amount of toxic compounds in milk in Pakistan

2. Amount of Toxic
Compounds in
Milk in Pakistan
Submitted By
Muhammad Saad Haleem
(15-ARID-5100)
IFNS
PMAS-Arid Agriculture
University, Rawalpindi
2

3. Topics of discussion
INT RODUCT ION
T YP E S OF COM P OUNDS
HE AVY M E TALS
P E S T ICIDE RE S IDUE S
AFLATOXINS
ANT IBIOT IC RESIDUES
CONCLUS IONS
3

4. Introduction
Chemical contaminants in milk comprise
chemical hazards that may introduce during:
– Milk production
– Dairy processing
– Packaging
4

5. Types of toxic compounds
• Toxic compounds or chemical contaminants
found in milk in different areas of Pakistan
are:
 Heavy Metals
 Pesticide Residues
 Aflatoxins
 Antibiotic Residues
5

6. Heavy Metals
Heavy metals are those metals which, in their
standard states have:
Specific gravity greater than 4.0
 Density five 5 g/cm3
 Atomic weights ranges from 63.546 to 200.590
(Aslam et al., 2011)
6

7. List of toxic heavy metals
found in Milk
1. Lead (pb)
2. Nickel (Ni)
3. Cadmium (Cd)
4. Copper (Cu)
5. Cobalt (Co)
6. Arsenic (As)
7

8. “Inhalation and ingestion
are two routes of entry of
these heavy metals in human
body “
(Tripathi, Raghunath, &
Krishnamoorthy, 1997)
8

9. 01
02
03
Nickle also causes severe oxidative stress and may
cause neurotoxicity (Doreswamy et al., 2004)04
Cobalt is likely cancer-causing complex
(Marselos and Vainio, 1991)
Health Risks due to Heavy Metals
Lead poisoning include abdominal pain,
constipation, headaches, irritability and tingling in
the hands and feet
Cadmium cause kidney problems and also a leading
cause of liver and skeletal problems

10. Flame atomic
absorption
spectrophotometer
(Thermo Scientific 3000
Series, Waltham, MA,
USA) was used for the
quantification of heavy
metals in milk samples.
Milk from
cows, buffalos and
goats were
collected during
July 2014 to
February 2015.
location
• Dera Ghazi Khan
• Sahiwal
• Rahim Yar Khan
• Bahawalnagar
Sample collection
Instrument
Study
No.1

11. Cow
Buffalo
Goat
(Ismail et al., 2017)
 The heavy metal absorptions in milk samples
of diverse animal classes were in the direction
of cow > buffalo > goat
11

12. Heavy metals (µg/g) in milk samples from different districts of Punjab Province.
12

13. (Iftikhar et al.,2014)
Location
The study was conducted on the
dairy milk in milk farms of
Peshawar. The samples were
collected during June – July of
2010.
Instrument
Flame atomic
absorption
spectrophotometer
Study No 2
Element
s
Mean Urban Mean
Rural
Cd 0.69 0.037
Cu 1.40 0.09
Ni 0.806 0.024
Pb 2.234 2.082
Mean Concentration is in mg/L

14. Results
Study No 1
A. Milk samples in the study were found to have Pb
and Cd levels higher than the allowed limits
B. while the levels of Co, Ni and Cu were in usual or
harmless zones
Study No 2
A. The concentrations of toxic metals in milk were
higher than the maximum permissible limits as
given by WHO
B. The concentrations in milk samples of urban areas
were greater than rural area 14

15. Pesticide Residues in milk
“ Pesticides are chemicals used to eliminate or
control a variety of agricultural pests that can
damage crops and livestock and reduce farm
productivity”
Types of Pesticides
 Organophosphate Pesticides
 Organochlorine pesticideticides
 Carbamate Pesticides
 Pyrethroid Pesticides
15

16. The most commonly
applied pesticides are:
 Insecticides (to kill insects)
 Herbicides (to kill weeds)
 Rodenticides (to kill rodents)
 And fungicides (to control fungi,
and mold,)
Total % of insecticides used in
Pakistan in 2017 16

