Determination of Heavy Metals in Cow Milk

Determination of Heavy Metals in Cow Milk The cow milk feeding concept of infants, in the larger parts of Lake Victoria basin of Kenya, for about the first six months after birth, has immensely invigorated the interest in investigating the presence and levels of some heavy metals in cows milk. Toxicity of a heavy metal depends on its fractional bioavailability and concentration in the environment, therefore its speciation is of great importance. The knowledge of concentrations of toxic heavy metals like cadmium, chromium, copper, iron, lead and zinc in cows milk is hence very necessary. Fresh milk samples from lactating cows will be obtained by self milking into sterilized polyethene bottles and labeled according to time, date, location and replicate. Other parameters that will be collected from the three locations on the basis of 7-day interval and thereafter investigated include: grass feeds, sediments, water, soil, and lactating cows faecal drops and urine. This information therefore gives a suitable background for assessing and determining the concentrations of heavy metal contaminants on cows and their subsequent intake by human. Few drops of 0.1 M trichloroacetic acid will be added to the sample for coagulation and the aqueous layer heated at 500 °C for one hour. Digestion will be done with 0.5 M nitric acid as presence and concentration of heavy metals analyzed using an Atomic Absorption Spectrophotometer, AAS. Statistical analysis will be conducted using MSTATC two factor complete randomized block design, with the heavy metal concentration as the main factor with the locations as the sub treatment. The package will perform analysis of variance (ANOVA) at P †°Ã‚ ¤ 0.05 with two factor experiment and students T-test at P †°Ã‚ ¤ 0.05. The mean, standard deviation, range and linear correlation co-efficient on the measured parameters will be determined. The study is expected to give an indication of the exposure of mothers and infants in the region to the heavy metals and also ascertain the safety of absolute milk feeding of infants. This study will be conducted in Kisumu city, at the shore of Lake Victoria, Kenya. Key words: Heavy metals, infants, cows milk and contamination. 2.0 INTRODUCTION 2.1 Background Human and animals have been exposed to heavy metal toxicity for an immeasurable time. The industries have dramatically increased the overall environmental load of the toxins to levels that they are present in every area of modern consumerism. Therefore, it is necessary to know the environmental fate of all xenobiotics so as to predict their persistence and possible effects on non-target organisms (Kengara F.O: 2004). Anthropogenic activities appear to play an important role in this study since, in the past, solid wastes have been dumped by residents in their respective localities. In an apparent attempt to keep the environment clean, in Kisumu city, the local authorities, in the past four years have collected and dumped the solid wastes at a new site, Nyalenda Kachok, Kisumu. Similarly lorry-loads from all over the citys supermarkets, industrial set-ups, petrol stations, residences and markets dump theirs solid wastes at the site. Cadmium, chromium, copper, iron, lead and zinc are among the most common heavy metals known as contaminants in the environment and therefore come affront as hazardous substances to both human and animal health (Roberts J.R, 1999). This is due to wide spread environmental pollution by materials containing them: like batteries, paints, pipes, soldering rods, pesticides, fungicides, gasoline, engine oils, chemical fertilizers or when they occur in high amounts in air, soil, water, plants and other compounded animal feeds. They therefore increase concentrations of heavy metals in air, water, soil and subsequently taken by plants and animals into their food chain (Ahmad, W.M.S, 2002). The presence of heavy metals in cows milk may be attributed to contamination of the original one, which may be due to exposure of lactating cow to environmental pollution or consumption of contaminated feeding stuffs