Research in Chemistry and Environment. Determination of Specific Heavy Metals in Fruit Juices Using Atomic Absorption Spectroscopy (AAS)

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1 International Journal of Research in Chemistry and Environment Available online at: ISSN Research Paper Determination of Specific Heavy Metals in Fruit Juices Using Atomic Absorption Spectroscopy (AAS) Havanur Priya Pramod and *Haware Devendra J. Food Safety & Analytical Quality Control Laboratory, CSIR-Central Food Technological Research Institute, Mysore , Karnataka, INDIA (Received 31 st January 2014, Accepted 18 th June 2014) Abstract: Fruit Juices and portable water are some of the most widespread beverages in the habitual diet, and they contribute significantly to trace element dietary intake. Food can be contaminated with heavy metals by ingestion of contaminated or polluted food and water. The concentration of heavy metals in fruit juice products varies depending on their origin, storage condition and processing technologies. There is currently little information available on the content of heavy metal in fruit juices, both imported and locally produced in India. In the present study, the level of heavy metals in Apple, pineapple & mango fruit juices consumed in Mysore district are determined. Samples were randomly collected from Mysore market from the month of July to November This study quantitatively determined the composition of heavy metals like Pb, Cu, Mn, Ni, Zn & Fe in 24 different packaged fruit juice samples. Analysis of fruit juice samples for the presence of heavy metals were carried out using Atomic Absorption Spectrophotoscopy (AAS). The mean value of different metals in analyzed fruit juices were, for Pb ppm, Cu ppm, Mn ppm, Ni ppm, Zn ppm & Fe ppm. The result shows high level of some of the heavy metals, although they are within the legal limits. Keywords: Toxic metals, Health hazard, Environmental contamination, spectrometer IJRCE. All rights reserved Introduction The term HEAVY METAL refers to elements with densities greater than 5.0g/cm 3 & usually indicates metals and metalloids; the term also includes elements that are required by organisms at low concentration [1]. Heavy metals are potential environmental contaminants with the capability of causing human health problems if present in excess in the food we eat. They are given special attention throughout the world due to their adverse effects even at very low concentration [2].Atmospheric contamination like excessive use of fertilizers & pesticides, sewage sludge, irrigation with residual water are among the causes of contamination of raw food stuffs [3]. As the result of soil, atmosphere, underground and surface water pollution, our food and beverages are also getting contaminated [4]. Heavy metal composition of food is of interest because of their essential and toxic nature, for example Fe, Zn, Cu, Cr, Co & Mn are essential while Pb, Cd, Ni & Hg are toxic at certain levels [5]. Although many heavy metals are essential for animal tissue metabolism, the ranges between beneficial and toxic level are usually small [6]. Pb has been cited as one of the 3 most toxic heavy metals that has long term negative impact on health causing anemia, encephalopathy, hepatitis, and nephritic syndromes. Mn is an essential metal, at excessive levels in the brain, produces extra pyramidal symptoms similar to those in patients with Parkinson's disease, decreased learning activity in school-aged children, and increased propensity for violence in adults [7]. An adult body contains ppm Cu, above which it will be toxic [8]. Toxicity due to excessive intake of Copper causes live cirrhosis, dermatitis and neurological disorders. Deficiency of Cu includes bone demineralization, depressed growth, depigmentation, and gastro-intestinal disturbances [9]. Zinc constitute about 33 ppm of an adult body weight and is essential as a constituent of many enzymes involved in several physiological functions, such as protein synthesis and energy metabolism. Zinc deficiency, resulting from poor diet, alcoholism, and malabsorption, causes dwarfism, hypogonadism, and dermatitis, while the toxicity of zinc due to excessive intake may lead to electrolyte imbalance, nausea, anaemia, and lethargy [10]. Iron (Fe) deficiency anemia, for example, affects one third of the world population. On the other hand, excessive iron intake has been associated with an overall increased risk 163

