FACULTY OF HEALTH AND MEDICAL SCIENCES UNIVERSITY OF COPENHAGEN PhD thesis 2015 • Tran Minh Phu Chemical use, antimicrobial quality and withdrawal time in striped catfish (Pangasianodon hypophthalmus) aquaculture in the Mekong Delta, Vietnam Chemical use, antimicrobial quality and withdrawal time in striped catfish (Pangasianodon hypophthalmus) aquaculture in the Mekong Delta, Vietnam Tran Minh Phu Section of Food Safety and Zoonoses Department of Veterinary Disease Biology Faculty of Health and Medical Sciences University of Copenhagen Denmark This thesis has been submitted to the Graduate School of the Faculty of Health and Medical Sciences, University of Copenhagen February 2015 Supervisors: Professor Anders Dalsgaard (principal supervisor) Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark Professor Nguyen Thanh Phuong College of Aquaculture and Fisheries, Can Tho University, Can Tho, Vietnam Professor Marie-Louise Scippo Department of Food Sciences, Faculty of Veterinary Medicine, University of Liège, Belgium Assessment Committee: Associate Professor Leon Brimer (chairman) Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark Professor Siska Croubels Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Belgium Dr Mags Crumlish Institute of Aquaculture, Stirling University, Stirling, Scotland, UK Photographer cover photo: Tran Minh Phu PhD thesis 2015 © Tran Minh Phu Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbojlen 4, 1870 Frederiksberg C, Denmark Submitted: February 2015 ISBN… Printed by SL grafik, Frederiksberg C, Denmark (www.slgrfik.dk) Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS ABBREVIATIONS .4 SUMMARY SAMMENDRAG 10 SUMMARY (IN VIETNAMESE) 14 OUTLINE OF THE THESIS 17 INTRODUCTION AND STATE OF THE ART 19 1.1 Project framework 19 1.2 Striped catfish aquaculture in Vietnam 19 1.2.1 Historical development and production 19 1.2.2 Striped catfish aquaculture 22 1.2.3 Processing 24 1.2.4 Certification standards in striped catfish aquaculture 26 1.3 Fish health management 28 1.3.1 Diseases in striped catfish aquaculture 28 1.3.2 Chemical use in striped catfish aquaculture 29 1.4 Occupational health hazards in aquaculture 30 1.5 Quality of antimicrobials used in aquaculture 31 1.6 Elimination and withdrawal time of antimicrobials 31 1.7 Legislation in importing countries 34 1.8 Regulations and advisory services on chemical use in aquaculture in Vietnam 34 SPECIFIC OBJECTIVES AND HYPOTHESES OF THE THESIS 36 METHODOLOGY 37 3.1 Fish health management practices and occupational health hazards in striped catfish (Manuscript I) 37 3.1.1 Study sites 37 3.1.2 Survey design and data collection 37 3.1.3 Data analysis 38 3.2 Quality of antimicrobial products used in striped catfish aquaculture (Manuscript II) 38 3.2.1 Sample collection 38 3.2.2 Sample analysis 38 3.3 Elimination of enrofloxacin in striped catfish following on-farm treatment (Manuscript III) 39 3.3.1 Experimental set-up 39 3.3.2 Sampling methods 39 3.3.3 Analytical methods 40 3.4 Withdrawal time for sulfamethoxazole and trimethoprim following treatment of striped catfish and hybrid red tilapia (Oreochromis mossambicus x Oreochromis niloticus) (Manuscript IV) 40 3.4.1 Study design 40 3.4.2 Fish sampling and processing 41 3.4.3 Sulfamethoxazole and trimethoprim residue analysis 42 3.4.4 Method validation 42 OVERVIEW OF RESULTS AND DISCUSSION 44 4.1 Fish health management practices (Manuscript I) 44 4.1.1 Bacterial disease 44 4.1.2 Disease diagnosis between small-scale and large-scale farms 45 4.1.3 Chemical use in striped catfish 47 4.2 Quality of antimicrobial product and medicated feed 54 4.3 Farmer health risks associated with chemical use 57 4.4 Antimicrobial withdrawal time 58 4.4.1 Elimination of enrofloxacin in striped catfish following on-farm treatment 58 4.4.2 Withdrawal time for sulfamethoxazole and trimethoprim following treatment of striped catfish and hybrid red tilapia 62 CONCLUSIONS AND FUTURE PERSPECTIVES 67 5.1 Conclusions 67 5.2 Future perspectives 68 REFERENCES 71 ANNEX MANUSCRIPTS 83 Manuscript I Acknowledgements I would like to express my sincere gratitude to my principal supervisor, Prof Anders Dalsgaard, for his enthusiastic guidance of my academic research as well as the care he has shown during my stay in Copenhagen I would also like to thank Prof Nguyen Thanh Phuong and Prof Marie-Louise Scippo for their considerable support during the planning, fieldwork planning, sample analysis, preparation of the manuscript and thesis writing of this PhD I learnt a considerable amount from my supervisors on how to be a researcher The study was made possible through the support, financial and otherwise, of the EU-funded FP7 project “Sustaining Ethical Aquaculture Trade” (SEAT project number 222889; www.seatglobal.eu) and I would especially like to thank Prof David Little, SEAT project leader, for successfully coordinating the SEAT