http://reposit.library.du.ac.bd:8080/xmlui/xmlui/handle/123456789/156 2026-04-08T20:24:34Z http://reposit.library.du.ac.bd:8080/xmlui/xmlui/handle/123456789/4678 Development of methods for analyzing and monitoring antibiotic and pesticide residues in fish and shrimp 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. This thesis is submitted for the degree of Doctor of Philosophy. 2025-07-07T00:00:00Z http://reposit.library.du.ac.bd:8080/xmlui/xmlui/handle/123456789/4330 Rearing of carps by using bio-gas plant slurry and other ingredients in ponds and large water body Huda, Md. Nurul This thesis is submitted for the degree of Doctor of Philosophy. 2025-05-04T00:00:00Z http://reposit.library.du.ac.bd:8080/xmlui/xmlui/handle/123456789/4329 Importance of fish pass and fish friendly structure in inland open water fish production Hossain, Md. Motaher This thesis is submitted for the degree of Doctor of Philosophy. 2025-05-04T00:00:00Z http://reposit.library.du.ac.bd:8080/xmlui/xmlui/handle/123456789/2832 Molecular Characterization of Pathogenic Strains of Bacteria Causing Motile Aeromonas Septicemia in Heteropneustes fossilis: Implications for Potential Drug Development 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. A dissertation submitted to the University of Dhaka in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Fisheries. 2023-12-05T00:00:00Z