monitoring of antibiotic residues in poultry feed, water and eggs and its public health significance

112 Pages·2015·1.58 MB·English

MONITORING OF ANTIBIOTIC RESIDUES IN
POULTRY FEED, WATER AND EGGS AND ITS
PUBLIC HEALTH SIGNIFICANCE

Thesis

Submitted to the Guru Angad Dev Veterinary and Animal Sciences University
in partial fulfillment of the requirements for the degree of

MASTER OF VETERINARY SCIENCE
in
VETERINARY PUBLIC HEALTH AND EPIDEMIOLOGY
(Minor Subject: Veterinary Pharmacology and Toxicology)

By
C. Lalawmpuia
(L-2013-V-60-M)

Department of Veterinary Public Health and Epidemiology
School of Public Health and Zoonoses
College of Veterinary Science
© Guru Angad Dev Veterinary and Animal Sciences University
LUDHIANA – 141004
2015
CERTIFICATE – I

This is to certify that the thesis entitled,“ MONITORING OF ANTIBIOTIC
RESIDUES IN POULTRY FEED, WATER AND EGGS AND ITS PUBLIC
HEALTH SIGNIFICANCE” submitted for the degree of M.V.Sc., in the subject of
Veterinary Public Health and Epidemiology (Minor subject: Veterinary
Pharmacology and Toxicology) of the Guru Angad Dev Veterinary and Animal
Sciences University, Ludhiana, is a bonafide research work carried out by
C. Lalawmpuia (L-2013-V-60-M) under my supervision and that no part of this
thesis has been submitted for any other degree.
The assistance and help received during the course of investigation have been
fully acknowledged.

______________________________
(Dr. J.S. Bedi)
Major Advisor
Assistant Professor
School of Public Health and Zoonoses
Guru Angad Dev Veterinary
and Animal Sciences University
Ludhiana – 141004 (India)
CERTIFICATE – II
This is to certify that the thesis entitled, “MONITORING OF ANTIBIOTIC
RESIDUES IN POULTRY FEED, WATER AND EGGS AND ITS PUBLIC
HEALTH SIGNIFICANCE” submitted by C. Lalawmpuia (L-2013-V-60-M) to
the Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, in partial
fulfillment of the requirements for the degree of M.V.Sc., in the subject of
Veterinary Public Health and Epidemiology (Minor subject: Veterinary
Pharmacology and Toxicology) has been approved by the student‟s Advisory
Committee after an oral examination on the same, in collaboration with an external
examiner.

_________________________ ___________________
(Dr. J.S. Bedi) (Dr. N.K. Mahajan)
Major Advisor External Examiner
Professor and Head
Department of Public Health
and Epidemiology
LUVAS, Hisar

