Objectives:
 

1. To assess the state-of-the-art of pesticide residues of lipophilic nature in meat & meat products in India by conducting biological monitoring of pesticides in meat & meat products of popular edible animals.
2. To elucidate the residue dynamics of persistent pesticides, chlorinated groups commonly used in existing agricultural practices in the body tissues of edible animals.
3. To help the meat and meat products industry of the country in understanding the international food standards with regard to pesticide residues and undertaking effective measures and technologically proceeding to ensure the quality of meat & meat products in order to sustain the export potential of this vast national resource.
4. To focus the attention of policy makers, plant protection agencies, consumers and their agencies involved with the welfare of human and animal health about the potential hazards faced by the chlronic exposure of toxic pesticides through meat & meat products in human and animals.
5. To identify the least and maximum susceptible edible animal with regard to lipophilic pesticides.

Pesticides demands have been influenced by more awareness of the farmers for better crop yield. Pesticide usage for the cultivation of food crops among the different states of India showed a mixed pattern. Tamil Nadu consumes 1.2 to 2 kg/ha of pesticide which is followed by Andhra Pradesh and Punjab where 0.8 to 1.2 kg/ha is the rate of consumption, Gujarat, Jammu & Kashmir show pesticide consumption at the rate of 0.6 to 0.8 kg/ha, whereas states like Kerala, West Bangal and Assam use 0.4 to 0.6 kg/ha for plant protection. Interestingly, the rate of pesticide consumption is almost the same (0.3 to 0.4 kg/ha) in the northern states such as Rajasthan, Himachal Pradesh, Haryana, Uttar Pradesh and Bihar, Meghalaya in north east and Karnataka in the south. Similarly, Maharastra, Madhya Pradesh and Orissa consume 0.2 to 0.3 kg/ha of pesticide for crop protection. Information is not available regarding the status of pesticide consumption for Sikkim, Nagaland, Manipur and Tripura. Pesticide consumption in Arunachal Pradesh and Mizoram is 32 and 21.5 million tons, respectively in the entire states, as per figures available from the Toxicological Atlas of India prepared by ITRC/CSIR (121).

The figure reveals wide variations in pesticide consumption in various states of India as per the Toxicological Atlas of India for Pesticides (121). This necessitates fixing up of a standard for pesticide usage per hectare of land, depending on the pest type and nature of the crop protection desired. With an increased cropping intensity and advanced farming techniques, the farmers have to be educated in the proper uses of pesticides as well. Usually, myth prevails in the field "more is better", hence, there is a tendency to overuse pesticides. But excessive use may cause toxicity to various plants, soil fauna and increase in residue creates burden in the food chain.

Statewise Environmental Load of Pesticides in India

The environmental load of pesticides in India in term of kg/sq.km. land area presents an interesting picture. Maximum pesticide load is found in the environment of Punjab, Haryana, Delhi and Tamil Nadu. The pollution load in these areas ranges from 70-100 kg/sq.km. land area. Andra Pradesh and West Bengal fall into the second category where the load of pesticides in the environment is between 30-70 kg/sq.km. land area, whereas the load of pesticides in the environment ranges between 10-30 kg/sq.km.. Interestingly substantial areas in Rajasthan, Madhya Pradesh, Orissa and Assam. Carry the pesticide load of 7-10 kg/sq. km. In contrast to this, the load of pesticide in Himachal Pradesh ranges between 3.7 kg/sq. km. land area, while Jammu & Kashmir, Meghalaya. Nagaland and Manipur indicate the pesticide load of 1.3 kg/sq. km. There is no data available regarding pesticide load in the environment in Sikkim, Arunachal Pradesh and Mizoram (87).

This data may provide a guideline to study whether or not such increased or decreased usage of pesticides/ sq. km. land area may have a direct correlation with increased or decreased residue build-up vis-a-vis persistence in the environment. It may also help to consider whether or not greater than the required amount of pesticides are being used per sq. km. Appropriate curtailment of the amount used may help considerably to decrease the pesticide burden in our environment.

