part of the CSAM intervention in India. This study will contribute to evidence on effective strategies to manage children with uncomplicated SAM in India. The study protocol has all necessary ethical approvals. Written informed consent will be obtained from caregivers of the children. TRIAL REGISTRATION: The study is registered with Clinical Trial Registration of India (Registration No.: CTRI/2020/09/028013 ) Date of registration 24/09/2020.

Assessment of Cytokine and Herpesvirus Level in Peri-implantitis and Healthy Patients.

BACKGROUND: The present study was conducted to assess level of cytokine and herpesvirus in peri-implantitis and healthy patients. METHODOLOGY: Fifty patients with healthy dental implant (Group I) and dental implant with peri-implantitis (Group II) were enrolled. The level of interleukin (IL)-1 ß, IL-2, IL-4, IL-6, MCP-1 and tumor necrosis factor-α (TNF-α), and herpesvirus was estimated. RESULTS: The mean IL-1 ß level was 1.54 in Group I and 5.12 in Group II, IL-2 was 0.05 in Group I and 0.02 in Group II, IL-4 was 0.018 in Group I and 0.0021 in Group II, IL-6 was 0.83 in Group I and 0.92 in Group II, MCP-1 was 64.5 in Group I and 23.1 in Group II, and TNF-alpha was 1.54 in Group I and 1.05 in Group II. There was a significant higher level of TNF-alpha in virus + patients (P < 0.05). CONCLUSION: The cytokine level was increased in patients with peri-implantitis as compared to patients with healthy dental implants. Virus-positive patients exhibited higher cytokine levels than virus-negative patients.

Can Increased Metabolic Status be a Grading Tool for Oral Squamous Cell Carcinoma? A Glucose Transporter 1 Immunoexpression Study.

BACKGROUND: Glucose transporter-1 (GLUT-1) is a GLUT protein whose expression is upregulated in malignant cells where enhanced uptake of glucose is observed. AIM: The aim of this study is to evaluate the expression of GLUT-1 protein in oral squamous cell carcinoma (OSCC) tissue sections using immunohistochemistry and to describe the relationship between increased metabolic status and the grades of OSCC. MATERIALS AND METHODS: This is cross-sectional study with 76 formalin-fixed paraffin-embedded tissue blocks of OSCC, obtained from the archives of the department. All the cases were scored using Bryne's grading system by three oral pathologists independently. The tissue sections were then stained using immunohistochemistry with anti-GLUT-1 rabbit monoclonal antibody. RESULTS: Staining intensity and localization of positively stained slides were evaluated. Overall, a significant correlation between Bryne's histopathological grading system for OSCC and GLUT-1 immunohistochemical expression was observed. Thus, high GLUT-1 expressions are observed with increasing grades of OSCC. CONCLUSION: This study shows that a significant positive correlation exists between GLUT-1 immunoexpression and histological grading of OSCC. Thus, GLUT-1 expression can be used as a diagnostic adjunct and prognostic marker for OSCC patients.

Persistence, dissipation, and risk assessment of a combination formulation of trifloxystrobin and tebuconazole fungicides in/on tomato.

Multi-locational supervised field trials were conducted in different agro-climatic regions in India to study dissipation of trifloxystrobin and tebuconazole in tomato after spraying a combination formulation (trifloxystrobin 25% + tebuconazole 50%, 75WG) at recommended doses: (i) single (trifloxystrobin 87.5 g a.i. ha-1 + tebuconazole 175 g a.i. ha-1) and (ii) double (trifloxystrobin 175 g a.i. ha-1 + tebuconazole 350 g a.i. ha-1). Fruit samples were extracted with ethyl acetate using a modified QuEChERS method. The residues (parent fungicides + metabolite) were analyzed and confirmed by GC-ECD and GC-MS, respectively. The half-life (t1/2) of trifloxystrobin and tebuconazole in tomato varied from 1.08 to 1.72 and 1.13 -to 1.64 days at single; and 1.27 to 2.13 and 1.24 to 1.96 days at double dose, respectively. Since maximum residue limit (MRL) at pre-harvest interval (PHI) of 5 days is impractical, as tomato is usually harvested and consumed almost everyday after the last spray, the risk assessment was performed at minimum PHI of 1 day. Accordingly, on the basis of supervised field trial data and using OECD MRL calculator, MRL of 0.5 and 1.5 mg kg-1 at single dose were proposed for trifloxystrobin and tebuconazole in/on tomato, respectively.