_________________________
(Dr. J. P. S. Gill)
Director

_________________________
(Dr. Simrat Sagar Singh)
Dean, Postgraduate Studies
ACKNOWLEDGEMENT
At the very outset, I bow my head with reverence to the
“Almighty God” for His benign blessing hand and bestowing a creative
and healthy environment throughout my academic and research period.
I express my deep sense of gratitude and wish to place on record
my sincere and heartfelt regards to my revered Major Advisor Dr. J.S.
Bedi, Assistant Professor, School of Public Health and Zoonoses,
GADVASU for his sagacious guidance, marvelous affection, constant
moral support, constructive & valuable suggestions, friendly behaviour
and thoughtful criticism throughout my association with him.
With great regard and with due decorum, I acknowledge my
sincere thanks to Dr. J.P.S. Gill, Dean PGS Nominee(Advisory
Committee) Director, School of Public Health and Zoonoses, GADVASU.
He has always encouraged and motivated me in research work. He has
helped me to see life and science in full depth and taught me how to
appreciate the good scientific work. He has enlightened me through his
wide knowledge of intelligence.
I owe my sincere thanks to the members of my advisory
committee Dr. R .S Aulakh and Dr. Rajdeep Kaur for their ever-willing
help, scholastic guidance and constructive criticism throughout the
pursuance of this study.
I express my special thanks to Dr. Simrat Sagar Singh, Dean PGS,
GADVASU and Dr. S.N.S. Randhawa, Ex Dean PGS, for encouragement
and providing help during the study.
Words are compendious to express my sincere and whole hearted
thanks to the other faculty members of the department of Veterinary
Public Health and Epidemiology Drs. Randhir Singh, B B Singh, Rajnish
Sharma and Simranpreet Kaur for their sincere help, moral support and
constant encouragement.
With a deep sense of gratitude, I wish to convey my thanks to Drs.
Navdeep Singh (VO), Pradeep Singh (VO), Dr. Navdeep Singh Kihnda
(VO) and Mandeep Singh (Research Fellow) for helping me in collection
of samples for this study.
I have great pleasure in expressing my sincere thanks to my
batchmates Drs. Pranita, Rashmi, Amandeep, Mohit and Prateek for
their love and support throughout my study. I am also thankful to my
juniors in the department Drs. Yashodhara, Sarmistha, Neharika,
Shalini, Shalinta, Poonam, Vishal and Prithvi for their help during my
research work. I feel elated in expressing thanks to Phd students Drs.
Pallavi, Reshmi, Reeti, Anupama, Deepali, Lokesh, Abdul and Bhaskar
for their expert advice and cooperation from time to time in conducting
my research work. I am highly grateful to Dr Shilpa and Research
Fellows Mr Amit and Mr Pravez for their endless assistance, co-
operation and infallible patience. Without them, none of this would have
been possible. I also wish to thank all the laboratory and office staff
members of department Ms. Ranjana, Mr. Amardeep, Mr. Nirpinder,
Mr. Makhan Singh and Mr. Baljinder of the department. Their guidance
and support helped a long way in completion of this study.
I owe a debt of gratitude to my friends Drs. Ripamchi, Mimi,
Nancy, Jerry, Zodina, Sawmkima, David, Ponil, Suiding, Gilsang,
Saptak, Morchi, Sarat, Angam, Subhra De, Michael Solo, Rosanga,
Mary, Angaihi, Mawitei, Rengpuii, Sangtea, Mesak, Ramtea, Vantei,
MS-i, Mapuia, Duata, Jerome, Tetea, PC-a, Chhana, Kima, Sachin, Patel,
Shriram, Shabnam, Divya, Chandan, Brahma, Projolita, Jenny and
Mary Varte, Ramtei, Tleipuii, Pa Biaka, Pa Rama, Nu LN-I, Nu
Makimi, U MC-a, U Hrili, U Duhawmi, Ruthi, Deli, Duhawma, Judy
Sampata and Tetei for their firm support and spontaneous help
throughout this endeavour.
I would like to take this opportunity to thank my beloved parents,
Mr. Lalrambeiseia and Mrs. Lalrozami who have been my biggest
inspiration and who have made me what I am today. Their love,
blessings, prayer and constant encouragement have always been with
me throughout my life. My gratitude for them is beyond words.
Words are woefully inadequate to express my sense of gratitude to
my other family members, my brothers Mr. Zothanpuia, Mr.
Lalhlimpuia, my sister Ms. Sarah Vanlalhriatpuii and my aunt Ms.
Lalrinmawii for their love, blessings and sacrifices they make for me
every day.
I sincerely thank all those who led me to accomplish this task with
earnest effort. All may not have been mentioned but none is forgotten.

Date: (C. Lalawmpuia)
Title of the Thesis : MONITORING OF ANTIBIOTIC RESIDUES
IN POULTRY FEED, WATER AND EGGS
AND ITS PUBLIC HEALTH SIGNIFICANCE
Name of the student : C. Lalawmpuia
Admission No. : L-2013-V-60-M
Major Subject : Veterinary Public Health and Epidemiology
Minor Subject : Veterinary Pharmacology and Toxicology
Name and Designation of : Dr. J.S. Bedi
Major Advisor Assistant Professor
Degree to be Awarded : M.V.Sc.
Year of award of Degree : 2015
Total Pages of Thesis : 96 + Annexure + VITA
Name of University : Guru Angad Dev Veterinary and Animal Sciences
University, Ludhiana – 141004 (India)

