Logistics and Health Risk Assessment of Veterinary Drug and Additive in Animal Feed after ASEAN Economic Community (AEC)

Presented by Preechaya Yodrasing

ID 51216325 ENTM/M

1 Introduction

2 Literature Review

3 Methodology

Objectives Expected Results

Page 2

Introduction

Agricultural and food products

Asean+3Page 3

Introduction

Page 4

Page 5

Introduction

“Food Safety”

Page 6

Objective

To determine the level of residue veterinary in terms of livestock product, within ASEAN+3 and the current domestic market.

To evaluate the risk for contaminated residue of veterinary infeed of used animal.

To find out a environment that suitably for measurement the quantity of residue veterinary in terms of livestock product, and its measuring method will perform by ultrasonic-assisted extraction technique (UAE) - SPE - LC-MS/MS.

Page 7

Expected Results

To understand the overview of livestock product not only in-house and outer market but also imported product that may conceal a residue of veterinary.

To be model of management and monitoring a residue, that may cause a risk to consumer especially after the AEC was formally announce.

To know a dispersion trend of residue after the AEC was formally announce and a quantity of residue in the country.

Page 8

0

2,000,000

4,000,000

6,000,000

8,000,000

10,000,000

12,000,000

14,000,000

16,000,000

18,000,000

Feed consumption in 2004-2014(Thai Feed Mill Association, 2014)

Literature Review

Volume (Tons)

Page 9

Thailand’s total of animal feed import from ASEAN (2010-2013)(Division of Animal Feed and Veterinary Products Control, 2013)

0

50,000

100,000

150,000

200,000

2010 2011 2012 2013

0

1,000

2,000

3,000

4,000

5,000

6,000

2010 2011 2012 2013

Volume (Tons)

Value (Million Bath)

Page 10

Thailand’s total imports of animal feed from ASEAN in 2013

Raw material66.40%

Mixed material9.35%

Supplementary20.28%

Other feed3.97%

Raw material17.70%

Mixed material49.97%

Supplementary27.39%

Other feed4.93%

Ratio of quantity and animal feed imported value, segmentation by type

Countries Volume (Ton)

Value (million bath)

Vietnam 53.67 4.23

Malaysia 116.14 6.45

Singapore 4,719.02 548.82

Indonesia 6.54 2.45

China 8,603.36 939.38

Korea 1,439.68 78.91

Japan 1,520.96 143.27

Ratio of quantity imports

Ratio of value imports(Division of Animal Feed and Veterinary Products Control, 2013) Page 11

48 locals customs facilitations (Customs Facilitations, 2014)

Related Agencies of animal feed import.

1. Customs Department, Ministry of Finance.

Page 12

3. Division of Animal Feed and Veterinary Products Control, Department of Livestock Development, Ministry of Agriculture and Cooperatives.

Proceeding to evaluation, control and preserve for animal feed entrepreneur (Producer, importer and trader) follow the Feed Quality Control Act, 1982.

2. Division of Veterinary Inspector and Quarantine, Department of Livestock Development, Ministry of Agriculture andCooperatives.

Responsibly for animal quarantine, both of domestic and international, also control the movement of animal carcasses and livestock products. There are 43 outposts for in and out animal quarantine control.

Regulations The Act of Drug B.E. 2510 (1967) The Act of Feed Quality Control B.E. 2525 (1982) and revised B.E. 2542 (1999)

Related Agencies of animal feed import (Cons.)

Page 13

Import Entry

Import permit for animal feed

A license to import for animal feed into Thailand

Importer

Division of Animal Feed and Veterinary

Products Control

Imported process of animal feed is divided into two phases: pre-import and date of import.

Phases I: pre-import

Customs Facilitation

Page 14

Phases II: date of import

Inspection of the permit and other documents

Sampling for disease inspection and residue

Seizure or Withholding for further inspection

Issue the permit

Return the country of origin and legal actions

Inspect and release products

Animal Quarantine

Customs Facilitation

- DNA Ruminant- Melamine

Bureau of Quality Control of Livestock Products

The Act of Feed Quality Control

- Kasetsart University- Chulalongkorn University- Central laboratory

pass

Consideration

Not passed

Passed

by

Page 15

The outpost that mainly imported animal feed.