Estimation of Salivary and Serum Lactate Dehydrogenase in Oral Submucous Fibrosis.

AIM AND OBJECTIVES: The objective of the present study was to estimate the serum and salivary lactate dehydrogenase (LDH) levels in patients with oral submucous fibrosis (OSMF) and to study the association between serum and salivary LDH levels and mouth opening, frequency of habit, and duration of habit in patients with OSMF. MATERIALS AND METHODS: Participants were divided into two groups: Group I - case group diagnosed with OSMF and Group II - the control group. Unstimulated whole saliva was collected from all the participants. The International Federation of Clinical Chemistry method was carried out with the help of LDH (P-L) kit (Crest Biosystems, Goa). Collection of blood sample was done under aseptic precautions and processed for LDH. The data were analyzed using SPSS software 2010 (version 19, IBM, Armonk, NY, USA). Descriptive statistics including mean, range, standard deviation, and percentage were used along with unpaired t-test and Pearson's correlation test. RESULTS: All the participants were male and were in the age range of 18-60 years, with a mean age of 28.63 ± 10.39 years. The mean salivary LDH levels in patients with OSMF were 1057.30 ± 640.12 µg/dl and in the control group were 668.25 ± 498.45 µg/dl. The mean serum LDH level in patients with OSMF was 408.35 ± 158.35 µg/dl as compared to the control group was 313.05 ± 82.69 µg/dl. The Pearson's correlation coefficient between serum LDH and frequency of habit, duration of habit, and mouth opening was found to be 0.55, 0.53, and 0.69, respectively. The Pearson's correlation coefficient between salivary LDH and frequency of habit, duration of habit, and mouth opening was found to be 0.33, 0.04, and 0.13, respectively. The Pearson's correlation coefficient between salivary LDH and serum LDH was found to be -0.18. CONCLUSIONS: There was a significant increase in the serum and salivary LDH in OSMF patients as compared to controls; the salivary LDH did not seem to correlate with frequency of the habit, duration of the habit, or mouth opening of OSMF patients. However, serum LDH was found to correlate directly with frequency of the habit and mouth opening in OSMF patients. Hence, based on the results of the present study, it can be hypothesized that serum LDH is a better biological marker than salivary LDH in the evaluation of OSMF.

Estimation and risk assessment of flubendiamide on fodder berseem clover (Trifolium alexandrinum L.) by QuEChERS methodology and LC-MS/MS.

The residues of flubendiamide from berseem were extracted and cleaned up by the QuEChERS approach and analysed by liquid chromatography/mass spectrometry (LC-MS/MS). The dissipation studies on berseem were carried out by application of flubendiamide at five different dosages i.e. 24, 36, 48, 72 and 96 g a.i. ha(-1). The residues reached below determination limit (BDL) of 0.01 mg kg(-1) in 7, 10, 10, 15 and 15 days for 24, 36, 48, 72 and 96 g.a.i. ha(-1)dosages, respectively. Half-life (t1/2) of flubendiamide on berseem was observed to be 1.08, 1.88, 1.94, 2.26 and 2.27 days, respectively, at 24, 36, 48, 72 and 96 g a.i. ha(-1). Theoretical maximum residue contributions (TMRC) values reached below the maximum permissible limit (MPI) for 3, 5 and 7 days in treatments at 24, 36 and 48 g.a.i. ha(-1), respectively, whereas in case of higher dose treatments i.e. 72 and 96 g a.i. ha(-1), the residues of flubendiamide reached below MPI after 10 days of treatment. These data could provide guidance for the proper and safe use of this pesticide on berseem in India.

Degradation pattern and risk assessment of chlorantraniliprole on berseem (Trifolium alexandrinum L.) using high performance liquid chromatography.

