PhD Thesis

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    Development of methods for analyzing and monitoring antibiotic and pesticide residues in fish and shrimp
    (© University of Dhaka, 2025-07-07) Islam, Sk. Farzana
    Background: Aquaculture has seen significant growth in recent years, especially in fish and shrimp production. This expansion is driven by increasing global demand for seafood, prompting substantial advancements in the fish and shrimp industries and higher production levels for both species. Bangladesh boasts an expansive wetland region that supports a wide array of aquaculture practices. The aquaculture industry contributes substantially to the economy of Bangladesh with regards to employment, foreign exchange earnings, nutrition and income. In aquaculture, antibiotics are widely used from the larval stage through to the growth stage for disease prevention and treatment purposes. The extensive use of antibiotics in aquaculture can result in residues remaining in shrimp products, which are consumed globally and may lead to various toxic effects. Similarly, pesticides are widely used in modern agriculture worldwide, albeit in varying amounts. While pesticides can boost crop yields, they pose significant environmental, health, and safety concerns for both terrestrial and aquatic ecosystems. Aquatic creatures, including fish, are exposed to pesticides and their residues through multiple pathways, such as run-off and spray drift from agricultural fields. Bangladesh exports shrimp and fish to various countries around the world. To ensure the safety and quality of fish and fish products, it is mandatory to submit a pre-export test report and health certification before shipping the consignment. The objective of the research was to develop sensitive, economical, and straightforward methods for determining and quantifying antibiotic and pesticide residues in shrimp and fish. Methods: Three methods were developed for the analysis of antibiotic and pesticide residues. In the first experiment, a modified QuEChERS technique was developed to simultaneously analyze tylosin (Tyl) and metronidazole (MNZ) residues in shrimp samples using LC-ESI-MS/MS. In the second experiment, A sensitive and effective method was developed and validated using QuEChERS extraction for detecting 19 organochlorine pesticides in tilapia fish. In the third experiment, a novel method was developed and validated for the first time to quantify three pesticide residues (chlorpyrifos, cypermethrin, and acetamiprid) in tilapia fish using QuEChERS extraction combined with GC-ECD. xi Result: The first method demonstrated excellent linearity for both analytes over the concentration range of 0.5–50 µg/L. The coefficients of determination (R²) for Tyl and MNZ were 0.9997 and 0.9998, respectively. The recovery rates were 81-85% for Tyl with a relative standard deviation (RSD) ≤ ± 4.9%, and 85-88% for MNZ with RSD ≤ ± 4.07%. The limits of quantification (LOQ) were determined to be 1 µg/kg for Tyl and 0.9 µg/kg for MNZ. The limits of detection (LOD) were found to be 0.4 µg/kg for Tyl and 0.3 µg/kg for MNZ. The second method employed linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) to validate the procedure. The recoveries of the 19 organochlorine pesticides ranged from 81.03% to 102.02%, with relative standard deviations (RSD) ≤ 6% across three different levels of fortification. The third method demonstrated excellent linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) during method validation. The recoveries for chlorpyrifos, cypermethrin, and acetamiprid at fortification levels of 0.01, 0.02, and 0.05 mg/kg ranged from 85% to 95%, with relative standard deviations (RSDr) ≤ 4.5%. The matrix-matched calibration curve exhibited good linearity (r² ≥ 0.998). The LOD ranged from 2.9 µg/kg to 3.1 µg/kg, and the LOQ was 10.00 µg/kg for all analytes. Conclusion: To our knowledge, this is the first time in Bangladesh that the modified QuEChERS extraction method has been developed for the determination of Tyl and MNZ antibiotic residues in shrimp muscles. The second method allows for the simultaneous detection of nineteen organochlorine residues in tilapia fish. Additionally, the third method, developed for the first time in Bangladesh, utilizes GC coupled with an Electron Capture Detector (ECD) for the determination of chlorpyrifos, cypermethrin, and acetamiprid residues. These three methods can be employed for the routine analysis of antibiotic and pesticide residues in shrimp and tilapia fish, contributing to enhanced food safety standards in Bangladesh. This improvement in food safety will also bolster the country's exports.