A=Bangkok PortB=Laem Chabang PortC=Ladkrabang Cargo ClearanceD=Suvarnabhumi Airport Cargo Clearance

AB

CD

Laos

Vietnam

Cambodia

China

Myanmar

Page 16

Antimicrobial agent used in feed

(Frank, 2010)

• Antimicrobials for the production of medicated feed is confirmed by national feed manufacturers’ associations.

13

2

Therapy

prevention

Growth promoter

Page 17

Prohibition of using chemical product in feed additive and its object Act.

Consist of

1. Avoparcin 3. Chloramphenicol 4. Carbadox5. Olaquindox 6. Nitrofurans gr.7. Nitroimidazole gr. 8. Diethylstilbestrol9. Dioxin 10. Melamine Ammeline

Ammelide Cyanuric acid

2. Beta-agonist

Page 18

The contaminated in situation that residues drug and other additives in animal feed of AEC.

In Malaysia, according from the study of Veterinarian services in 2012 found that, the amount of chicken fed up to 50% are resistant to antibiotics, Ampicillins, Sulfonamides and Tetracycline. And for an imported products, were found to be resistant to both Ampicillin, Streptomycin and other antibiotics in proportions ranging from 50 to 87% The most obviously pathogens in food such as. Listeria, Salmonella and E. coli. That is detrimental to control food quality. From the workhouses industry and manufacturing to consumers in the long term. (TheFishSite, 2014)

In China, they have been found to have used beta-agonist for a while, for example in2006, there are 336 people were taken to hospital after ate pork and its entrails(pathogens to clenbuterol) and in 2009. Police officers arrested 9 suspects who sellingpork meat those pathogens to beta-agonist. That caused more than 70 customers inGuangdong got sick. And in 2011, Chinese media have presented a farmer, in theprovince of Henan, was mixed clenbuterol into pig feed. (ASTV, 2011)

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**Contaminated assessment of residues drug in beef*** (2013) From 314 beef samples, found that there are had 128 samples contaminated

which are 40 percentage of a sampling (range of amount is 174.81 – 39,092.74 ppb)

Residue monitoring results in 2010 - 2012

Department of Livestock Development, 2013

The contaminated in situation that residues drug and other additives in animal feed of Thailand.

Page 20

The contaminated in situation that residues drug and other additives in animal feed of Thailand.

Year Type ofsample

Samplingplaces

SampleQuantity

DetectedQuantity

Type of residues

2011 Poultry feed Poultry farms 304 - -

Pig feed Pig farms 1,297 15 Tetracyclines, Sulphonamides

2012 Poultry feed Poultry farms 315 - -

Pig feed Pig farms 1,340 17 Chlortetracycline, Oxytetracycline, Tylosin A, Sulfamethazine (SMZ)

Department of Livestock Development, 2013Page 21

Effected of veterinary drug residues to the consumers

Antibiotic

Beta-agonist

Page 22

Literature Review

Pleadin et al. (2010) were determine the level of clenbuterol residues in muscle tissue of pigs after repeat administration in a growth-promoting dose. An anabolic dose of clenbuterol (20 μg/kg body mass per day) was administered orally to experimental group (n= 12) for 28 days. Determination of residual clenbuterol was by enzyme-linked immunosorbent assay (ELISA) as a screening method and liquid chromatography tandem mass spectrometry (LC-MS/MS) as a confirmation method. Result that the highest clenbuterol content in the muscle of treated animals was recorded on day 0 of treatment cessation (4.40± 0.37 ng/g) and significantly (p<0.05) exceeded the maximum residue limit (MRL) of 0.1 ng/g. On day 3 of withdrawal, it was 0.49±0.22 ng/g and on day 7 0.10±0.02 ng/g (at MRL); on day 14 of treatment discontinuation, clenbuterol content was below the limit of detection (<0.1 ng/g) in all samples.

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Wijayanti et al. (2011) were investigate the effect of doxycycline (a tetracycline derivative)administered at disease-prevention dose given daily in the feed and drinking water on the residue level in the broiler-chicken meat. Doxycycline at concentration of 100 ppm was mixed in the drinking water (1 g of doxycycline in 10 L of drinking water) and feed (1 g of doxycycline in 10 kg of feed). Analysis of doxycycline level was performed using high performance liquid chromatography with extraction method referring to the standard of Association of Official Analytical Chemistry. The result showed that the highest residue level was found at the first week in the group given doxycycline in drinking water (0.96±0.15 μg/g),while the highest was found at the third week in the group given doxycycline in feed (0.15±0.02 μg/g). The level of doxycycline residue exceeding the level permitted by the Indonesian National Standard (0.1 μg/g). It can be concluded that doxycycline is not recommended to be given daily both through water or feed to commercial chickens as a disease-prevention agent.