2 of colorectal cancer [11].The consequences of toxicity of nickel includes, higher chances of development of lung cancer, nose cancer, larynx cancer and prostate cancer, sickness and dizziness after exposure to nickel gas, lung embolism, respiratory failure, birth defects, asthma, chronic bronchitis and allergic reactions [12]. Fruit juices are the usual beverages, liked by all polulation and used in most of the festivals and celebrations in India. Fruit juices are served in lunch with pastries or biscuits and some times with breads. Fruit juices found in retail market are mostly derived from citrus fruit. After expression in reamer the juice is strained, flash, pasteurized, filled in sterilized bottle and sealed [13]. Heavy metal contamination has become a matter of public health concern, but still has not received much research attention in India, especially in case of fruit juices. The objective of this study is to determine the concentration of heavy metals (Pb, Ni, Cu, Mn, Zn & Fe) in fruit juices available commercially in Mysore district (India), using Atomic Absorption Spectrophotoscopy (AAS). AAS is most widely used technique for elemental determination owing to its simple setup, low running cost, robustness & good sensitivity [14]. Material and Methods Twenty four varieties of packaged fruit juices were investigated for the presence of heavy metals, brought from retail market of Mysore from the month of July to November 2013, out of which 8 where mango juices, 6 apples, 2 pineapples. One sample each of grape, pomegranate, orange, lime and peach fruit were colleced. The selection was specially made to reflect the popular juice types consumed by different income groups. Thermo Fisher Scientific (ice 3000) Atomic Absorption Spectrophotoscopy was used for quantitative determination of heavy metals, employing Air Acetylene oxidizing flame. This instrument was controlled by a personal computer using ice thermofisher software. Atomic Absorption Spectrophotometer standard solution for Pb, Mn, Cu, Ni, Fe & Zn were purchased from Merck (NIST traceable). The Nitric acid used for preparation of standards and sample solution was 69% pure, having a density of approximately 1400 mg/ml. Heavy Metals were analyzed using calibration curves made up of minimum of three standards with the minimum values of determination coefficient (R 2 ) of Pb, Cu, Ni, Mn, Fe & Zn Standard curve being , , 1.0, 1.0, 1.0 & respectively. The readings were made at absorption wavelength of 217.0, 324.8, 232.0, 279.5, & for Pb Cu, Ni, Mn, Fe & Zn respectively. Different parameters are employed for elemental analysis of heavy metals. The flame fuel used is air-acetylene mixture. The sensitivity of the instrument is The band width for Pb and Cu is 0.5 nm, for Mn, Fe & Zn is 0.2 nm, and for Ni it is 0.1 nm. Appropriate quality assurance procedures and precaution were carried out to ensure reliability of the result. Samples were carefully handled to avoid contamination [6]. Standard and Sample Preparation: Working standards solutions were prepared by diluting Atomic Absorption Spectroscopy Stock Solutions. All solutions were prepared using Milli Q water. All Stock Solution were of 1000 ppm, from this 100 ml of 0.5, 1.0, 2.0 & 4.0 ppm working solutions were prepared using 1% nitric acid. The dry ashing method was used for sample preparation in the present study. All glass wares were washed with demineralised water rinsed with milliq water followed by 1% Nitric acid. Fruit juice was homogenously mixed before samples were taken for analysis. 