Objectives:
 

1. To detect  the heavy metal contaminants in vegetable oils, especially the hydrogenated vegetable oils manufactured in India. 
2. To assess the quality of the mass consumer product (Vegetable oil) regularly consumed by every Indian as a sole cooking medium.
3. To outline the toxicological and safety limit values of the heavy metals in edible vegetable oils of the country.
4. To prepare a base line data on the state of the affairs prevailing in the country in relation to heavy metal contamination in vegetable oils and suggests remedial measures toward the vulnerability of Indian mass consumers to the toxicological hazards posed by the consumption of contaminated vegetable oil.
5. To trace the source of heavy metal contamination in vegetable oil and outlining the measures needed to reduce them.

13 (a) Literature survey:

Literature survey for nickel heavy metal which was found to be widely present in edible oils was extensively carried out to provide a comprehensive picture of the nickel contamination in mass consumer products. A detailed outline is given below.

THRESHOLD LIMIT VALUES

TWA-OSHA           1 mg/m (metal & soluble compounds) 0.001 ppm. 
                                (0.007 mg/m) (nickel carbonyl)
NIOSH                    0.015 mg/m/10 hours (metal & inorganic 
                                compounds)
TLV-ACGIH           1 mg/m (metal) 0.1 mg/m (soluble compounds-as
                                 metal)
                                 1 mg/m (nickel sulphate roasting fume and dust-as
                                 metal)
                                 0.05 ppm (0.35mg/m) (nickel carbonyl-as metal)
STEL-ACGIN          0.3 mg/m (soluble compounds-as metal)
HSE-recommended   1 mg/m (metal & insoluble compounds-as metal) limit                           1 mg/m (soluble compouds-as metal)
                                 0.5 ppm (0.35 mg/m) (nickel carbonyl-as metal)
MAC values inFood  0.1 mg/m in mild products; 
                                 0.5mg/Kg in meat products; Fish product 
                                 0.5 mg/Kg; Cereals 3mg/Kg; Vegetables/Fruits
                                 0.5 mg/Kg; Other Products 0.2-8.0 mg/Kg; 
                                 Beverages 0.3 mg/kg (Applies to nickel and its 
                                 compunds, calculated as nickel as enforced by the
                                 Hygienic regulations of Ministry of health of CSR 
                                 endorsed by the IRPIC vide REC-NR; 24086 
                                 CMEA: REC: Food: MPC entered in IRPIC on 
                                 October, 1985.
MXL Values             Maximum authorised conc. in a composite sample 
                                 0.75 mg/l & pH>5.53; Maximum authorised 
                                 monthly arithmatic mean conc 0.5 mg/I & 
                                 pH>6.02. These standards have been fixed by the 
                                Canadian authorities as confirmed in Canada 
                                Gazette Part II 1981 * 115 (721), 2783, 1981 
                                goted by IRPIC vide REC-NR: 41077 entered 
                                into IRPIC on April, 1988.
An occupational exposure limit for nickel and its compounds in different countries is appended as Annexure-1
Ni on absorption by the human body tends to localize in the connective tissue, kidney & lungs (Oskarson & Tjalve, 1977). Ni concentration in parenchymal tissue is generally quite low, usually less than 25 ug/kg. in lung &10 up/kg. in liver. Insoluble Ni compounds on inhalation tend to accumulate in the nasal mucosa & lungs (Torjussen & Andersson, 1979). The human adult body contains about 10 mg Ni, about 18% present in the skin and the rest distributed in other tissues. Human foetuses contain traces of Ni. Ni in the plasma of healthy subjects averages 2.1 ug/1. Whole blood conc. are approximately twice those of plasma or serum (Sunderman 1977).
The presence of Ni in human body demands a thorough discussion about the toxic, biochemical & epidemiological effects of Ni. To discuss the Ni toxicity the first & most toxic effect of Ni is carcinogenicity.
CARCINOGENICITY.

A number of studies have taken place into the carcinogenicity of Ni & its compouds (7, 12-15) and Ni carbonyl in particular (7,14). The overall conclusion has been that airborne Ni in refinery dusts should be considered carcinogenic to lungs & masal tracts. Ottolenghi et. al. reported that and excess of lung cancer in rats exposed daily for 14 months to inhalation of Ni sulphate at the conc. of 1 mg/cubic meter.