PESTICIDE RESIDUES IN THE ENVIRONMENT

Pesticide residue means "any substance or substances in food for humans or animals resulting from the use of pesticide". It also includes any specified derivatives, such as degradation and conversion products, metabolites and reaction products which are considered to be of toxicological significance. The term pesticide means any substance or mixture of substances intended for preventing or controlling any pest and includes any substance or mixture of substances intended for use as plant growth regulator, deferent, or desiccant. The term excludes fertilizers and antibiotics or other chemicals administered to animals for other purpose such as to stimulate their growth or to modify their reproductive behaviours.
It is essential to reveal that pesticide residues occur in agricultural commodities due to :
a) intentional use of pesticides for protection or growing crops or stored products or on animals
b) unintentional exposure to pesticide such as would occur in crop     grown in soil treated previously or contaminated by foliar treatment of other crops grown earlier in the rotation.
c) unintentional accumulation in animals from the ingestion of feeds containing pesticide residues and 
d) contamination of crops or animals exposed to chemicals in the environment.

FATE OF PESTICIDES IN THE ENVIRONMENT

The fate of pesticides applied for crop protection and vector control in public health is discussed here. The compartmentalization in the environmental components is facilitated by the  manner of application (Sprays mostly) and the chemical nature of the compounds. Pesticides contamination of the environment is directly related to their persistent nature. The two most important degradative forces operating on chemicals in the environment are micro-organisms and sunlight. This conclusion has been derived on the basis of the following observation:-

• Most pesticides end up in soil. 
• They are spread in the environment in very low concentrations.
• Soil and aquatic media are generally rich in micro-organisms.
• Depending on their exposure to sunlight many pesticides escape   by volatilization into the atmosphere.

Meat and meat products are the one of the major components of  Indian dietary habits. Therefore, with the growing population, the consumption of meat & meat products in India has also increased considerable over the last few years. Indian production of meat & meat products is estimated to be around 1.03 million metric tonnes as per survey. The meat & meat products samples were procured from meat stalls of local markets, from five regions. Chandigarh (North), Madras (South), Calcutta (East), Bombay (West), Lucknow (Central) from the local market.

One hundred samples of meat & meat products (forelimbs, L.D. and round muscles) of each edible animal species viz. Buffalo, Goat, Sheep, Chicken, Pork were respectively collected from the local vendor/slaughter houses in three seasons in thermo-cooled containers properly jacketed with the mixture of ice and sodium chloride to maintain suitable temperature, needed for the transfer of such samples from field to the laboratory as being practiced normally for similar studies. Adequate care and precaution were also observed to avoid contamination of pesticides during transfer by way of observing good laboratory practices as stipulated under the guidelines prescribed under the global environmental monitoring surveillance programme for the dietary intake of chemical contaminants by the World Health Organization, 1990. The samples thus, collected were kept in deep freeze before being processed for the analysis of pesticide residues and were analysed within 48 hours. The procedure adopted for the multi residue determination of pesticides strictly followed the standardized method of Saxena et. al. as described in our earlier report communicated to ICAR which is briefly restated under relevant sections of report.

The samples were wrapped in pre-washed aluminium foil, immediately after collection. Before wrapping in aluminium foil, the meat samples were thoroughly washed with 0.85% normal saline to maintain the isotonicity of the tissue. The samples thus, collected were kept in thermo-cool boxes having provision to maintain temperature using ice cubes mixed with granular sodium salt to ensure safe transportation of the sample during transit period. After the samples were taken into laboratory, the same were kept in deep freezer at 4°C till the time of analysis, normally within 48 hours. Each sample was segregated into triplicate number and allowed suitable time to maintain room temperature before being processed for extraction and clean-up procedure.