and water (Carl M, 1991). This occurrence can lead to considerable concentrations in human body since they are not metabolized therefore poses a serious risk to human health when consumed even in small amounts (Selinger B, 1979). Most of them, like cadmium, lead and mercury persist in the body and exert their toxic effect by combining with one or more reactive groups essential for normal physiological functions of the cells thus causing cellular disturbances or clinical manifestation. The adverse toxic effects caused by lead, cadmium mercury are widely recognized (Friberg, L. and Elinder, C.G, 1988). The major clinical signs in animals and man for lead and copper poisoning include, among others, deviations of the hematological parameters due to their direct effects on hematopoiesis, reduced integrity of red blood cells membrane leading to intravascular haemolysis, anemia and dehydration (Radostits O. M. et al., 1994). Therefore hematological parameters have diagnostic value in animals suspected of heavy metal toxicity (Mlay P.S and Migumia Y.O, 2008). Man becomes at risk by eating food and drinking fluids contaminated with heavy metals,  through air, direct contact with the metals like in people working in car wash or body spraying industries or factories dealing with heavy metals and their derivatives (Farr G, 2001). Kisumu city is endowed with relatively many but small enterprises dealing with metal works, car maintenance and repair (Jua Kali Sheds), construction works that pose a risk of contamination to the environment with hazardous substances including heavy metals. Subsistence farming and husbandry are quite enhanced in its neighborhoods. The foregone information prompts the desire to investigate the presence of some heavy metals in cows milk. The data generated will assist the concerned city planners, institutions and bodies charged with environmental control and surveillance to formulate measures and policies that would firmly govern the dumping of solid wastes, re-locate the site and authoritatively bar animals from feeding on the wastes. 2.2 STATEMENT OF THE PROBLEM The presence and concentration of heavy metals in water, sediments, soil and cows milk urine and faecal drops are unknown. This means that both man and animals likely assimilate the heavy metal contaminants unabated, thus endangering their lives. 2.3 JUSTIFICATION OF THE RESEARCH The toxic heavy metals from the possible sources as stated above continue to get into the environment and the biota. This phenomenon is a real threat to the human life therefore it is an urgent issue that the study needs to address. It is important to note that with the known concept of milk feeding of infants for the first six months after birth, either through mothers breasts or other sources like cow milk, more so in the rural set-ups, presents a possible lethal exposure route of heavy metal poisoning. The clinical manifestations that un-permissible levels of the heavy metals cause to both man and animals are fatal and expensive to treat thus jeopardize the economic progress of the affected community. The dumping point at the present site has continued to pollute the air due to organics releasing poisonous gases when they decompose and burnt. The constant burning of the wastes has hindered visibility, caused breathing difficulty and eye-aches to the road users as the site is at the high way and the entrance to the city. It is therefore a rude welcome to the tourists visiting the city. Therefore, the study will give an indication of the exposure of mothers and infants in the region to the heavy metals and also ascertain the safety of absolute milk feeding of infants. 2.4 HYPOTHESIS (i) The solid wastes dumped at the dumping site at Kachok, Kisumu city, contain toxic heavy metals like cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), and zinc (Zn) . (ii) The animals that feed on the wastes take-up the heavy metals into their body systems. 