ABSTRACT
The presence of residues of antimicrobial agents in food of animal origin in excess of the
established maximum residue limits (MRLs) indicates that good veterinary practices are not being
respected. Consequently, it is necessary to establish simple, sensible, and reliable analytical
methods for the determination of multi-residues of different classes of antimicrobials in food and
particularly in eggs. Therefore, the present study was conducted to determine the antibiotic
residues in poultry feed, water and eggs, evaluate the effect of various heat processing techniques
on these residues in eggs and to assess the risk associated with consumption of eggs having
residues. Two samples each of poultry feed and water and three egg samples from 27 different
poultry farms were collected. Out of these, 7/81 (8.6%) and 7/54 (12.9%) eggs and feed samples
were found positive for oxytetracycline, respectively while, for chlortetracycline, 4/81 (4.9%) and
2/54 (2.5%) eggs and feed samples were found positive, respectively. The level of eggs and water
positive samples for enrofloxacin were 4/81 (4.9%) and 2/54 (3.7%), respectively whereas for
ciprofloxacin, 2/81 (2.5%) and 1/54 (1.9%) eggs and water samples were found positive,
respectively. Among the 106 commercially available market eggs, 5/106 (4.7%) and 3/106 (2.8%)
samples were positive for oxytetracycline and chlortetracycline residues, respectively. While, for
enrofloxacin and ciprofloxacin, 2/106 (1.8%) and 2/106 (1.8%) egg albumin samples were found
positive, respectively. Whereas, none of the egg albumin sample was found positive among the
sulfonamide groups including sulfadiazine and sulfamethoxazole. Meanwhile, 2/106 (1.8%) and
1/106 (0.9%) samples were positive for oxytetracycline and chlortetracycline, respectively. While,
for enrofloxacin and ciprofloxacin, 1/106 (0.9%) and 1/106 (0.9%) egg yolk samples were found
positive, respectively. The effect of heat treatment on antibiotic residues was evaluated. The types
of heat treatment given to the egg samples included boiling and frying. The percentage reduction
of antibiotic residues was recorded for each heat treatment. The percentage reduction of
oxytetracycline at 100 and 500 µg/kg ranged from 52.3-68.0% and 50.4-59.5%, respectively
whereas the percentage reduction of chlortetracycline at 100 and 500µg/kg ranged from 61.2-
69.2% and 59.4-52.3%, respectively. Meanwhile the percentage reduction of enrofloxacin at 100
and 500µg/kg ranged from 10.9-23.6% and 9.03-18.5%, respectively whereas the percentage
reduction of ciprofloxacin at 100 and 500 µg/kg ranged from 10.8-9.4 and 9.9-7.9%, respectively.
Based on the mean values of antibiotic residues, the Hazard Quotient was evaluated for
oxytetracycline, chlortetracycline, enrofloxacin and ciprofloxacin in positive egg samples and at
the current mean levels of antibiotic residues in eggs and the availability or per serving of egg,
these residues pose no significant health effects on consumer health.
Keywords: Antibiotics, HPLC, Tetracyclines, Fluoroquinolones, Sulfonamides, Hazard Quotient