Extraction Methodology for Multi Residues of Pesticides in Meat Samples

The tissue was finely chopped with the help of sharp edged knife and was spread gently over Whatman filter paper over which another filter paper of the same dimension was kept and pressed with light pressure to remove the blood by soaking. An aliquot (1 gm) of the tissue was accurately weighed and transferred to a 50 ml Borosil conical flask. To this was added 5 ml of formic acid (AR grade) and 3 ml of hexane (GLC grade). The mouth of the flask was tightly covered with the help of aluminium foil. The contents of the flask were shaken at 37°C for half an hour in a metabolic shaker to allow the tissue to be dissolved in formic acid and facilitate the solvent to come in contact with the pesticides covalently bonded with the fatty acid of the tissue. The contents were then transferred to graduated centrifuge tubes with the help of additional amount hexane (2 ml) to ensure quantitative transfer to the solvent. The contents were subjected to centrifugation for 10 minutes at 6000 RPM at 20°C. The upper layer of hexane was collected and the reminder was again extracted with 2 ml of hexane. The procedure was repeated and the hexane fraction were pooled.

Clean-up Methodology:

While extracting the pesticides, fat is also co-extracted. As such the extracted samples are subjected to exhaustive clean-up method prior to their analysis on GLC & HPLC not only to ensure better results but also to prevent the damage to the column. Therefore, clean-up of the extracted samples, in the qualitative & quantitative estimation of pesticides, is an important consideration and should be followed very precisely. For the standardization of the methodology, the clean-up was accomplished as follows:

The pooled hexane fraction was transferred to a test tube containing 1 ml of A.R. grade sulphuric acid (65% v/v). The contents were gently shaken  for 2 minutes and then allowed to rest. The sulphuric acid would turn yellow (since it reacts with fat and forms yellow colour complex). Collect the upper hexane layer with the help of disposable suction pipette and transfer it to another test tube containing one ml of A.R. grade sulphuric acid and repeat the same treatment as was done earlier till the sulphuric acid does not turn yellow, which is an indication that the co-extracted fat has been completely removed from the pesticide extracted hexane. Finally, the hexane fraction is transferred to the test tube containing 0.5 ml double distilled water for the complete removal of sulphuric acid from the hexane fractions. After this the hexane fraction is kept in glass stoppered GLC specimen bottle with few crystals of anhydrous sodium sulphate to absorb the moisture. The cleaned up samples were analysed for the qualitative & quantitative determination of pesticides employing the following techniques.

Gas Chromatographic Analysis:

The Gas Chromatographic Analysis of the samples was carried out on Nucon-5700 Gas Chromatograph equipped with electron capture detector (Ni⁶³). the operating condition of the equipment were as follows:

Carrier gas:                   Nitrogen (99.5% ultra pure IOLAR Grade-1)
Flow rate:                     4 ml/min
Gas pressure:                65 p.s.i.
Column:                        Glass column 4' length;OD¼",Internal diameter 
                                    3 mm filled with OV-17 with 80-100 mesh size.
Oven Temperature:       240°C.
Injector Temperature:   280°C.
Detector Temperature: 300°C.
Detector Source:         Ni⁶³
Attenuation:                   1 x 32, 1 x 64.
Sample size:                  2 to 5 µl.
Recorder Output:          1 mV.

The sample were analysed on GLC and each batch of analysis was performed using freshly prepared standards of multiple residues of pesticides, which were run in the same operating conditions before and after the analysis of each batch of samples. Retention time was taken into consideration for the qualitative analysis. The peak height and area was calculated for the quantitative analysis. The pesticide standards were obtained under the ICMR programme & were used to confirm & determine the actual concentration of pesticides in the samples. Thus, the recovery of multiple residues of pesticides in different parts of various edible spices were checked in natural and fortified samples and the results of the gas liquid chromatographic analysis revealed the levels present in meat & meat products.

However, in order to re-confirm the presence of detected pesticides and avoidance of artefacts, a High Performance Liquid Chromatographic analysis is used.