3.0 LITERATURE REVIEW Heavy metals are elements with specific density of 5gcm3, at least five times as the specific gravity of water (Florea T et al., 2006) and (Steven, D, 2003). They have been found in human breast milk and shown to affect health in infants. This may be due to mothers being susceptible to chemicals mostly in foods. Inhalation and dermal routes are possible though they are insignificant. Heavy metals, mercury, lead, arsenic, cadmium, bismuth, antimony most often disrupt immune function, neurological and endocrine functions. Some common effects of heavy metal toxicity include brain fogginess. Insomnia in children, memory loss, dementia tremors delay development (Molin J, 2000). Due to their toxic nature, the human body upon assimilation begins to get rid of them through the organs such as the skin, liver, kidney and through urine and sweat. However, this process is quite strenuous thus burdens and damages the organs (Bentum J.K, et al., 2010). Unfortunately human milk is one of the routes of elimination this burden, and therefore a source of exposure to infants (Oskarsson A, 1998). Some of these metals are stored in the mothers bones and are extracted from her to provide calcium for the development of the childs bones. As a result, they enter the maternal blood and breast milk during pregnancy and lactation, thus exposing the fetus and infants to risk (Sonawane R.B, 1994). However, at permissible levels, some of them are essential for normal physiological functions in animal tissues (Ahmed, E.E.K, et al., 1999). Dietary deficiencies of copper, zinc, calcium, iron, protein and excess fats cause an increase in the absorption and toxicity of lead (Goldfrank, L.R. et al., 1990). While copper is a trace element in various metabolic functions in the body, lead and other heavy metals have no function in the body and can be highly toxic due to interference directly in metabolic pathways or indirectly by causing deficiencies of other trace metals (Farr G, 2004). Excessively higher levels of the metals in milk and tissues of animals suggest an exposure either from the air, soil, water or feeds or all of these sources (Farr G, 2001) and (Dupler D, 2001). Animals can tolerate elevated levels of these metals though at certain levels clinical signs of toxicity manifest which can be acute or chronic when there is low exposure for a long time since these metals bio-accumulate in the body (IARC, 1997) and (Allcroft R, 1951). Heavy metals like cadmium, lead and mercury have been detected in breast milk in many parts of the world and have different means and ranges (Appendix 6.1). In many parts of the world, they exceed the recommended limits (Oskarson A et al., 1995) while in others lead has been found in breast milk between 5-20 ppb (Rabinowitz M et al., 1985). This may be attributed to the fact that sources of lead exposure are numerous ranging from ceramic and pottery glazed with lead, electronic works, welding and solders, jewelry making and repairing, certain hair dyes, automobile repairs (ATSDR, 1990). The presence of cadmium has been detected in breast milk as 0.28 ÃŽÂ ¼g/litre. It is found in many components of vehicles and in electrical and electronic equipment (Honda R et al., 2003). Cadmiums levels in breast milk have also been associated with cigarette smoking. Arsenic has not been thoroughly studied in breast milk but is however known to cause cancer in humans (Radisch B and Luck W, 1987). METHODOLOGY 4.1 Sampling and Sampling Design A Two factor completely randomized block design will be employed in sampling where one of the locations will be considered as a block. They will be spread out within 8 km apart. The samples will be taken within an interval of seven days. Table2 shows the experimental design detailing the number of samples per location and the sampling intervals of 7 days, 14 days and 21 days (Table 2): Key: D= Days; Re= Replicates; Lo= Locations: 4.2 Study Area The study area will be at the shore of Lake Victoria, Kisumu city and its environs within the area limits of 00 51 South and Longitude 0041 North and longitudes 330 20- 35020 East and an altitude of 528m above the sea level. The following locations will be picked for the study: Location 1: Nyalenda-Kachok: whose animals feed and graze at the dumping