project I would also like to thank to associate professors Truong Quoc Phu, Do Thi Thanh Huong, Tran Thi Thanh Hien, Tran Ngoc Hai, Tu Thanh Dung and Dang Thi Hoang Oanh of the College of Aquaculture and Fisheries, Can Tho University, for their support, encouragement and advice during my PhD work I also thank Dr Andreu Rico, Dr Francis Murray, Prof Henry Madsen, Dr Vo Nam Son, Dao Minh Hai, Nguyen The Dien, Nguyen Thanh Phong and other SEAT staff, BSc students Tran Che Linh, Huynh Phuoc Vinh, Cao Thi Kieu Tien and other students, and those who worked with me in the field in the Mekong Delta for their considerable support with data input and analysis and sample collection and analysis during my PhD work I would like to thank the SEAT PhD students Gazi Md Noor Uddin, Nguyen Thanh Lam, Jesper Hedegaard Clausen, Andreu Rico and Li Kang for their support and guidance and sharing information on many aspects during my PhD study I would especially like to thank Nguyen Quoc Thinh, Caroline Douny, Le Tran Truong Thuy and Tran Le Cam Tu for the wonderful time sharing ideas and for their support I also thank my friends (Chi, Quan, Ngoc, Duy, Ninh, Bac Ot, Steffen etc.) in Copenhagen for the time we spent together and also thanks to Nora Ottens, Lars-Espen Tøsse, Finn Dønning and the staff of the Department of Veterinary Disease Biology for their support and discussions I would very much like to thank my parents, brothers, sister’s in-law and lovely nieces for their never-ending love and moral support Most importantly, I am enormously grateful to my wonderful wife, Hong Sen, for sharing her life with me when times have not always been easy Last, but not least, thanks to anyone not mentioned above, many of my friends and colleagues, for your help and advice Abbreviations AMX Amoxicillin AOZ 3-amino-2-oxazolidinone, a metabolite of furazolidone ASC Aquaculture Stewardship Council BAP Best aquaculture practices BMP Better management practices BNP Bacillary necrosis of Pangasius CAP Chloramphenicol CIP Ciprofloxacin CPF Chlorpyriphos DOX Doxycycline ENR Enrofloxacin EU European Union FAO Food and Agriculture Organization FFC Florfenicol GAA Global Aquaculture Alliance GlobalGAP Global good agriculture practice HPLC-UV High-performance liquid chromatography with UV detection ISO International Organization for Standardization IVER Ivermectin LEX Cefalexin LMG Leucomalachite green MARD Ministry of Agriculture and Rural Development MAS Motile Aeromonad septicaemia MG Malachite green MRL Maximum residue limit NAFIQAD National Agro-Forestry-Fisheries Quality Assurance Department NEO Neomycin NRMP National aquaculture residue monitoring programme PAD Pangasius aquaculture dialogue QuEChERS Quick, easy, cheap, effective, rugged and safe extraction method RASFF Rapid Alert System for Food and Feed SEAT Sustaining Ethical Aquaculture Trade SEM Semicarbazide SDZ Sulfadiazine SMX Sulfamethoxazole SMZ Sulfamethazine TMP Trimethoprim TRIFLU Trifluralin UPLC-MS/MS Ultra high-performance liquid chromatography mass spectrometry USDA United States Department of Agriculture US-FDA US Food and Drug Administration US$ US dollars VASEP Vietnamese Association of Seafood Exporters and Producers VietGAP Vietnam good aquaculture practice WP Work package Summary Striped catfish (Pangasianodon hypophthalmus) is a dominant aquaculture species in the Mekong Delta of Vietnam The industry grew rapidly from 93,000 tonnes in 2000 to 1.2 million tonnes in 2012 Intensive production practices and open farming systems, where influent and effluent water sources are frequently shared and used by closely located farmers, are seen as a main cause of frequent disease outbreaks where mortality rates commonly range from 30 to 60 % Bacillary necrosis of Pangasius (BNP), caused by Edwardsiella ictaluri, and Motile Aeromonad septicaemia (MAS) are the most important diseases in striped catfish farming There has not yet been widespread vaccination against these bacterial diseases and farmers therefore apply medicated feed when disease outbreaks occur This thesis consists of studies that collectively contribute to enhancing knowledge about the use of chemicals in striped catfish aquaculture, with an emphasis on fish health management practice, the quality of the antimicrobials used and their withdrawal periods In order to evaluate fish health management practices in striped catfish (Pangasianodon hypophthalmus) farming, semi-structured interviews were conducted with 83 aquaculture enterprises (15 hatcheries, 32 nurseries and 36 grow-out farms) The grow-out farms were classified as small-scale (26) and large-scale farms (10) Information was collected on the educational background of the respondents and the farm infrastructure (e.g pond area, volume and stocking density) as well as details of antimicrobial, disinfectant and probiotic usage during the last crop, and whether these compounds were