High Performance Liquid Chromatographic Analysis

Chromatographic separation assumes great significance in the quantitative and qualitative analysis of organic substances, especially in the case of pesticides which are quite complex otherwise. Unlike the GLC where the substance is separated between gas-liquid phases, HPLC uses high pressure instead of high temperature for the separation techniques. This assumes critical significance, especially in the case where the substances under investigation are thermally susceptible and labile. Thus, to avoid the decomposition of the substances during the analysis, the technique uses high pressure and the separation takes place between liquid - liquid. This technique is often termed as corroborative techniques to confirm the presence of the detected substances and reduces the chances of artefacts which are quite common during analysis unless extra precaution is taken.

The samples extracted for gas liquid Chromatographic analysis were further purified by passing through the membrane filter cartridges supplied by micro filter, USA for 0.2 microns to allow the detention of particles more than HPLC. The HPLC techniques used to confirm the pesticides detected over GLC, were performed on "SCHIMADZU- LC - 9A", Japan. The operating conditions of the instruments are as follows:

1.  Mobile phase:                        Methanol: n hexane.
2.  Pump:                                   Micro volume double plunger pump 
                                                 (10µl / strokes) 
3.  Flow rate:                             1 ml / min. ± 2%
4.  Constant Pressure Feeding:   76/77 p.s.i.
5.  Suction Filter:                        5 m mesh.
6.  Line Filter:                             2 mesh capacity 200 µl.
7.  Detector:                               U.V. at 234 nm.
8.  Temperature:                         25°C.
9.  Column:                                Length 150 mm; I.D. 3.1 mm;
                                                 Shim Pack ODS Steel column.
10. Sample size:                        10-20 µl.
11. Signal recording:                  Integrated CR6 A Chromatopac 
                                                 recorder with computer programming 
                                                 data output with built in pesticide
                                                 reference library.

Results and Discussion

Results of biological monitoring conducted on various specified animal species from the five zones of the country during rainy, summer & winter seasons allowing different portion of the meat products to show their respective susceptibility for the residues of organochlorine pesticides have been undertaken. The analytical results are based on GLC & HPLC analysis of the isomers and metabolites of the respective pesticides to allow meaningful interpretation and exhibit the true state of affairs prevailing in the country. The study was based on 100 meat sample of the edible animals from each of the five zones to draw meaningful inference. The marginal variation in three seasons reflects that although there exists some changes but these are not statistically significant. This may be due to the fact that the market animals are transported from one place to another as per demand and supply position. This may be true for Chicken, Goat and Sheep but may not be valid for buffalo & pork. Interestingly, the pattern of seasonal variation in the organochlorine pesticides residues in buffalo and park was found to be more distinct!

Similarly, the country wide variation in the over all toxic load of organochlorine pesticides in meat samples of different animal origin was also found to be within reasonable limit but did not exhibit drastic changes in the levels of important pesticides.