17. Animals pesticides
Bif
ent
hrin
Chlorpy
rifos
carbof
uran
deltame
thrin
imidacl
oprid
cyhalot
hrin
Buffalo 0.5
2
0. 80 0.60 0.75 0.99 0.71
Goat 0.7
1
0.76 0.74 0.71 0.80 0.69
Camel 0.7
2
0.75 0.55 0.72 0.68 0.72
Maximum residue levels
(MRL)
 Pesticides are tested and
approved for use by the
EPA, which establishes
“tolerances,” or
maximum residue levels
(MRL)
 describe the amount of
a given pesticide that
can safely remain in or
on a food
(Shahzadi et al., 2013)17

18. Pesticides Risks and
Benefits
• Benefits
 Crop protection
 Food preservation
 Material preservation
 Disease control
• Risks
o Toxic to Humans
o Have impact on environment and ecosystem
18

19. Study No 1
Location
Samples were
collected from
agricultural areas
of South Punjab
where cotton is the
major cash crop
Instruments
The pesticides
detection was
conducted on
HPLC system,
Shimadzu, LC
20AT (Japan)
with reversed-
phase sorbent
RP-C18 column
Merck(Germany)
Sample
Collection
A total of 150
raw milk samples
were collected for
the
Organochlorine
and pyrethroid
pesticides analysis

20. Pesticides Mean
Concentration
(µg/ml)
DDT 0.0073
Endosulphan 0.1326
DDE 0.038
Aldrin 0.6771
Pesticides Mean
Concentration
(µg/ml)
Bifenthrin 1.7681
Cypermethrin 0.2277
Deltamethrin 0.2095
Permethrin 1.2353
Organochlorine pesticides
Mean concentration
Pyrethroid pesticides
Mean concentration
(Ul Hassan et al 2014)
20

21. STUDY NO 2
Location
All milk
samples were
collected from
Sahiwal region
because
Sahiwal is well
known for milk
production
Instruments
These
pesticides
were
confirmed by
using Gas
chromatograp
hy-mass
spectrometry
(GC-MS)
Sample
Collection
Milk samples
(250 mL) are
collected from
local markets
of Sahiwal.

22. CONCENTRATION OF PESTICIDE RESIDUES
IN MILK.
variable Range µg/kg
DDT 0.05-4.6
DDE 0.21-15.61
Dieldrin 2.08-18.45
ɣ-HCH 0.19-10.18
α -Endo 0.75-228.6
β -Endo 2.4-297.1
Endosulphan sulphate 1.5-19.6 (Ishaq & Nawaz 2018)
22

23. Results
Study No 1
 150 samples analyzed, 70% of total samples were
contaminate with either of the one or more than one
of the pesticides
 About 35% of the milk samples were polluted with
aldrin
Study No 2
1. In Sahiwal Cotton is the most cultivated crop
2. Cattle are fed on agro-industrial by-products such as
cotton & wheat
3. This may transfer chemicals like pesticides to cattle
and ultimately in milk. 23

24. Aflatoxins
“Moulds produce numerous poisonous
compounds known as mycotoxins”
• Aflatoxins AFM1 is found only in milk of the
animals that are fed on aflatoxin contaminated
feed
(Veldman et al,1992)
24

25. Fungal Species
 Aspergillus flavus
 A. parasiticus
 A. nomius
Aflatoxins production is dependent on
Environmental factors such as:
• Moisture & temperature
• Plant density
• Poor harvest practices
• Improper grain storage
25

26. Classification of Aflatoxins
There are more than 20 known
aflatoxins, but the four main ones are:
 aflatoxin B1 (AFT-B1)
 aflatoxin B2 (AFT-B2)
 aflatoxin G1 (AFT-G1)
 aflatoxin G2 (AFT-G2)
AFTs B
series are
produced by
A. Flavus
A. Parasiticus
A. nomius
26

27. www.presentationgo.com
Health Consequences
• Acute and chronic
aflatoxicosis
• Aflatoxin-related
immune
suppression
o Adverse pregnancy
outcomes
Intrauterine growth
restriction
o Premature delivery
Nutrition-related
problems in
children such
as stunted growth Mutagenesis