The persistence pattern of chlorantraniliprole on berseem leaves and its risk assessment for the safety of cattle were studied. QuEChERS method was used for the extraction and cleanup of samples and the residues of chlorantraniliprole were estimated using high performance liquid chromatography (HPLC) and confirmed by Liquid Chromatograph-Mass Spectrometer (LCMS-MS). The dissipation studies on berseem were carried out by application of chlorantraniliprole at five different dosages i.e. 11.6, 17.1, 23.1, 34.7 and 46.2 g a.i. ha(-1). Average initial deposits of chlorantraniliprole were found to be 0.47, 0.61, 0.78, 1.15 and 1.31 mg kg(-1), respectively. The residues reached below determination limit (BDL) of 0.01 mg kg(-1) in 5, 7, 7, 10 and 10 days for 11.6, 17.1, 23.1, 34.7 and 46.2 g a.i. ha(-1) dosages, respectively. Half-life (t1/2) of chlorantraniliprole on berseem was observed to be 0.93, 1.14, 1.06, 1.00 and 1.33 days, respectively, at 11.6, 17.1, 23.1, 34.7 and 46.2 g a.i. ha(-1). It was found that the theoretical maximum residue contributions (TMRC) values reached below maximum permissible intake (MPI) on 0 day in berseem samples treated with chlorantraniliprole. These studies, therefore suggest that the use of chlorantraniliprole formulation at different dosages does not seem to pose any hazards to the consumers and a waiting period of one day is suggested to reduce the risk before consumption of berseem leaves. These data could provide guidance for the proper and safe use of this pesticide on berseem in India.

Risk assessment of ß-cyfluthrin and imidacloprid in chickpea pods and leaves.

Dissipation of ß-cyfluthrin and imidacloprid in chickpea pods and leaves was measured following three applications of Solomon 300 OD (ß-cyfluthrin 9 percent +imidacloprid 21 percent) at 200 and 400mLha(-1). Residues of ß-cyfluthrin on chickpea pods and leaves were found to be below its limit of quantification (LOQ) of 0.01mgkg(-1) after 7 days at both the dosages. Similarly, imidacloprid residues were found to be below its LOQ of 0.01mgkg(-1) at 10 days. Half-life periods on chickpea pods and leaves for ß-cyfluthrin were found to be 1.06 and 0.58 days, whereas for imidacloprid these values were observed to be 2.07 and 1.75 days at recommended dose. As the theoretical maximum residue contributions on chickpea pods and leaves are found to be less than the maximum permissible intake values even on 0 day, therefore consumer health risks are minimal at both the dosages on chickpea.

Estimation of quizalofop ethyl residues in black gram (Vigna mungo L.) by gas liquid chromatography.

Quizalofop ethyl, a phenoxy propionate herbicide is used for post emergence control of annual and perennial grass weeds in broad-leaved crops in India. The experiments were designed to study the harvest time residues of quizalofop ethyl in black gram for two seasons. At harvest time, the residues of quizalofop ethyl on black gram seed, foliage and soil were found to be below the determination limit of 0.01 mg kg(-1) following a single application of the herbicide at 50 and 100 g a.i. ha(-1) for both the periods. Application of the herbicide is quite safe from a consumer and environmental point of view.

Persistence of thiacloprid residues on brinjal (Solanum melongena L.).

Thiacloprid, a neonicotinoid insecticide with novel mode of action, is found to be effective against several lepidopteran as well as hemipteran pests. The present studies were carried out to observe the persistence pattern of thiacloprid on brinjal and to suggest suitable waiting period for the safety of consumers. Persistence of thiacloprid in brinjal was studied following three applications of thiacloprid (Alanto 240 SC) at 180 and 360 g a.i. ha(-1) at 7 days interval. Residues of thiacloprid in brinjal were estimated by high-performance liquid chromatography and were confirmed by liquid chromatography-mass spectrometry. The average initial deposits of thiacloprid were observed to be 0.48 and 1.05 mg kg(-1) on the brinjal fruit following third application of thiacloprid at recommended and double the recommended dosages, respectively. Half-life periods for thiacloprid were found to be 0.47 and 0.50 days at single and double the application rates, respectively. Residues of thiacloprid declined below its limit of quantification of 0.05 mg kg(-1) after 3 and 5 days, respectively, at recommended and double the recommended application rates. Therefore, the use of thiacloprid does not seem to pose any risk hazard and a waiting period of 1 day is suggested for safe consumption of brinjal fruits.