Epidemiological studies of Ni workers concludes an excess risk of cancer of the nasal cavity & lungs, it is concluded that Ni in some form is carcinogenic to man. Lung carcinomas of the epidermoid & anaplastic type are the ones which are most common, but the highest ratio of cancer is found in nasal cavities. An increased risk of laryngeal cancer has been found. Gastric cancer is also suggested by Burges(1980). Tor fussen et. al. (1979) found that epithelial dysplasia in the nasal mucosa cause more common in Ni exposed workers than in non- industrially exposed persons. In Ni plating industry exposure to Ni containing vapours has been reported to be associated with asthma. NIOSH (National Institute of Occupational Safety & Health) constituted a review committee on the subject of carcinogenic potential of nickel which in its report titled Criteria for a Recommended Standard - Ocupational Exposure to Inorganic Nickel, U.S. Dept. of Health, Education & Welfare (May 1977) concluded that ".....in the absence of evidence to the contrary, nickel metal and all inorganic nickel compounds, when airborne, should be considered carcinogens." (Documentational of TLV's & Biological Indices, ACGIH, 1986, pp. 423).
EFFECT ON RESPIRATORY TRACT
Isolated case reports have been published in which nasal cancer has been attributed to Ni exposure (2,18-19). Epidemiological studies in electroplaters & other workers exposed specifically to soluble Ni have not been reported. Other respiratory effects have been reported as case reports in workers exposed to Ni dusts & fumes including asthma (10,25) pulmonary fibrosis (8,29) & pulmonary edema in welders using Ni alloys (5,9).

SKIN EFFECTS
Nickel and its compounds have a strong sensitizing effects on the skin. Contact dermatitis caused by nickel is an important problem for general population. 5-31% of population with eczenatous skin manifestation are sensitized through contact with nickel.
EFFECT ON LIVER
Nickel concentration in liver of a man was found to be in between 5.2 to 13.2 ng/g. Effect of nickel on liver includes changes in the over all appearence of the liver, ultra structural changes in the form of mitochondrial swelling and cystic changes of the endoplasmic reticulum as well as a distinct decrease in the oxidation of glycrol phosphate. No data available at present show that long term exposure to nickel has caused systemic effects in human beings.
SUBCELLULAR TOXICITY: INTERACTION WITH DNA/RNA
According to Webb and co-workers (1972) 70-90% of the nickel in nickel-induced rhabdomyosarcomas is found in the nucleus, being about equally distributed between nucleolus and nuclear sap plus chromatin fraction. Within the nucleus, direct binding to DNA & RNA has been shown by Heath & Webb (1967), which lends support to the data of Beach & Sunderman (1970) showing nickel binding tothe RNA polymerase chromatin complex obtained from rat liver nuclei with exposure to nickel carbonyl.
The effect of nickel ion on DNA conformation have been reported only in-vitro and at present their biological significance is unknown. There are significant concentration of nickel present in DNA &RNA (Wacker & Vallee, 1959). Sirover & Loeb (1976) found that nickel chloride affects the fidelity of DNA synthesis, nickel carbonyl inhibits DNA dependent RNA polymerase.