28. 28

29. Samples were analyzed
for AFM1 using
fluorescent detector of
High performance
liquid
chromatography
(HPLC).
Acetonitrile (HPLC
grade) of Sigma-
Aldrich (Steinheim,
Germany) was used for
AFM1 analysis
Eighty four samples of
raw milk (250 ml each)
were collected
randomly from the
markets of
 Gulberg Town
 Ravi Town
 Gungbuksh Town
 Shalimar Town
during April
2007 to July,
2007
Sample collection
Instrument
Study
No.1

30. Area Mean (µg/L)
Gulberg Town 19.75
Ravi Town 15.43
Gungbuksh
Town
12.13
Shalimar Town 22.31
(Muhammad et al., 2010)

31. The analysis was
carried out by using
Immuno
affinity
columns and
Fluorometer.
 The concentration
of M1 is measured
in fluorometer
Raw milk samples from
fourteen districts of the
Punjab province,
Pakistan, were
 Total 168 milk
samples were
analyzed
 The samples were
collected throughout
2005 in the middle
of Each month
Sample collection Instrument
Study
No.2

32. Months Mean
January 0.503
February 0.466
March 0.404
April 0.398
May 0.323
June 0.351
July 0.329
August 0.199
September 0.328
October 0.345
November 0.403
December 0.403
(Hussain & Anwar 2008)

33. Results
Study No 1
 Eighty one percent milk samples contained AFM1
levels exceeding the American and European
tolerance limits
 The mean value of AFM1 was 17.38µg/L ranged
from 0.69 to 100.04 µg/L
Study No 2
 The level of aflatoxin M1 in 96.4% samples was
below the US tolerance limit of 0.5 μg/L
 Only 3% samples showed AFM1 concentration
higher than the US maximum limit
33

34. Antibiotics
“A medicine (such as penicillin or
its derivatives) that inhibits the
growth of or destroys
microorganisms”
Antibiotic Residues
“Include the parent compounds
and/or their metabolites in any
edible portion of the animal
product”
34

35. 35

36. Antimicrobials used in
dairy cattle
 Beta-lactams
– Penicillin
– Cephalosporin
 Tetracyclines
– Oxytetracycline
– Tetracycline
– Chlortetracycline
36

37.  Amino glycosides
– Streptomycine
– Neomycin
– Gentamycin
 Macrolides
– Erythromycin
 Sulfanomides
– Sulfamethazines
Mitchell et al., 1998 37

39. Effect of Residues
a) Anaphylactic reaction
b) Allergy Drug resistant organism
c) Therapy failure
d) Toxicity
e) penicillin highly allergic
39

40. www.presentationgo.com
(Hubbert etal., 1996)
Routes of drug Administration in Animals
Orally
Intra-mammary
Injection route
 Intrauterine
infusions
 Skin

41. The present study was
conducted to evaluate
the extent of ß - lactam
Bacillus subtilus Field
Disc Assay
High Performance
Liquid Chromatography
(HPLC)
(Khaskheli etal., 2008)
A total of 137
unprocessed market
milk samples were
analyzed during the
year 2006
location
Milk samples were
randomly collected
from
 Hyderabad city
 Latifa bad
 Qasimabad
Sample collection Instrument
Study
No.1

44. STUDY NO 2
Location
 Hyderabad
 Mirpur khas
 Sukkur
 Larkana
 Karachi
Instruments
• Bacillus subtilus
Qualitative Field
Disc
Assay(microbial
inhibitor)
• Some of samples
were further
using Twin
SensorBT
(KIT020) method
Sample Collection
A total of 400
samples from the
vicinities of above
said divisions (80
from each division)
were collected into
clean sterile bottle,
and transported under
refrigeration for
analysis

45. Division β-lactum
%
Tetracycline
%
Other /unknown
antibiotics %
Hyderabad 40 25.71 34.29
Mirpurkhas 37.17 20 42.86
Sukkur 34.29 17.14 48.57
Larkana 42.86 20 37.14
Karachi 37.14 22.86 40
Overall 38.29 21.14 40.57
(Mangsi et al., 2014)
45
Percent of milk samples positive to ß-lactum and
Tetracycline residues