Development and validation of QuEChERS method for estimation of chlorantraniliprole residue in vegetables.

An easy, simple and efficient analytical method was standardized and validated for the estimation of residues of chlorantraniliprole in different vegetables comprising brinjal, cabbage, capsicum, cauliflower, okra, and tomato. QuEChERS method was used for the extraction and cleanup of chlorantraniliprole residues on these vegetables. Final clear extracts of ethyl acetate were concentrated under vacuum and reconstituted into high performance liquid chromatograph (HPLC) grade acetonitrile, and residues were estimated using HPLC equipped with PDA detector system, C(18) column and confirmed by liquid chromatograph mass spectrometer (LC-MS/MS), and high performance thin layer chromatograph (HPTLC). HPLC grade acetonitrile:water (80:20, v/v) was used as mobile phase @ 0.4 mL/min. Chlorantraniliprole presented distinct peak at retention time of 9.82 min. Consistent recoveries ranging from 85% to 96% for chlorantraniliprole were observed when samples were spiked at 0.10, 0.25, 0.50, and 1.00 mg/kg levels. The limit of quantification of this method was worked out to be 0.10 mg/kg.

Dissipation pattern of flubendiamide in/on brinjal (Solanum melongena L.).

Residues of flubendiamide and desiodo flubendiamide were studied following three applications of flubendiamide 480 SC at 7 days interval at 90 and 180 g a.i. ha(-1) in/on brinjal fruits. An average initial deposit of 0.33 and 0.61 mg kg(-1) of flubendiamide was observed respectively after application at single and double dosages. The residues of flubendiamide dissipated quickly at both the dosages, and after 3 days, the extent of dissipation was found to be about 76% and 79% at the single and double dosages, respectively. Brinjal fruit samples analysed at different time intervals did not show the presence of desiodo flubendiamide. The half-life of flubendiamide was observed to be 0.62 and 0.54 days at single and double dosages, respectively. The limit of determination of flubendiamide and desiodo flubendiamide was observed to be 0.05 mg kg(-1). Soil samples analysed after 15 days of the last application did not reveal the presence of flubendiamide and desiodo flubendiamide at their determination limit of 0.05 mg kg(-1). An assessment of the total intake of flubendiamide resulting through the consumption of brinjal fruits and its comparison with acceptable daily intake seems to be quite safe from consumer point of view.

Persistence of trifloxystrobin and tebuconazole on grape leaves, grape berries and soil.

Following four foliar applications of Nativo 75 WG (trifloxystrobin 25% + tebuconazole 50%) on grapes @ 175 and 350 g/ha, resulting in active applications of trifloxystrobin @ 43.75 and 87.5 g a.i./ha and that of tebuconazole @ 87.5 and 175 g a.i./ha, the average initial deposits of trifloxystrobin were observed to be 7.76 and 15.53 mg/kg, respectively, at single and double the application rates. These residue levels dissipated to >85% after 10 days of its application at both the dosages. Similarly, the average initial deposits of tebuconazole were observed to be 13.84 and 26.55 mg/kg at single and double the application rate, respectively. These residue levels dissipated to >90% after 10 days of application at both the dosages. The half-life (t(1/2)) periods of trifloxystrobin on grape leaves were observed to be 2.92 and 3.48 days at single and double application rates, respectively, whereas these values were 2.68 and 3.96 days for tebuconazole. Ripe grape berries and soil samples collected at harvest which happened to be 34 days after the last application, did not show the presence of residues of trifloxystrobin and tebuconazole at their determination limit of 0.01 and 0.02 mg/kg, respectively.