25ml of fruit juice was taken directly into crucible and evaporated completely by placing on water bath at 100 º C. The thick mass was charred on hot plate & kept in muffle furnace (450 º C) till it becomes complete ash. The crucible with ash was put in dessicator for cooling. The ash was dissolved in 2ml Nitric acid & Milli Q water & finally made upto 30ml with milli Q water. Data Validation The accuracy of the method was evaluated by the percentage recovery of heavy metal at different spiking level in the fruit juices. A recovery test of the total analytical procedures was carried out by spiking analysed samples with aliquots of metal standards and then reanalyzing the sample. The result of recovery study for Pb, Cu, Ni, Mn, Fe & Zn was 85%, 100%, 91.5%, 97.5%, 100.6% & 94.3% respectively. Recovery study was done at 3 ppm level of spiking for Pb, Zn and Fe, at 2 ppm level for Cu, Ni and Mn. Repeatability of analysis was expressed in the term of Standard deviation and % RSD. Limit of detection of Instrument for Pb, Ni, Mn, Fe & Zn is 0.5 ppm and for Cu it is 0.25 ppm. Results and Discussion The range of linearity of concentration versus absorbance graph is of great importance in determination of elemental concentration in fruit juices [15]. The calibration graphs for all heavy metals studied are shown in Figure 1. The linear equation for Pb, Cu, Ni, Mn, Fe & Zn are y= X , X , X , X , X & X respectively. In fruit juice samples the mean value detected for Pb ppm, Cu ppm, Mn ppm, Ni ppm, Zn ppm & Fe ppm. The maximum value for Pb was found in sample FJ-13. The Pb content was found maximum in apple juices, on and average of 0.15 ppm. Apple juices also showed the maximum content of Ni, and it was found maximum in FJ-12 sample. Apple juices had an average of 0.11 ppm Ni content. Mango juices showed maximum amount of Copper content, compared to other fruit juice types, it showed an average of 0.09 ppm. FJ-10, one of the mango juices showed highest concentration of copper. The fruit juices derived from Pineapple had high amount of Mn in them that is 0.96 ppm. It was maximum in FJ-18. Fe was found in almost all the fruit juices, its presence was predominant in juices of grape fruit. The average value of Fe in grape fruit found to be 7.67 ppm. Similarly Zn was found maximum in FJ-20. Juices derived from pineapple and pomegranate showed high amount of Zn that is 0.29 ppm on an average. Different heavy metals in analyzed fruit juice samples were found in the range of, Pb ppm, 164

3 Cu ppm, Ni ppm, Mn ppm, Fe ppm & Zn The range and arithmetic mean concentration of Pb, Cu, Ni, Mn, Fe & Zn in different fruit juices are also given in table 1 as average of three replicates of the individual fruit juices. The Concentration of Fe was found to be highest in fruit juices and Pb was found having least concentration. The distribution chart of different heavy metals in fruit juice is shown in Figure 2. The order of heavy metals concentration in terms of magnitude was found to be Fe>Mn>Zn>Ni>Cu>Pb in fruit juices. On analysis of levels of Pb, Cu, Ni, Mn, Fe & Zn in fruit juices, we found that the concentration of heavy metals varied considerably between each sample and the values were compared with WHO, BIS and FSSAI limits. Some samples Fruit juices analyzed Haware et al. Int. J. Res. Chem. Environ. Vol. 4 Issue 3 ( ) July were below the recommended safety limits, others within this and some exceeding the threshold limits established by BIS, WHO and FSSAI. The level of Pb recorded in this study was found to be within the safety limits set by BIS (1 ppm for apple and orange juices and 0.3 ppm for mango juices). Similarly the level of Fe was found to be within the limits set by BIS (10 ppm & 15 ppm for apple and mango juice respectively). The level of Zn set by BIS is 5 ppm for apple and mango juice & 9 ppm for orange juice. Our study shows that the concentration of these heavy metals in fruit juices is much lower than permissible limit. The level of Cu in apple and mango juices are much below the safe limit set by BIS (5 ppm). The different between mean value of Cu, Zn & Pb in sample and FSSAI safe limit value (5 ppm, 5 ppm & 1 ppm respectively) are statistically significant. Table 1 Concentration of heavy metals in different fruit juices in ppm Pb Cu Ni Mn Fe Zn FJ ± ± ± ND ± ± FJ ± ± ± ± ± ± FJ3 ND ± ± ± ± ± FJ ± ± ± ± ± ± FJ5 ND ± ± ± ± ± FJ6 ND ± ± ND ± ± 0.0 FJ ± ± ± ND ± ± 0.0 FJ ± ± ND ± ± ± 0.0 FJ ± ± ± ± ± ± FJ ± ± ± ± ± ± 0.0 FJ ± ± ± ± ± ± FJ ± ± ± ± ± ± FJ ± ± ± ND ± ± FJ ± ± ± 0.0 ND ± ± FJ15 ND ± ± ± ± ± FJ16 ND ± 0.0 ND ± ± ± FJ ± ± ND ± ± ± FJ18 ND ± ± ± ± ± FJ19 ND ± ± ± ± ± FJ ± ± ± ± ± ± FJ21 ND ± 0.0 ND ND ± ± 0.0 FJ ± ± ND ± ± ± 0.0 FJ ± ± 0.0 ND ND ± ± 0.0 FJ ± ± ± ± ± ± Range Mean ND: Not Detectable