site- suspected to be polluted with the metals. Location 2: Mamboleo: 8 km north eastern outskirts of the city with relative high animal husbandry. Location 3: Chiga: 8 km- eastern outskirts of the city with subsistence and light animal husbandry. The choice of the sampling areas 2 and 3 is based on the fact that the cows graze freely in their areas but can not reach the dumping site where only those from location 1 access for herbage. All samples will be collected from 5 (hence five replicates) randomly selected points from each of the three locations; 1, 2 and 3 and immediately taken to the laboratory for preparation, digestion and analysis at Chemistry Laboratory, Maseno University, Kenya. 4.3 SAMPLES COLLECTION 4.3.1 Cow Milk Sample By self milking into sterilized polyethylene bottles, about 50ml fresh milk samples will be collected from five lactating cows from randomly selected homes in each location on a three day milking interval in the morning (i.e 50ml x 5cows x3 locations x 3 milking intervals = 2,250 ml will be collected in total. The samples will then be packed into ice-bags and labeling will be done with respect to time, date, location and replicate. 4.3.2 Urine Sample 50ml urine sample will be randomly collected from each lactating cow (whose milk is sampled) from its shed. The samples will then be wrapped and tied with sterilized polythene papers, packed into ice-bags and labeled according to time, date, location and replicate. 4.3.3 Faecal Drops Sample In each location, approximately 100g of faecal drops will be randomly collected from each of the five lactating cows (whose milk is sampled) from their sheds, wrapped and tied with sterilized polythene paper, packed into ice-bags and labeled according to time, date, location and replicate. 4.3.4 Water Sample About 50ml of water samples from five different points in each location will be placed in clean unused 100ml plastic bottles with screw caps: The points are across the swamp and dam for locations 1 and 3 while five equidistant spots along the stream that passes through the grazing area of location 2. All the samples will then be packed into ice-bags and labeling will be done with respect to time, date, location and replicate. 4.3.5 Grass Feeds and Soil Sample Grass feeds will be cut at approximately1 cm height from the ground in an area of 1x1m2, (within five randomly selected cattle grazing ground per location), bundled, labeled and packed in clean polythene bags. At the centre of the 1x1m2 area where grass feeds are taken, the topsoil will be dug to 12 cm depth at an area of 24x24cm2. The soil will then be put in clean polythene bags and labeled according time, date, location and replicate. 4.3.6 Sediment Sample Five sediment sub-samples will be randomly taken in each location. Approximately top 2 cm surface layer will be collected with a strainer and the samples packed and labeled with respect to time, date, location and replicate. 4.4 PREPARATION, DIGESTION, ANALYSIS AND QUANTITATION 4.4.1 Cow Milk Samples Five drops of 0.1 M trichloroacetic acid will be added to the cow- milk sample to precipitate the proteins, and the aqueous layer of the milk separated by centrifugation. 5 ml of the aqueous layer will be placed in porcelain crucible and heated in a furnace at a temperature of 500 0C for about 45 minutes. Thereafter, 3 ml of 0.5M nitric will be added and then filtered through Whatman filter paper (No 40) into a 10ml measuring cylinder. Further 0.5M nitric acid will be added to the 10 ml mark of the measuring cylinder. The concentrations of Cd, Cr, Cu, Fe, Pb, and Zn in blank and the milk samples will be analyzed with an AAS. 4.4.2 Urine and Water Samples 100ml of each sample will be boiled till complete dryness. 10ml of conc. nitric acid will be added to the sample and boiled close to dryness then diluted to 20 ml with de-ionized water. The solution will be filtered and the filtrate taken for AAS analysis for Cd, Cr, Cu, Fe, Pb, and Zn. 4.4.3 Quantitation of heavy metals in milk, urine and water samples: Concentrations of Cd, Cu, Fe, Pb, Mn, Se and Zn in examined samples will be calculated according to the following equation:- Mg/kg in examined samples = AxB/W A= mg/kg of metal in prepared samples (obtained by calibration). B= final volume of prepared sample in ml. W= weight of samples in grams. 