EFFECT ON HEME METABOLISM
The catalytic activity of the enzyme-amino levulinic acid dehydratase (ALAD) involve in heme metabolism is inhibited by nickel in various organs (Maines & Kappas, 1977) especially in kidney. Nickel also enhances the rate of cellular heme degradation. It is one of the most potent inducers of heme oxygenase, the rate limiting enzyme in heme degradation in kidney. The potency of HO induction varies from one tissue to another, and this induction is found in liver, heart, lung, intestinal mucosa and the skin.
PLACENTAL TRANSFER/EMBRYO TOXICITY FETOTOXICITY
Passage of nickel across the placental barrier in man has not been conclusively established, although Stock et. al. (1976) reported that nickel levels in teeth from 25 cases of still birth and neo death showed a mean value of 25 ppm. Dentition from four foetuses had nickel levels ranging from 11-19 ppm.
Prenatal exposure to nickel is not known to disrupt development in humans. Detectable amounts have been identified in human foetal tissues (Casey & Rabinson, 1978), and are somewhat higher than conc. in full term infants. The embryo toxicity and teratogenic effects of Ni have been recently reviewed by Sunderman et al (1983). Malformations have been reported inhamsters, mice and rats, anomalies included ocular, skeletal and neural defects. Basrur and Gilman (1967) showed that addition of Ni subsulfide to cultured embryonic muscle cells inhibit mitotic activity and induces abnormal mitotic figures. This suggests that Ni may interfere with gene replication and with the control of cell division. There are no reports indicating that exposure to Ni has caused malformation in human beings. No effect on human exposure, neither any teratogenic effects have been reported in connection with Ni exposure. Ferm (1972) found that Ni exposure in pregnant hamsters, a dose related increase in the number of resorbed embryos as well as few unspecified malformation in surving embryos. Administration of Ni to pregnant rodents at dosage that do not produce maternal mortality are sufficient to produce fetal mortality and to impair intra-uterinal growth.Sunderman et al (1977) showed that 63 Ni can enter the products of conception after administration to pregnant rats on the 8th or 18th day of gestation. 
GENOTOXICITY
DNA strand breakage, DNA protein cross links, and mutagenicity have been demonstrated in hamster embryo cells exposed to either crystalline or soluble Ni salts and increased sister chromatid exchange has been demonstrated in cultured human lymphocytes exposed in vitro.(Newman et at, 1982).
However, studies on chronic exposuer to Ni in human are not available  to pronounce the actual and potential hazards of regular Ni ingestion. But if results of epidemiological surveys and animal studies are to be taken into consideration then sufficient evidence have already been adduced to implicate Ni as highly toxic substance debarring its presence in human body. 
ILLEGAL MECHANISMS & RECOMMENDATIONS OF IRPIC:
International Registary of Potentially Toxic Chemicals, known as IRPTC, under the U.N.Environmental Programme in its document on Nickel has classified Nickel as highly dangerous substance and included as CARCINOGENIC WORKING MATERIAL PROVEN THROUGH EXPERIENCE WITH HUMANS. NO MAK VALUE ESTABLISHED.
IRPIC on the recommendations of State Standard of USSR listed Nickel as a substance included in the list of Carcinogenic Working Material a 2A Group (Sufficient Evidence for Probable Carcinogenicity to Humans)
IRPIC on the recommendation of Code of Federal Regulation quoted that chemical agents for use as a Dispersing mixture (nitric acid: sulphuric acid: 6:1) until the solution turned colourless. The flasks were cooled, 5 ml hexane was added for the removal of fatty substances, shaken and centrifuged. Hexane layer was decanted. Now 3 ml of conc.i sulphuric acid was added to the contents and digested till the appearance of white fumes of sulphur-tri-oxide. To this 5 ml of citrate buffer (0.BBM pH 9.3) and few drops of phenophthalene solution were added. The contents were neutralizd with conc. ammonium hydroxide (29% w/v) and 3 ml of each dimethylglyoxime (0.1% w/v) in 20% ethanol and chloroform were added, the flasks were shaken on metabolic shaker at room temperature for 5 minutes. The chloroform layer was removed by suction pipette and the contents of the flasks were extracted was 3 ml of chloroform. Both the chloroform fractions were pooled and shaken with 1 ml of 0.5 N HCI and centrifuged at 3000 r.p.m. for 5 min. The nickel contents were estimated in aqueous layer by taking absorbance at the wavelength of 352 nm on GBC-901 Atomic Absorption Spectrophotometer. 
The principle involved in the analysis of atomic absorption spectrophotometer is based on the estimation of experimental solution in which the atoms are chemically bonded. On reaching the flame, the solvent is removed and the chemical bond is broken to form free atoms under the stream of respective cathode lamps. These free atoms absorb as definite amount of radiation for a specific element. This absorbed energy provides a qualitatives as well as quantitative determination of a particular element in the matrix.
13 (c) Testing/ survey work carried out:
Approximately over 100 million tonnes of edible oils are being consumed annually in India. Out of this between 10-15% is the share of hydrogenated vegetable oils and the remaining is being unequally distributed between different categories of oils. Thus, vegetable oils constitute an important mass consumer product with which the public at large is seriously associated. The presence of toxins even in small quantities may have profound effect on the health and well being of the common masses. Therefore, this project was undertaken to spell out the heavy metal contamination in edible oils, especially the chemically processed ones like Hydrogenated vegetable oils & Refined oils.
Popular brands of HVO viz. Rath, Ruchi,Gagan, Dalda, Panghat, Suhagin, No.1, Postman, Vital, Crystal, Saffola, Dalda refined, Dhara etc. were collected randomly from the local market belonging to different batch numbers and subjected to testing/screening for the presence of heavy metals. The results or Surface collecting agent for spills of oil will not be considered for use under the National Pollution Contingency Plan unless technical product data have been provided to & accepted to EPA. The data must include the conc's or upper limits of this substance in the product. Included in IRPIC vide REC-NRO 8572
Further recommendations and TLV's are being appended as annexure2.
CONCLUSION
The prevalent practices in the clearance of drugs/chemicals to be considered safe/unsafe is based on the extrapolation of animal results and the epidemiological studies. On the basis of the results obtained animal experiments using different concentration, duration of exposure and the time of administration which are the three major factors considered in the toxicological evaluation vis-a-vis the findings on human exposure to nickel, inter-alia the considered opinion of the NIOSH (National Institute of Occupational Safety & Health) under obligation of International Labour Organisation (ILO) that nickel in some form should be treated as carcinogenic unless proved otherwise.
More studies are urgently required to thresh out the safety limits  of nickel in edible food items, if its use can not be abandoned in the process of hydrogenation of vegetable oil, which is regarded as the major source of chronic exposure in humans to nickel.

Latest Developments:

Following the discovery of high nickel residual contents in the edible vegetable oils including hydrogenated vegetable oils, the Indian Prevention of Food  Adulteration Act has been ammended to incorporate the fixing of maximum upper limit of Nickel (1.5 ppm) in the vegetable oils. This has come into effect from 1995.