_______________________ _____________________
Signature of Major Advisor Signature of the Student
CONTENTS
CHAPTER TOPIC PAGE NO.
I INTRODUCTION 1 – 5
II REVIEW OF LITERATURE 6 – 27
III MATERIALS AND METHODS 28 – 47
IV RESULTS AND DISCUSSION 48 – 82
V SUMMARY 83 – 87
REFERENCES 88 – 96
ANNEXURE i – iii
VITA
LIST OF TABLES
Table Title Page
No. No.
1 List of antibiotic standards used in present study 29
2 Specification details of High Performance Liquid Chromatography 30
HPLC
3 HPLC conditions for tetracycline residues 36
4 HPLC conditions for fluoroquinolone residues 41
5 HPLC conditions for sulfonamide residues 45
6 Various heat processing temperature and time combinations 46
7 Method Validation parameters for detection of tetracycline antibiotic 51
residues using HPLC
8 Tetracycline residues (µg/kg) detected in eggs, feed and water 52
collected from different poultry farms in Punjab
9 Method Validation parameters for detection of fluoroquinolones 54
antibiotic residues using HPLC
10 Fluoroquinolones residues (g/kg) detected in eggs, feed and water 56
collected from different poultry farms in Punjab
11 Method Validation parameters for detection of sulfonamides 60
antibiotic residues using HPLC
12 Concomitant presence of antibiotic residues (µg/kg) in eggs, feed and 61
water collected from 27 different poultry farms in Punjab
13 Antibiotic residues detected in egg albumin collected from 64
commercially available markets in Ludhiana, Punjab
14 Antibiotic residues detected in egg yolk collected from commercially 65
available markets in Ludhiana, Punjab
15 Effect of heat treatment (% reduction) on oxytetracycline residues by 69
boiling and frying
16 Effect of heat treatment (% reduction) on chlortetracycline residues 69
by boiling and frying
17 Effect of heat treatment (% reduction) on enrofloxacin residues by 69
boiling and frying
18 Effect of heat treatment (% reduction) on ciprofloxacin residues by 69
boiling and frying
19 Estimation of risk assessment based on Hazard Quotient for eggs 75
samples from different poultry farms (from mean concentration of
positive samples)
20 Estimation of risk assessment based on Hazard Quotient for eggs 76
samples from different poultry farms (from absolute mean
concentration of samples)
Table Title Page
No. No.
21 Estimation of risk assessment based on Hazard Quotient for eggs 77
samples (albumin) from commercial market in Ludhiana (from mean
concentration of positive samples)
22 Estimation of risk assessment based on Hazard Quotient for eggs 78
samples (albumin) from commercial market in Ludhiana (from
absolute mean concentration of samples)
23 Estimation of risk assessment based on Hazard Quotient for eggs 79
samples (yolk) from commercial market in Ludhiana (from mean
concentration of positive samples)
24 Estimation of risk assessment based on Hazard Quotient for eggs 80
samples (yolk) from commercial market in Ludhiana (from absolute
mean concentration of samples)
LIST OF FIGURES
Fig. No. Title
1 Flow diagram for extraction and clean up of tetracycline residues from
poultry egg samples
2 Flow diagram for extraction and clean up of tetracycline residues from
feed and water samples
3 Flow diagram for extraction and clean up of fluoroquinolones residues
from poultry eggs
4 Flow diagram for extraction and clean up of fluoroquinolone residues
from poultry feed and water samples
5 Flow diagram for extraction and clean up of sulfonamide residues from
poultry eggs
6 Flow diagram for extraction and clean up of sulfonamide residues from
poultry feed and water samples
7 Standard curve for oxytetracycline standard at different concentrations
8 Standard curve for chlortetracycline standard at different concentrations
9 HPLC chromatogram of oxytetracycline and chlortetracycline standard at
different concentrations
10 HPLC chromatogram of oxytetracycline and chlortetracycline standards
11a&b HPLC chromatograms of oxytetracycline and chlortetracycline positive
samples
12 Standard curve for enrofloxacin standard at different concentrations
13 Standard curve for ciprofloxacin standard at different concentrations
14 HPLC chromatogram of enrofloxacin and ciprofloxacin standards
15 HPLC chromatogram of enrofloxacin positive samples
16 HPLC chromatogram of ciprofloxacin positive sample
17 Standard curve for sulfonamide standards at different concentrations
18 HPLC chromatogram of sulfadiazine positive samples
19 HPLC chromatogram of sulfamethoxazole positive samples
20 Comparative positive means of different antibiotic residues detected in
egg albumin and yolk
21 Comparative percentage positive of different antibiotic residues detected
in egg albumin and yolk
22 Heat treatment of Oxytetracycline by boiling and frying at 0.1 ppm and
0.5 ppm