4.4.4. Quantitation of Heavy Metals in Faecal Drops, Grass, Sediments Soil Samples The samples will be rinsed with de-ionized water several times and separately air-dried on open plastic bags for 24 hours, ground in a mortar to obtain small particles of uniform size, thus large surface area. Conventional aqua regia digestion will be performed in 250ml glass beakers covered with watch glasses. A well-mixed sample of 0.50 g each of the samples will be digested in 12ml of aqua regia on a hot plate for 3 h at 110 °C. After evaporation to near dryness, the sample will be diluted with 20 ml of 2% (v/v with H2O) nitric acid and transferred into a 100-ml volumetric flask after filtering through Whatman ® filter paper grade 40 and diluted to 100 ml with de-ionized distilled water thereafter analyzed for levels of Cd, Cu, Fe, Pb, Mn, Se and Zn using AAS. 5.0 REFERENCES 1. Ahmad, W.M.S. (2002): Studies on heavy metal pollution in poultry farms in relation to production performance; Ph.D. Thesis-Faculty of Vet. Medicine. Zag. University. 2. Ahmed, E.E.K, Haleem, H.H. and Aly, A.A. (1999): Effect of copper and ascorbic acid in restriction of cadmium toxicity. J. Egypt. Vet. Med. Ass., 59 (5): 1549-1573. 3. Allcroft R. 1951: Lead poisoning in cattle and sheep. Veterinary Record 63:583-593. 4. ATSDR Case study in environmental medicine: Cadmium toxicity: U.S Department of Health and Human Services. Atlanta G.A, 1990. 5. Roberts J R, 1999: Metal toxicity in children. In Training Manual on Pediatric Environmental Health: Putting It into Practice 1999 Jun. Emeryville, CA: Childrens Environmental Health Network. 6. Bentum J.K, Sackitey O.J, Tuffuor J.K., Essumang D.K, Koranteng-Addo E. J, and Owusu-Ansah E., 2010: Cadmium and Arsenic in breast milk of lactating mothers in Odumanse-Atua community in Manya Krobo district of eastern region of Ghana. 7. Carl, M. (1991): Heavy metals and other trace elements. Monograph on residues and contaminants in milk and milk products. Special Issue 9101, pp. 112-119. International Dairy Federation IDF, Belgium. 8. Dupler D. 2001: Heavy metal poisoning Gale Encyclopedia of Alternative Medicine. Farmington Hills, MI: Gale Group. 9. Farr G 2001: The Hair Tissue Mineral Analysis. 10. Farr G 2004: Why Heavy Metals are a Hazard to Your Health. 11. Florea T, Sarolta O.B and Gheorghe C, 2006: Heavy metals in fresh cow-milk and cheese. 12. Friberg, L. and Elinder, C.G. 1988: Cadmium toxicity in humans. Essential and toxic trace elements in human health and disease, edited by A.S. Prasad (New York: A.R.Liss), pp. 559-587. 13. Goldfrank, L.R.; Osborn, H. and Hartnett, L, 1990: Lead. In: Goldfrank, L.R.; Flomentbaum, N.E.; Lewin, N.A.; Weisman, R.S. and Howland, M.A. (Eds.): Goldfranks Toxicological Emergencies. 4th edition. pp. 627-637. Prentice-Hall International Inc. New Jersey, USA. 14. Honda R; Tawara K; Nishyo M; Nakagawa H; Tanebe K; Saito S, Toxicology 2003;186(3) 255-259. 15. IARC (International Agency for Research on cancer) 1997: Monograph of carcinogenic risk to human. Lyon. Supplement. 7:230-231. 16. Kengara F.O, 2004: Analysis of organo-chlorine pesticides in Nyando catchments of Lake Victoria and fate studies of atrazine and glyphosate in soil using the radioisotope tracer technique: MSc Thesis-Faculty of Science, Department of Chemistry, Maseno University, Kenya. 17. Mactaggart D.L and Farewell S.O: Analytical use of regression. Part 1: Regression procedures for calibration and quantitation, 1992, Journal of AOA International, 75 594-606. 18. Mlay P.S and Mgumia Y.O, 2008: Levels of lead and copper in plasma of dairy cows, pastures, soil and water from selected areas of Morogoro suburbs. (Department of Physiology, Biochemistry, Pharmacology and Toxicology, Tanzania). 19. Molin J: Journal of occupational and environmental medicine; 2000; 42(11) 1070-1075. 