Literature Review (cons.)

Page 24

Summary of analytical methods for monitoring of Beta-agonist

Compounds Target species for

feed use

Sample treatment Analytical technique

Method performance Ref.

Extraction procedure

Clean up Detection system

Recovery (%)

Limit (ng/g)

SalbutamolRactopamineClenbuterol

Pig vortex SPE (C18 cartridges)

LC-MS 83-110 LOD:10LOQ: 50

Zhang, 2009

7 Beta-agonist Not specified

sonication SPE (polymericcartridge)

LC-MS/MS 82-110 LOD: 0.87 LOQ: 0.5-

0.92

Juan, 2010

CimaterolSalbutamol TerbutalineRactopamine

Not specified

shaking+centrifuge

SPE (MCXcartridge)

UPLC 90.1-101.4 LOD: 5-6.5LOQ: 16.7-

21.7

Qu, 2011

12 Beta-agonist Not specified

mechanicalshaking

SPE (mix-mode cartridge)

UPLC-Q/TOF MS

89.2-103.3 LOD: 1-5LOQ: 5-50

Xiu-Juan, 2013

20 prohibited veterinary drugs

Not specified

vortex+sonication

PSA sorbent LC-MS/MS 56.7-103 LOD: 0.42-5.74 LOQ 5.70-9.81

Zhang, 2013

Page 25

Summary of analytical methods for monitoring of antibiotics

Compounds Target species for

feed use

Sample treatment Analytical technique

Method performance Ref.

Extraction procedure

Clean up Detection system

Recovery (%)

Limit (ng/g)

OxytetracyclineTetracyclineChloramphenicol

Not specified

Subcritical water extraction

None LC-UV 82.1-90.0 Not reported

Wang, 2008

Multi-class CattleLambPig

shaking+sonication

PSA sorbent LC-MS/MS 51-116 LOQ:3.8-65.0

Boscher, 2010

Multi-class PigPoultry

vortex+mechanical shaking

LLE, Extraction solvent: hexane

LC-MS/MS 89.2-103.3

Not reported

Cronly, 2010

16 Sulfonamides Pig shaking+centrifuge

SPE (alumina) LC-MS/MS 80-120 LOQ: 0.5-20

Liu, 2011

ChlortetracyclineOxytetracyclineTetracyclineDoxycycline

PigPoultry

vortex+ultrasonic bath

SPE (Oasis HLB)

LC-MS/MS 80.9-119.5

LOD: 3-20LOQ: 10-50

Guo, 2012

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Methodology

Frameworks of study

Select sampling point of animal feed

Health risk assessment

Beta-agonist Antibiotics

Solid-Phase Extraction (SPE)

LC-MS/MS

Ultrasonic-assisted extraction (UAE)

Tetracyclines

TetracyclineOxytetracyclineChlortetracycline

Doxycycline

Sulfonamides

SulfadiazineSulfamethazine

SulfamethoxazoleSulfaquinoxaline

ClenbuterolSalbutamol

Ractopamine

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Sampling point

1-2 farms

1-2 farms

1-2 farms

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Analytical method

Sample preparation

-Blended and a portion of 2 g powdery feed was weighed to continue.

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Extraction(UAE)

Clean-up SPE (SCX)

LC-MS/MS

- Sample 2 g - Add Milli-Q 20 mL and 1 M HCl 2 mL

- Shaker 350 mot/min, 40 min- Ultrasonic 15 min - Centrifuge 4,000 rpm, 20 min- Filtration membrane filter, GF/B

- Precondition: MeOH 3 mL andMilli-Q 3 mL

- Wash: Milli-Q 2 mL and 2% Formic acid in water 2 mL

- Elute: 5% Ammonia in MeOH 5 mL- Dry nitrogen gas 40°C

Reconstitution: 0.1% Formic acid in water:ACN (90:10) 1 mL

Beta-agonist analysis

Page 30

Extraction(UAE) - Shaker 400 mot/min, 1 hr.