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    Importance of fish pass and fish friendly structure in inland open water fish production
    (© University of Dhaka, 2025-05-04) Hossain, Md. Motaher
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    Molecular Characterization of Pathogenic Strains of Bacteria Causing Motile Aeromonas Septicemia in Heteropneustes fossilis: Implications for Potential Drug Development
    (©University of Dhaka, 2023-12-05) Sultana, Shirin
    Background: Dysbiosis of harmonized microbial communities of fish are crucial to identify for better health management of aquaculture species. In addition to classical methods, high-throughput sequencing techniques are now frequently used to analyze the 16S rRNA gene amplicon to assess the shifting of microbial populations as biotic stressors for fish. This study elucidated the different microbiome community by both culture dependent and culture independent methods isolated from healthy and diseased stinging catfish (Heteropneustes fossilis) and associated environment. Moreover, the isolated pathogenic Aeromonas hydrophila strains from diseased fish skin were identified and confirmed by pathogenic gene amplification and experimental infection. In addition, an attempted was made to design potential inhibitor (drug like compound) against virulent serine protease. Methods: Healthy and diseased stinging catfishes, soil and water samples were collected from fifteen locations of Bangladesh. Bacterial community from fish tissues and environmental samples were isolated and identified by conventional culture methods and then confirmed by 16S rRNA gene sequencing. Different microbiome community were identified and compared by 16S rRNA gene specific metagenomics approach. Pathogenic Aeromonas hydrophila was isolated from diseased fish skin lesions using selective Rimler Shotts medium with novobiocin supplement. Pathogenicity of isolated A. hydrophila was confirmed by PCR amplification of five pathogenic genes. After confirming virulence factors, two isolates (AGM2 and AYN7) were selected for experimental infection. The pathogens were later identified based on pathogenic gene specific markers and characterized by antibiogram profiling. One of the pathogenic genes coded for the serine protease was selected for identification of effective drug using in silico drug designing bioinformatics tools. Results: Higher bacterial community was evident in healthy fish tissue than diseased fish; and found similarities with the communities of soil and water samples by culture dependent method. Significant difference of bacterial load was observed between healthy and diseased fish tissues. Similarly, the richer and diversified microbiome communities were found in healthy fish skin than diseased fish skin by culture independent 16S rRNA metagenomics. At the genus level, Pseudomonas had the highest richness in healthy groups and was almost completely absent in the diseased groups, but Flavobacterium was quite prevalent in diseased fishes. The diseased group harbored the Aeromonas hydrophila as minor dominant which might reflect their opportunistic characteristics. Two isolated of A. hydrophila (AGM2 and AYN7) showed highest resistance in antibiogram test (9 and 6 antibiotics, respectively) against 11 antibiotics. In Mueller-Hinton (MH) agar media, the growth of these two isolates was inhibited by ceftriaxone, cefepime, gentamycin, azithromycin, and ciprofloxacin. Challenge test with AGM2 showed higher pathogenicity than AYN7 in post injection of healthy stinging catfish. Pathological signs and symptoms (lesions) and excess mucus (typical signs of MAS) were observed in injected fish with both isolates. The virulent gene ahpA encoded for serine protease was characterized and the homology modelling was performed to find potential inhibitor against the protein. An important parameter Ramachandran plot exhibited maximum amino acid residues (88.1%) in the favorable region which was also supported by the accepted G-factor value (overall score 0.38). The molecular docking of 3D structure showed the highest affinity (-7.6) with ceftriaxone from six selected compounds. Conclusion: Fish health can be impacted by the highly varied microbial communities associated with skin across populations and species. Several factors can alter the community structure to make dominant as single or mixed pathogens. The findings of this study will improve the understanding of microbiota modulation and would act as a baseline information to develop possible drug (s) like compound that would be helpful to avoid indiscriminate use of antibiotic in aquaculture sector.
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    Chemical contaminants in selected fish, fisheries products and important foodstuffs of Bangladesh
    (University of Dhaka, 2019-01-14) Ali, Md. Mohsin