used for disease prevention or treatment Questionnaire interviews were conducted together with in-depth discussions to collect information on the types and frequency of diseases and the farmers’ understanding of the clinical symptoms of diseases in striped catfish Farmers were also asked about the perceived health hazards associated with their work Results showed that a total of 24 different antimicrobials were used for the treatment of BNP and MAS, and that a variety of disinfectants, probiotics and nutritional supplements were applied In contrast to small-scale farmers, all large-scale grow-out farmers studied were certified according to the GlobalGAP aquaculture standard and therefore had higher levels of formal education and specialised aquaculture training to diagnose and treat diseases All the farmers prepared their own medicated feed, which resulted in a high risk of treatment failure, a negative environmental impact from released antimicrobials, and resistance development Small-scale farmers were exposed to particular occupational health risks when handling antimicrobials and other chemicals, e.g mixing medicated feed with their bare hands The quality of antimicrobial products was investigated by obtaining 21 products commonly used by striped catfish farmers from so-called chemical shops located in the Mekong Delta, Vietnam The aim was to determine the actual concentration of antimicrobial products compared to the concentration declared on the product labels Eleven products contained a single antimicrobial and 10 products contained a mixture of two different antimicrobials Two different batches of 11 products were obtained for comparison analysis Ultra high-performance liquid chromatography mass spectrometry (UPLC-MS/MS) was used to analyse concentration of sulfonamides, trimethoprim, amoxicillin, cefalexin and ciprofloxacin, while concentrations of florfenicol and doxycycline were analysed using high-performance liquid chromatography with UV detection (HPLC-UV) The information on product labels was also evaluated Analytical results showed that only four of the 11 products with a single antimicrobial and two of the 10 products with a mixture of antimicrobials contained active substances within ±10 % of the concentrations declared on the product labels Two products with antimicrobial mixtures did not contain any of the declared antimicrobials In the comparison of two batches, an analysis of 11 products revealed that only one product contained a concentration of active compound that varied by less than 10 % in both batches Several product labels provided inadequate information on how to calculate therapeutic dosages and furthermore stated the withdrawal times, despite the lack of pharmacokinetic data on antimicrobials in striped catfish The poor quality of antimicrobial products has direct implications on the effective treatment of bacterial diseases and the development of antimicrobial resistance, e.g by administering sub-therapeutic doses In order to establish the withdrawal period for enrofloxacin (ENR) following treatment of striped catfish, an on-farm trial in three ponds was undertaken Striped catfish were treated daily for five consecutive days with medicated pelleted feed containing ENR (10 mg/kg body weight) following the farmers’ normal procedures Sampling of striped catfish muscle/skin for residue analysis was performed one day before the first feeding with medicated feed, as well as three times during and five times after (7, 15, 30, 45 and 200 days) application of the medicated feed ENR and its metabolite ciprofloxacin (CIP) residues were analysed by liquid chromatography coupled to mass spectrometry Results showed that a withdrawal time of 45 days was sufficient for striped catfish treated with ENR, i.e below the maximum residue limit (MRL) accepted by the European Hatchery farms (1) In some cases the active ingredient content is not displayed as a % Instead, some products just mention e.g oxytetracycline-HCl 500mg In this case one must calculate the % of active ingredient based on the whole weight or volume of the package Example1: if the package contains 1kg of formulated product and the label says oxytetracycline-HCl 500g, then the % of active ingredient is calculated as (500g/1000g)x100 = 50% Example2: if the package contains 1L of formulated product and the label says oxytetracycline-HCl 200g, then the % of active ingredient is calculated as (200g/1000g)x100 = 20% (assuming density of 1, so 1L = 1kg = 1000g) (2) A=pellet, B=solid (powder), C=liquid ; (3) A= pond cleaning, B= water treatment, C= nutritional supply, D = diseases prevention, E = disease treatment, F = routinely at stocking to prevent stress (can be more than one); (4) A=directly to water B=mixed with feed C= applied to water/sediment treatment after harvest D=applied to the in-flow water E=used to clean equipment; (5) mg, ml, L, g, kg (Please if the farmer reports a different unit, e.g table spoons, cups, keep record of the weight or volume and convert into a standard unit) (6) m2, For chemicals applied mixed with feed, you adjust the chemical dose to the cultured species mass in the pond? Yes/No If Yes, how you estimate the cultured species mass:………………………………………………………………………… Observation of medicated feed making when possible, taking picture Do the farmers keep records of amounts of chemicals used (including antimicrobials, probiotic, pesticides, disinfectants) during operation? Yes / No If yes, ask to see the records and compare the data with the information provided by the interviewer Do farmer have fish health management plan? Yes / No Brief look if possible Disease frequency? Name:…………………… Times/crop:……………………… Name:…………………… Times/crop:……………………… Name:…………………… Times/crop:……………………… Name:…………………… Times/crop:……………………… Hatchery farms D INFORMATION ON HEALTH RISKS ASSOCIATED WITH THE USE OF CHEMICALS Which chemicals/drugs have information described on the chemicals package and which information? Commercial name Information in different language 1Yes No % of active substance Yes No Dosage Yes No Safety instructions Yes No Toxicity Yes No 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Are drugs/chemicals administered according to: a safety instructions described on the package Which ones:………………… b by veterinarian/technicians c extensionist d experiences e friends/others Hatchery farms How are the chemicals/drugs stored? Commercial name Status of container Closed Opened Site of storage shelf cupboard others:… 2 3 2 3 2 3 2 3 2 3 Type of container glass solid container metal solid container plastic container plastic bag paper bag 3 Are the tools used for handling chemicals washed after the use? Circle if Yes Antimicrobials Disinfectants Pesticides Probiotics Is the any direct contact between the skin of the workers and antimicrobials, disinfectants and probiotics? Circle if Yes Antimicrobials Disinfectants Pesticides Probiotics Hatchery farms If Yes, which chemicals and when? Commercial name Type of handling Mixing into feed Loading and applying into water Disinfecting of tanks/equipments Others:………………… 4 4 Is there any direct contact between the skin of the workers and the water used after chemicals apply? Yes / No If Yes, when and how often? Type of handling Water quality control Feeding Harvesting Others Frequency Frequently Not frequently 2 2 10 Hatchery farms Do farm workers use any protection during handling of antimicrobials or disinfectants? Yes / No 10 If Yes, which types of protection which workers use during chemicals handling? Commercial name Protective measure (1=gloves, 2=rubber boots, 3=mask, 4=glasses = clothes) Type of handling (1=mixing into feed, 2=loading and applying into water, 3=disinfection) 2 2 2 2 2 Appropriate use Yes No 11 Do workers regularly clean their hands/take a shower after handling of chemicals or contact with water/feed containing chemicals? Yes/No 12 If Yes, how are the hands/body cleaned? With water With water and soap Other:……………… 11 Hatchery farms 13 Are hatchery workers instructed to safety handling of chemicals? Yes / No 14 If Yes, by whom? Owner/Hatchery manager Local officer Training Media (publications, TV etc.) Others 15 Are farm workers informed about the health and environmental risks associated with the use of chemicals? Yes / No 16 If Yes, who informed hatchery workers about such risks? Owner/Hatchery manager Local officer Training Media (publications, TV etc.) Others 17 Are farm workers informed about banned chemicals? Yes / No 18 If Yes, who informed farm workers about banned chemicals? Owner/Hatchery manager Local officer Training Media (publications, TV etc.) Others 12 Hatchery farms 29 Do some workers have common signs of illness/poisons? Yes / No 20 If Yes, which signs and how many workers? Skin lesions Headache Coughing Conjunctivitis Sneezing Abdominal pain Diarrhea Vomit Others Which are the main compounds causing these symptoms:………………………………………………………… 21 If skin lesions or any other signs of illness/poisons, affected workers used any medication to treat the lesions? Yes / No 22 If Yes, which medication treatment? ………………………………………………………………… ………………………………………………………………… ………………………………………………………………… 13 .. .Chemical use, antimicrobial quality and withdrawal time in striped catfish (Pangasianodon hypophthalmus) aquaculture in the Mekong Delta, Vietnam Tran Minh Phu Section of Food Safety and. .. withdrawal times in striped catfish aquaculture in the Mekong Delta, Vietnam 1.2 Striped catfish aquaculture in Vietnam 1.2.1 Historical development and production The striped catfish (Pangasianodon hypophthalmus). .. services and the private sector, e.g the role of chemical shops in disseminating information on the prudent use of antimicrobials and other chemicals 4.1.3 Chemical use in striped catfish 4.1.3.1 Antimicrobial