- Ultrasonic 15 min- Centrifuge 4,500 rpm, 20 min- Filtration by membrane filter, GF/B volume 10 mL And add Milli-Q 40 mL

Clean-up SPE (HLB)

LC-MS/MS

- Sample 2 g- Add MeOH:0.2M citric acid, pH3 (1:1, v/v) 30 mL

- Precondition: MeOH 2 mL and 0.04M citric acid, pH3 2 mL- Wash: 0.04M citric acid, pH3 2 mL and0.1M Sodium acetate 2 mL- Elute: MeOH 6 mL- Dry nitrogen gas 40°C

Reconstitution: MeOH +10mM Formic acid 0.5 mL

Antibiotics analysis

Page 31

Health Risk Assessment

The results obtained from this analytical

Calculated in a percentage of amount of the residue in animal products, referred from a research.x 100 = x%

y1 y2 X%

y2

y1

ba

x%

100%x a = b

bPage 32

Veterinary Drug Animal Species Tissue

Maximum Residue Limit (𝛍g/kg )

Ministry of Public Health

Department of Livestock

Development

EU CODEX Japan

Clenbutarol Not specified Edible offal - Not detected - - -

Cattle Muscle - Not detected 0.1 0.2 0.2

Liver - Not detected 0.5 0.6 0.6

Kidney - Not detected 0.5 0.6 0.6

Ractopamine hydrochloride

Cattle and Pig Muscle - - 10 10 10

Fat - - 10 10 10

Liver - - 40 40 40

Kidney - - 90 90 90

Tetracycline/Oxytetracycline/Chlortetracycline

Not specified Muscle 200 100 100 200 200

Liver 600 300 300 600 600

Kidney 1,200 600 600 1,200 1,200

Egg 400 200 200 400 400

Milk (Cattle, Sheep)

100 - 100 100 100

Page 33

MRL index

Veterinary Drug Animal Species Tissue

Maximum Residue Limit (𝛍g/kg)Ministry of Public

HealthDepartment of

Livestock Development

EU CODEX Japan

Doxycycline Pig and Poultry Muscle - 50 100 - 50Fat/Skin - 50 300 - 50

Liver - 50 300 - 50Kidney - 50 600 - 50

Other Poultry Muscle - 100 100 - 100Fat/Skin - 300 300 - 300

Liver - 300 300 - 300Kidney - 600 600 - 600

Cattle Muscle - - 100 - 100Liver - - 300 - 300

Kidney - - 600 - 600Sulfonamide Not specified Muscle 100 100 100 100 100

Fat 100 - 100 100 100Liver 100 100 100 100 100

Kidney 100 100 100 100 100Egg - 100 - - 10

Cattle, Goat and Sheep

Milk (μg/l) 25 - 100 25 10

Page 34

MRL index (cons.)

References• ASEAN Economic Community. (2013). ASEAN Economic Community History. Retrieved October 28, 2013, from Thai-AEC.com

Website: http://www.thai-aec.com/ประวตัคิวามเป็นมาประชา• ASTV. (2009). บรษิทัจนีฉาวอกี! เรยีกคนือาหารกล่องหลงัเนื้อเป้ือนสารเคม.ี Retrieved March 17, 2014, from Website:

http://www.manager.co.th/China/ViewNews.aspx?NewsID=9520000045726• ASTV. (2011). จนีเกบ็คนืผลติภณัฑเ์น้ือหม ูเหตุอือ้ฉาวปนเป้ือน “สารหมผูอม”. . Retrieved March 17, 2014, from Website:

http://www.manager.co.th/China/ViewNews.aspx?NewsID=9540000034654• Boscher, A., Guignard, C., Pellet, T., Hoffmann, L., and Bohn, T. (2010). Development of a multi-class method for the

quantification of veterinary drug residues in feedingstuffs by liquid chromatographt-tandem mass spectrometry. Journal of Chromatography A, 1217(41), 6394-6404.

• Cronly, M., Behan, P., Foley, B., Malone, E., Earley, S., Gallagher, S., Gallagher, M., Shearan, P., and Regan, L. (2010). Development and validation of a rapid multi-class method for the confirmation of fourteen prohibited medicinal additives in pig and poultry compound feed by liquid chromatography-tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 53(4), 929-938.