    Chemical contamination and adulteration in foodstuffs are quite alarming at present in Bangladesh. This PhD thesis investigates the chemical contaminants in 8 groups of fish, fisheries products and important foodstuffs of Bangladesh (total sample 1215) during April, 2008 to May, 2016. This study also investigates the migration of pesticides from soil and water to paddy in the experimental paddy fields. Samples were supplied to Pesticide Analytical Laboratory, Entomology Division, BARI, Gazipur for identification of residues level of harmful chemicals. BCSIR Laboratory was used for formalin test and proximate analysis of meat, fish, milk, mangoes. Institute of Marine Sciences and Fisheries Laboratory, University of Chittagong and Department of Fisheries Lab, University of Dhaka, were used for proximate analysis of cereal, meat and fish. Proximate analysis was carried on accordingly to the method of AOAC. Chemical residues identification was carried out by BARI laboratory manual 2009. For chemicals test, elute was injected in either run by GC with ECD/FTD/FID and GCMS-EI or GCMS-NCI modes depending on the nature of contaminants. Overall, 31.77% samples of different categories were contaminated with chemicals (e.g., pesticides or formalin). The highest pesticide residues was detected in fruits (in 66.67% sample) followed by fish and fisheries products (60.19%); meat, fat and liver (36.36%); water (26.67%); vegetables (20.98%) and cereals (6.45%). On the other hand, no chemical was detected in milk or eggs. This study also found no migration of pesticides from soil and water to paddy. The pesticide contaminations in fish and fisheries products samples varied geographically: DDT, Heptachlor, Diazinon, Chlorpyriphos, Acephate, Qunalphos, Carbaryl, etc. were abundant in Dhaka, Chittagong and Rajshahi divisions; in contrast, they were not detected in Khulna division. In fish and fisheries products the highest level of DDT was detected in Penaeus monodon (0.127±0.006 ppm) while the lowest level was detected in Pumpus chinensis (0.046 ppm). In the samples, Heptachlor ranged from 0 to 0.210±0.008 ppm and the chemical was only detected in Penaeus monodon from Chittagong among four divisions. Diazinon was found in Labeo rohita, Macrobrachium rosenbergii, Corica soborna, Herpodon nehereus, M. brevicornis, Pumpus chinensis, Pampus argenteus, Trichurus haumela, M. rosenbergii, and P. indicus fish species from different divisions. The highest level of Diazinon was detected in Labeo rohita (1.273±0.097 ppm) from Dhaka division, while the lowest level was detected in M. brevicornis (0.063±0.030 ppm) from Chittagong division. Chlorpyriphos was detected in P. indicus sampled from Chittagong division that was not detected in other divisions. The highest level of Acephate was detected in Labeo rohita (0.917±0.012 ppm) from Rajshahi division, while the lowest level was found in Tenualosa ilisha (0.086 ppm) from Chittagong division. Both Qunalphos and Carbaryl were only detected in fish and fisheries samples from Dhaka division. In most of the fish and fisheries products samples the contamination level exceeded the Acceptable Daily Intake (ADI) and Maximum Residue Limit (MRL) level of FAO and WHO. This study further found that 30.97% fish and shrimp were contaminated by formalin. However, Formalin concentrations varied amongst species and locality: 57.14% Kaski (Corica soborna), 54.29% Lotia (Herpodon nehereus), 54.29% Hilsa (Tenualosa ilisha), 51.43% Kakila (Xenentodon cancila), 48.57% Rupchanda (Pumpus chinensis), 48.57% Catla (Catla catla), 45.71% Mola (Amblypharyngodon mola) 42.86% Pabda (Ompok pabda), 42.86% Rui (Labeo rohita), 40% Chela (Chela cachius), 37.14% Tangra (Mystus vittatus), 37.14% Chapila (Gudusia chapra), 31.43% Prawn (Macrobrachium rosenbergii), 28.57% Shrimp (M. monoceros), 28.57% Vadha (Nandus nandus), 25.71% Sharputi (Puntius sarana), 20% Bowal (Wallago attu) samples from different markets of Chittagong and Dhaka City were contaminated. No formalin was detected in Shrimp (P. indicus), Tilapia (Oreochromis niloticus), Ayer (Mystus aor), Khalisha (Colisa faciatus) and Silver Carp (Hypopthalmichthys molitrix). In beef samples only Diazinon (0.387±0.0E-7 ppm), Quinalphos (0.371±0.021 ppm) and Acephate were detected. In parble (Trichosanthes dioica) only Diazinon (0.047 ppm) and Malathion (0.350 ppm) were detected. These concentrations of pesticides in both beef and parble were beyond the ADI and MRL level. Malathion was also detected as 0.253±0.077, 0.222±0.057, 0.265±0.044, 0.376, 0.293±0.002, 0.250, 0.175, in Solanum melongena, Abelmoschus esculentus, Vigna unguiculata, Momordica charantia and Solanum melongena, respectively. Canal waters were more pesticide contaminated than river water. In drinking water and other supply water the TDS was less than 50 ppm and pH ranged from less than 6.5 to 7.5. The highest level of TDS (332 ppm) was found in both WASA supply water and green coconut water, whereas, the lowest level (19 ppm) was found in Taiwan machine pure drinking supply water. Green coconut, rain water, AC release water and WASA supply water had pH less than 6.5 but Mum and Taiwan machine pure drinking supply water had pH 6.5 to 7.5. Water: River water, WASA water, drinking water, canal water are contaminated and adulterated alarming position because the results beyond of ADI and MRL level. AC water, coconut water, rain water and mum water were free from contamination. Air Condition water is newly invented for using in Bangladesh. Namely AC (1.5 tons) releases 2.25/liters H20 per hour. This type of water is pure and suitable for drinking and so it is a glorious invention for supporting us. The protein standard level (g/100g) was 21.8%, 19.1% and 17.1% in Hilsha (Tenualosa ilisha), Shrimp (Penaeus monodon) and Mola (Amblypharyngodon mola) respectively. Formalin and AlPH3 contaminated foodstuffs decreased protein values. This study concludes that a considerable amount of the samples were adulterated or contaminated by the chemicals which are harmful for human and ecosystem as many of them exceeded the ADI and MRL levels. To get rid of this a combined efforts of the government agencies, policymakers, sellers, farmers, scientists and consumers are needed.