Evaluation of acetylcholinesterase (AChE) activity and determination of organophosphorus pesticides (OPs) are of great importance for the clinical diagnosis of several serious diseases correlated with their variations in human blood serum. In this study, a highly selective and sensitive ratiometric fluorescent probe was innovatively fabricated for the evaluation of AChE activity and the determination of OPs in tap water and food on the basis of the inner filter effect (IFE) between nitrogen-doped carbon dots (N-CDs) and 2,3-diaminophenazine (DAP). N-CDs were synthesized via a one-pot hydrothermal method by using pancreatin and 1,2-ethanediamine as precursors. N-CDs showed excellent fluorescence properties and negligible cytotoxicity on human cervical carcinoma HeLa cells and human embryonic kidney 293T cells, suggesting their further biological applications. Upon the addition of AChE and choline oxidase, acetylcholine was catalyzed to produce choline that was further oxidized to produce H₂O₂. In the presence of horseradish peroxidase, o-phenylenediamine reacted with H₂O₂ to produce fluorescent DAP. Therefore, a ratiometric fluorescent probing platform existed via IFE between N-CDs with a fluorescence signal at 450 nm and DAP with a fluorescence signal at 574 nm. OPs irreversibly impeded the catalytic activity of AChE, finally leading to the decrease of DAP amount and the variation of ratiometric fluorescent signal. Under optimal conditions, such a fluorescent probe showed relatively low detection limits of 0.38 U/L for AChE, 3.2 ppb for dichlorvos, and 13 ppb for methyl-parathion. Practical application of this ratiometric fluorescent probe to detect OPs was further verified in tap water and food samples with satisfying results that were highly consisted with the results obtained by GC–MS.

The research “Use of pesticides in Fresh Water Aquaculture in the Mekong Delta, Vietnam, and impacts on environment and food safety” consisted of four sections. To evaluate the pressure of drugs and chemicals in the environment, two surveys were performed in the Mekong Delta. The first was focused on rice and rice-fish system and was conducted in 2009. The second, an in depth survey, conducted in 2013, focused on rice-fish, striped catfish and red-tilapia systems. Results showed that, for the rice-fish system, most rice fish farmers grew 2 rice crops and 1 fish crop per year. Pesticides were applied generally 3 times per crop. Farmers normally applied pesticides based on the recommended doses of producers. Chess 50WG (containing 500g/kg pymetrozine, produced by Syngenta, Vietnam), Bassa 50EC (containing 500g/L fenobucard, produced by Việt Thắng Bắc Giang (Vithaco), Vietnam) and Kinalux (containing 250g/L quinalphos, produced by United Phosphorus Ltd., India) were the most common used commercial pesticides in rice crop, rice-fish crop and distributors as well. According to the majority of the distributors, the use of pesticides will increase in future. The in-depth survey showed that much more active compounds were used in 2013 compared to 2009, but, all of the active compounds belonged to the approved list of Vietnamese government. Few farmers used chemicals during fish crop. Farmers reported their awareness towards the use of agrochemicals in terms of health effects. The survey showed that the farmers select an agrochemical based on their experience. The study on red-tilapia demonstrated that many different types of disinfectants and antimicrobials are used. Further, the cost-effectiveness of such pesticide use, especially for feed supplement products, antimicrobials and disinfectants, is questionable and should be assessed. There is an urgent need to improve the farmer’s knowledge and their access to advisory services on careful use of disinfectants and antimicrobials. All visited striped catfish farms applied drugs and chemicals with seven types of antibiotics during the fish production. Enrofloxacin, sulfamethoxazole and trimethoprim were reported to be the most used chemicals by farmers to treat Bacillary Necrosis of Pangasius (BNP). The survey and practical situation demonstrated that quinalphos, trifluralin and dichlorvos were commonly used in rice fish system and, consequently, may contaminate aquaculture products. A Gas chromatography – mass spectrometry (GC-MS) analytical method was developed and validated according to European guidelines (SANTE/11945/2015) for the determination of residues of those pesticides in water. The developed method was then optimized using a gas chromatography – electron capture detector (GC ECD) technique to make the method more applicable in Vietnam. The developed method was used to analyze water samples collected from the aquaculture system in April 2013, at the beginning of the rainy season. Results showed that only 9 % of the total water samples analyzed contained residues of quinalphos, but only in water from rice fish systems. The other two pesticides, trifluralin and dichlorvos, were not detected. A comparison between GC-MS and GC-ECD indicated that GC-ECD is less sensitive than GC-MS. However, for samples with concentrations detectable with both techniques, no significant difference was observed between the results obtained using both equipments GC-ECD and GC-MS. The next step was to determine the distribution and elimination of quinalphos, the active substance of a popular insecticide used in the Mekong Delta, according to the first survey. An experiment was set up in a rice-fish integrated system in Can Tho City, Vietnam. Quinalphos was applied twice in a dose of 42.5 g per 1000 m2, according to the producer recommendations. Samples (fish, water and sediment) were collected at time intervals and were analyzed by GC-ECD. The results showed that quinalphos residues in fish muscles were much higher than those in the water and the bioconcentration factor (logBCF) was above 2 for the fish. The half-lives, after the first and second quinalphos applications, were 12.2 and 11.1 days for sediment, 2.5 and 1.1 days for silver barb, 1.9 and 1.3 days for common carp, and 1.1 and 1.0 days for water, respectively. Finally, as a case study including 3 commonly used pesticides (quinalphos, trifluralin and dichlorvos), dioxins and one forbidden antibiotic (chloramphenicol), the risk for the consumer, linked to the chemical contamination of the aquaculture related environment was evaluated. Sediments samples were collected including 10 samples collected from catfish ponds in An Giang Province and 12 samples randomly collected from rice-fish systems in Can Tho City. Analytical results showed that 3 from the 13 water samples collected from rice field were contaminated with low levels of quinalphos (with concentrations of 0.11, 0.08 and 0.04 μg/L). The other investigated pesticides were not detected in any sample. For chloramphenicol (CAM) residues in fish samples, analysis was performed on 36 fish samples of catfish (18 samples included 9 from small scale and 9 from large scale systems), snakehead (9 samples) and climbing perch (9 samples) collected at the beginning, middle and at the end of culture period. Results showed that one sample of climbing perch and one sample of snakehead were contaminated with traces of CAM (concentrations of 0.17 and 0.19 µg/kg, respectively). It appeared that CAM was not detected in catfish samples neither from the beginning to the end of the crop, nor from small and large scale systems. Dioxins were not detected in any of the collected sediments samples. In order to assess the general risk for the Vietnamese consumer of fish, a survey was performed in Can Tho City, using a questionnaire designed to collect information. A large part of interviewees (77%) stated that they like to eat fish. The number of days of eating fish was 3.4 days per week. In this study, the average amount of fish consumption ranged between 90 and 140 g per day. It was shown that the daily intake of trifluralin of interviewed people was 0.05 µg/kg body weight/day. This level of exposure was much lower than the acceptable daily intake (ADI) (15 µg/kg/day) (EFSA, 2015). However, trifluralin has not been approved in EU, so the presence of residues of trifluralin in aquatic product, even if they cause no problem for the consumer, would be a problem for aquatic product export. | Deltaquasafe Project