• Customs Facilitations. (2014). Customs Office map. Retrieved July 14, 2014, from Customs Facilitations Website: http://www.customs.go.th/wps/wcm/connect/Library+cus501th/InternetTH/About+Us/customs_area/2B75CC5BC8A4956AC217A77595536FB9

• Division of Animal Feed and Veterinary Products Control. (2013). Comparison data between quantity and imported values of animal feed in 2012-2013. Retrieved March 18, 2014, from Division of Animal Feed and Veterinary Products Control Website: http://afvc.dld.go.th/forum.php?mod=viewthread&tid=161&extra=Array

• Division of Animal Feed and Veterinary Products Control. (2014). History of Division of Animal Feed and Veterinary Products Control. Retrieved March 18, 2014, from Division of Animal Feed and Veterinary Products Control Website: http://afvc.dld.go.th/forum.php?mod=viewthread&tid=74&extra=page%3D1

• Division of Veterinary Inspector and Quarantine. (2014). History of Division of Veterinary Inspector and Quarantine. Retrieved December 2, 2014, from Division of Veterinary Inspector and Quarantine Website: http://aqi.dld.go.th/th/index.php?option=com_content&view=article&id=174&Itemid=57

• Frank, A. (2010). Evaluation of the EU legislative framework in the field of medicated feed. Retrieved February 20, 2014, from Food Chain Evaluation Consortium (FCEC) Website: http://ec.europa.eu/food/food/animalnutrition/labelling/medicated_feed_report_20100224.pdf Page 35

• Guo, L., Chen, Y., Zhang, L., Yang, W., and He, P. (2012). Development and validation of a liquid chromatographic tandem mass spectrometric method for determination of chlortetracycline, oxytetracycline, tetracycline, and doxycycline in animal feeds. Journal of AOAC International, 95(4), 1010-1015.

• Juan, C., Igualada, C., Moragues, F., Leon, N., and Manes, J. (2010). Development and validation of a liquid chromatography tandem mass spectrometry method for the analysis of beta-agonists in animal feed and drinking water. Journal of Chromatography A, 1217(39), 6061-6068.

• Liu, R., He, P., Li, Z., and Li, R. (2011). Simultaneous determination of 16 sulfonamides in animal feeds by UHPLC-MS-MS. Journal of Chromatographic Science, 49(8), 640-646.

• Pleadin, J., Vulic, A., Persi, N., and Vahcic, N. (2010). Clenbuterol residues in pig muscle after repeat administration in a growth-promoting dose. Journal of Meat Science, 86(3), 733-737.

• Qu, C. H., Li, X. L., Zhang, L., Xi, C. X., Wang, G. M., Li, N. B., and Luo, H. Q. (2011). Simultaneous determination of cimaterol, salbutamol, terbutaline and ractopamine in feed by SPE coupled to UPLC. Journal of Chromatographia, 73(3-4), 243-249.

• Thai Feed Mill Association. (2014). Animal population and Feed Consumption. Retrieved February 20, 2014, from Thai Feed Mill Association Website: http://www.thaifeedmill.com/tabid/56/Default.aspx

• TheFishSite. (2014). Malaysian Consumers urge ban of antibiotics in animal feed. Retrieved March 16, 2014, from National Bureau of Agricultural Commodity and Food Standards Website: http://www.acfs.go.th/read_news.php?id=11863&ntype=07

• Wang, L., Yang, H., Zhang, C., Mo, Y., and Lu, X. (2008). Determination of oxytetracycline, tetracycline and chloramphenicol antibiotics in animal feeds using subcritical water extraction and high performance liquid chromatography. Journal of Analytica Chimica Acta, 619(1), 54-58.

• Wijayanti, A. D., Wihandoyo., and Rosetyadewi, A. W. (2011). The comparison of Doxycycline residue in meat of broiler chickens administered in feed and water. Journal of Animal Science and Technology, 34(3), 175-178.

• Xiu-Juan, W., Feng, Z., Fei, D., Wei-Qing, L., Qing-Yu, C., Xiao-Gang, C., and Cheng-Bao, X. (2013). Simultaneous determination of 12 beta-agonists in feeds by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Journal of Chromatography A, 1278, 82-88.

• Zhang, G. J., Fang, B. H., Liu, Y. H., Wang, X. F., Xu, L. X., and Zhang, Y. P. (2013). Development of a multi-residue method for fast screening and confirmation of 20 prohibited veterinary drugs in feedstuffs by liquid chromatography tandem mass spectrometry. Journal of Chromatography B, 936, 10-17.

• Zhang, L. Y., Chang, B. Y., Dong, T., He, P. L., Yang, W. J., and Wang, Z. Y. (2009). Simultaneous determination of salbutamol, ractopamine, and clenbuterol in animal feeds by SPE and LC-MS. Journal of Chromatographic Science, 47(4), 324-328.

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