20. Ongeri, D.M.K, 2008: Physicochemical parameters, heavy metal residue levels and their speciation studies in Lake Victoria basin; Ph.D. Thesis-Faculty of Science, Department of Chemistry. Maseno University, Kenya. 21. Oskarson A., Palminger H.I, and Sundberg: J. Analyst: 1995; 120(3) 765-770. 22. Oskarsson, A, Analyst 1998 123(1); 19-23. 23. Osweiler D. G, 1996: Toxicology. Williams and Wilkins USA 491pp. 24. Rabinowitz, M., Leviton A., and Needleman H., Archives of environmental health 1985; 40 (5) 283-286. 25. Radisch B and Luck W: Nav H Toxicology letters 1987; 36 147-152. 26. Radostits O. M, Blood D. C and Gay C. C, 1994: Veterinary Medicine A Textbook of the Disease of Cattle, Sheep, Goat and Horses 8th Edition. Paston press ltd, London, Norfolk, UK 1469-1499p. 27. Roberts J R, 1999: Metal toxicity in Children. In Training Manual on Pediatric Environmental Health: Putting It into Practice 1999 Jun. Emeryville, CA: Childrens Environmental Health Network. 28. Selinger B, 1979: Chemistry in the market place. 29. Sonawane R.B: Envronmental Health Perspective, 1994; 196. 30. Tsoumbaris, P. and Papadopoulou, T.H. 1994: Heavy metals in common food stuffs: Quantitative analysis. Bulletin Environ. Contamination Toxicology, 53: 61-66. 31. Stevens, D. 2003. CSIRO Land and waters Methods Manual. Impact of Heavy Metals on Sustainability of Fertilization and Waste Recycling in Peri-Urban and Intensive Agriculture in South-East Asia. Australian Centre for International Agricultural Research (ACIAR). 29. World Health Organization, (WHO, 1993). 6.0 APPENDICES 6.1: Table 1- WHO: Selected Concentration Mean Ranges of Heavy Metals, 1993. Heavy Metal Concentration, ppb Concentration Range, ppb Arsenic 0.3 0.10 -0.80 Cadmium 0.1 0.10 3.80 Lead 5.0 0.00 41.10 Mercury 2.7 0.64 257.10 Manganese 18.0 7.00 102.00 6.2: Table 2: Experimental Design and Sampling Record Table Key: D= Days; Re= Replicates; Lo= Locations and F/D- Faecal Drops Re. Lo Milk Urine F/Drops Water Soil Grass Sediments 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 1 2 3 2 1 2 3 3 1 2 3 4 1 2 3 5 1 2 3 6.3: Table 3- TIME SCHEDULE ACTIVITY PERIOD DURATION Proposal Writing and Presentation Jan March 2011 12 weeks Research Site Survey and Preparation April 2011 4 weeks Acquisition of Chemical Reagents May 2011 4 weeks 1st Sample Collection and Extraction June 2011 1 week 1st Experimentations and Analysis AAS June 2011 3 weeks 2nd Sample Collection and Extraction July 2011 1 week 2nd Experimentations and Analysis AAS July 2011 3 weeks 3rd Sample Collection and Extraction August 2011 1 week 3rd Experimentations and Analysis AAS August 2011 3 weeks Discussion and Statistical Interpretation September, 2011 4 weeks Thesis Writing and Submission Oct-Nov, 2011 8 weeks Total 1 year 44 weeks 6.4: Table 4 BUDGET Item Quantity Unit Price (KSh) Total Cost (KSh) Chemicals Universal Indicator 1 litre 1,350.00 1,350.00 Distilled Water 40 litres 1,200.00 48,000.00 Aqua Regia 5 litres 3,500.00 17,500.00 Nitric Acid 2.5 litres 3,500.00 8,750.00 Sulphuric Acid 2.5 litres 3,500.00 8,750.00 Hydrochloric Acid 2.5 litres 3,500.00 8,750.00 Ammonium Nitrate 500g 3,500.00 3,500.00 Ferrous Nitrate 500g 3,500.00 3,500.00 Lead (II) Nitrate 500g 3,500.00 3,500.00 Unhydrous Sodium Sulphate 500g 2,850.00 2,850.00 Copper (II) Nitrate 500g 3,500.00 3,500.00 Zinc Nitrate 500g 3,500.00 3,500.00 Trichloricacetic acid 2.5 litres 4,500.00 11,250.00 Chromium (II) sulphate 500g 3,500.00 3,500.00 Cadmium Nitrate 500g 3,500.00 3,500.00 Sub Total 131,200.00 Apparatus, Equipment and Others AAS Analysis Lamps 12 7,500.00 90,000.00 Polythene Bags 6 1,000.00 6,000.00 Brown PVC Bottles 180 100.00 18,000.00 Whatman Filter Papers grade 40 6 1,000.00 6,000.00 Thesis Preparation and Binding 20,500.00 Goggles 2 1,000.00 2,000.00 Disposable Gloves 4 Boxes 3,500.00 14,000.00 Ice Box 1 9,000.00 9,000.00 Labels 1 Packet 750.00 750.00 Spade 1 1,200.00 1,200.00 Sickle 1 600.00 600.00 Sub Total 168,050.00 Travels and Subsistence Subsistence during Sampling 5 3,500.00 17,500.00 Analysis 20 Days 500.00 10,000.00 Site Visits and Sampling 10 Trips 3,000.00 30,000.00 Enumerators,3loc.x5Repsx3Intervals 3x3x5 1,000.00 45,000.00 Sub Total 102,500.00 Grand Total 401,750.00