46. Results
Study No 1
The result was
i. Negative 63.50%
ii. Positive36.50% for ß - lactam antibiotics
Study No 2
 It was observed that out of total 400 milk samples,
199 (49.75%) numbers were observed as positive
for residues contamination
 Others are safe from contamination
46

47.  Contaminants in cow’s milk and
dairy products cause by accidents,
carelessness and overzealous use of
antibiotics
 Chemical contamination cannot
completely prevent, or eliminate
from milk and dairy products.
 The use of food safety and quality
assurance in farms can reduce
contamination it includes:
 A regulatory law implementation
 Long term planning training of
Personnel
 Good manufacturing practices
 Monitoring

48. References
 Aslam, B., Javed, I., & Khan, F. H. (2011). Uptake of heavy
metal residues from Sewerage Sludge in the Milk of Goat and
Cattle during Summer Season. Pakistan Veterinary
Journal, 31(1),75-77.
 Doreswamy, K., Srilatha, B., & Rajeshkumar, T. (2004).
Nickel‐induced oxidative stress in testis of mice: evidence of
DNA damage and genotoxic effects. Journal of
Andrology, 25(6), 996-1003.
 Hussain, I., & Anwar, J. (2008). A study on contamination of
aflatoxin M1 in raw milk in the Punjab province of
Pakistan. Food control, 19(4), 393-395.
 Hubbert, W. T., Hagstad, H. V., Spangler, E., Hinton, M. H., &
Hughes, K. L. (1996). Food safety and quality assurance:
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Press. 48

49. Cont…
 Iftikhar, B., Arif, S., Siddiqui, S., & Khattak, R. (2014). Assessment of
toxic metals in dairy milk and animal feed in Peshawar, Pakistan. British
Biotechnology Journal, 4(8), 883-893.
 Ishaq, Z., & Nawaz, M. A. (2018). Analysis of contaminated milk with
organochlorine pesticide residues using gas chromatography. International
Journal of Food Properties, 21(1), 879-891.
 Khaskheli, M., Malik, R. S., Arain, M. A., Soomro, A. H., & Arain, H. H.
(2008). Detection of ß-lactam antibiotic residues in market milk. Pak J
Nutr, 7(5), 682-685.
 Marselos, M., & Vainio, H. (1991). Carcinogenic properties of
pharmaceutical agents evaluated in the IARC Monographs
programme. Carcinogenesis, 12(10), 1751-1766.
49

50. Cont…
 Muhammad, K., Tipu, M. Y., Abbas, M., Khan, A. M., &
Anjum, A. A. (2010). Monitoring of aflatoxin M1 in
market raw milk in Lahore city, Pakistan. Pakistan Journal
of Zoology, 42(6), 697-700.
 Mitchell, J. M., Griffiths, M. W., McEwen, S. A., McNab,
W. B., & Yee, A. J. (1998). Antimicrobial drug residues in
milk and meat: causes, concerns, prevalence, regulations,
tests, and test performance. Journal of food
protection, 61(6), 742-756.
 Mangsi, A. S., Khaskheli, M., Soomro, A. H., & Shah, M.
G. (2014). Detection of antimicrobial drug residues in milk
marketed at different areas of Sindh province. IOSR J Agri
Vet Sci, 4, 65-9.
50

51. Cont…
 Shahzadi, N., Imran, M., Sarwar, M., Hashmi, A. S., &
Wasim, M. (2013). Identification of pesticides residues in
different samples of milk. Journal of Agroalimentary
Processes and Technologies, 19(2), 167-172.
 Tripathi, R. M., Raghunath, R., & Krishnamoorthy, T. M.
(1997). Dietary intake of heavy metals in Bombay city,
India. Science of the Total Environment, 208(3), 149-159.
 Ul Hassan, A., Tabinda, A. B., Abbas, M., & Khan, A. M.
(2014). Organochlorine and pyrethroid pesticides analysis in
dairy milk samples collected from cotton growing belt of
Punjab, Pakistan. Pakistan Journal of Agricultural
Sciences, 51(2), 321-325.
 Veldman, A., Meijs, J. A. C., Borggreve, G. J., & Heeres-Van
der Tol, J. J. (1992). Carry-over of aflatoxin from cows' food
to milk. Animal Science, 55(2), 163-168.
51