However, the changes in the organochlorine pesticides contents within the different portion of the same animal and from one species to another was found to be highly significant. This study may further corroborate the earlier studies that OCP's have tendency to accumulate in adipose due to their lipophilic nature. The higher organochlorine pesticide residues in pork & buffalo as compared to sheep, goat and chicken may be attributed to their increased dietary exposure to these pesticides by way of contamination. Further, the relatively higher quantity of diet consumed by pork & buffalo and the poor excretion of pesticides resulted in the higher body burden of pesticides in buffalo & pork. India contributes significantly in the export market of meat and meat products owing to its considerable live stock population. The contamination of pesticides in these food items is a serious matter and should be attached high priority. It would be worth mentioning that India has very large population of live stocks. Nearly 14% of the world's cattle and 50% of the world's buffalo population is in India. The statistics available from FAO corroborate this statement. Meat from cattle and buffalo is obtained from culled and unproductive animals. In domestic market the preference is for sheep and goat meat which constitutes about 45% of the total meat production of approximate 1.03 million M. Tonnes as per figures of 1985. the figures of 1998 would obviously be significantly high due to over all increase in population and the resultant demand and supply position. India exports a huge quantity of meat and meat products to various countries. India's export of meat and meat products during 1987-88 was in the order of 59,508 M. Tonnes out of which 51,540 M. Tonnes was buffalo and 7,968 M. Tonnes of sheep and goat meat. The significant increase attained in the export of these products may be attributed to the fact that Indian buffalo meat has many advantages over beef exported by EEC countries, Australia and New  Zealand. This may not be limited to our proximity to the meat importing countries but also in the rearing practices and quality of meat. This may largely be due the fact that Indian animals are raised on greens and natural fodder, unlike, in the advance countries where they are fed on artificial harmonic doses and synthetic nutrients. These practices may often allow the animal raiser ti gain extra weight and thus extra economy to their produce but at the cost of quality. On the contrary, the situation of India meat industry may not be that sound after the revelation of these results! Pesticides are noted contaminants and are defined as poison harmful to human health as per the Provisions of the Prevention of Food Adulteration Act (PFA Act) which governs the quality of food products manufactured / consumed by the Indian population. In accordance with the MRL values specified by the PFA Act for various pesticide compounds, the number of samples above these values from each zone and each animal under study have been calculated. Although, the study was limited to few hundreds of samples but the trend available from this study is quite apparent and should cause serious concern to all. The industry, planners, exporters and importers should be cautious of the fall out of our increased  vulnerability to the hazards posed by the higher susceptibility of the edible animals to such toxins and thus urgently requires remedial measures to ward off human vulnerability to these toxins as co-contaminants of human diet. It is therefore, suggested that the findings of this study should be considered very aptly and the issues focused herein above should be dealt with appropriately in a judicious manner to allow the industry to take corrective steps to improve the quality of their products and the government should also take adequate and effective measures to ensure that the problem does not aggravate further and reaches an alarming situation which could not only cause embarrassment to the government and law implementing agencies but may be viewed as gross violation of this existing rules and laws governing the republic of India in the area of public health! The international position in respect of the maximum permissible levels of persistent pesticides such as OCP is quite clear and the International Registry of Potentially Toxic Chemicals (IRPTC) -  an an organ of the United Nation Environmental Protection Agency, has clearly stipulated that the meat and meat products should not have more than the maximum permissible limits because of human safety and environmental angle.

As such the scrutiny of these edible food items would not be able to escape the international screening for such residues and may thus attract the punitive action between the international trade and commerce, should such an eventuality arises at any stage of items. The matter therefore, assumes greater significance owing to legal, commercial, bilateral relationship between the countries involved in export and import and on the top public health; and because of the multiple angles associated with the single issue of pesticide residues in meat and meat products, the issue deserves to be handled very critically and sensibly to avoid unnecessary multidimensional fall outs. The role of industry has now become more important after this significant revelation and they should consider the ways and means to overcome this menacing problem infesting their growth and development in national, international and keeping the interest of public health on the top of it.

It is sincerely  concluded that the findings of this research scheme shall find appreciation by the researchers and the Indian Council of Agricultural Research (ICAR) would take appropriate step at national level to chalk out suitable scientific programme so as to address this issue with wider perspective in national interest.

The lipophilic nature of these pesticides is an added disadvantages, as the cell walls in human beings & animals are also made of lipoproteins, the problem assumes a greater significance. ICAR, New Delhi sponsored a research project focussing on this issue with the following objectives:

• to assess the state-of-the-art of pesticide residues, of lipophilic nature, in meat & meat products by undertaking biological monitoring of pesticides.
• to study the residue dynamics of persistent pesticides, in particular the chlorinated group commonly used in farming/agriculture, in the body tissues of edible animals.
• to help the meat industry in the country, in understanding the international food standards with regard to pesticide residues so that they could take appropriate and effective measures to ensure the quality of meat & meat products and sustain the export potential.