4 Calibration Graph for Cu Calibration Graph for Mn Calibration Graph for Zn Calibration Graph for Ni Calibration Graph for Fe Calibration Graph for Pb Figure 1: Calibration Graphs 166

5 Figure 2: Distribution graphs of heavy metals in fruit juice The concentrations of these metals in our fruit juice sample are far below the permissible limit value. The maximum permissible limit for Cu & Pb is 0.01 ppm set by WHO. The present study shows that in 50% of samples, the Pb content is much above this safe limit and in around 95% of samples, the Cu content is much above this safe limit set by WHO. Conclusion Our data revealed that the Pb, Cu, Ni, Mn, Fe & Zn level found in all samples were within the FSSAI and BIS standard values. There are many research articles on heavy metals content in water and several foods in India. Our research is of important in view of its being first study of the heavy metal content in fruit juice samples. In several countries similar studies were previously reported. Over all study states that most of analyzed juice samples are safe, since the level are below maximum permissible level given by BIS and FSSAI. The present study provides addition data on metal pollution in India which may connote a potential threat to the health, security and safety of these food items to the unsuspecting consumers. This study also calls for mandatory testing of heavy metals in beverages and foods as a way of monitoring the extent of violation of guideline values. References 1. Adriano D.C, Trace Elements in the Terrestrial Environment. 2 nd edition, Springer-Verlag, NewYork (2001) 2. Das A, Metal ion induced toxicity & detoxification by chelating therapy. In first a textbook on medical aspects of bio-inorganic chemistry, CBS, Dehli. P (1990) 3. Demirozu B.Saldamli, Metallic contamination problems in a pasta production plant, Turkish J. of engg. & environ. sci., 26, (2002) 4. Krejpico Z, Sionkowski S, Bartela J, Safety of fresh fruit and juices available on the polish market as determined by heavy metals residue, Polish J. of environ. sci., 14, (2005) 5. Onianwa P.C., Adetola I.G., Iwegbue C.M.A., Ojo M.F., Tella O.O., Trace heavy metals composition of some Nigerian and food drinks. Food chem., 66, (1999) Chukwujindu M.A., Iwegbue, Nwozo S.O., Ossai E.K. and Nwajei G.E., Heavy Metal Composition of Some Imported Canned Fruit Drinks in Nigeria, Am. J. of Food Technol., 3, (2008) 7. Finley J.W., Does environmental exposure to manganese pose a health risk to healthy adult?, Nutr ReV., 62, Kies K., J. of food Nutr., 61, 15(1989) 9. Silvestre M.D., Lagarda M.J., Farre R., Martinez- Costa C, Brines J, Copper, Iron and Zinc determination in human milk using FAAS with microwave digestion, Food Chem., 68, (2000) 10. Onıonwa P.C., Adeyemo A.O., Idowu O.E., Ogabıela E.E., Copper and Zinc contents of Nigerian foods and estimates of the adult dietary intakes, Food Chem. 72, (2001) 11. Senesse P., Meance S., Cottet V., Faivre J., Boutron- Ruault M. C., High dietary iron and copper and risk of colorectal cancer: a case-control study in Burgundy, France, Nutr. Cancer., 49, (2004) 12. Kasprzak K. S., Sunderman FWJr, Salnikow K. Nickel carcinogenesis, Mutat. Res., 533, (2003) 13. Kirk, Ronald S. Pearson s composition and analysis of food, 9 th edition Longman, Singapore(1991) 14. AOAC official methods of analysis of AOAC International, 18 th edition, AOAC International Gaithersburg, Maryland, USA, (2005). 15. Kinsara A.A, Farid S.M, Concentration of trace Elements in hunman milk and animal milk in Jeddah, Saudi Arabia, Medical J Islam Acad Sci., 16, (2008). 16. Hayford Ofori, Margaret Owusu and George Anyebuno, Heavy Metal Analysis of Fruit Juice and Soft Drinks Bought From Retail Market in Accra, Ghana, J. of Sci. Res. & Reports, (2013).

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