The Centre for Science and Environment
The Centre for Science and Environment, a non-governmental organization based in New Delhi, has recently set up a laboratory to monitor pollution. Its main aim is to undertake scientific studies to generate public awareness about food contaminated by pesticides and heavy metals. It provides scientific services at affordable prices to communities that cannot obtain scientific evidence against polluters. This can be crucial at times, say, in a court case that a community might be fighting against a polluter in its area. Given the state of scientific research in India -- most of it is restricted to national defense and food security -- this is an effort to use science to achieve ecological security. The laboratory will also provide paid services to become financially self-sufficient. The funds to set up the laboratory have come from Deutsche Gesellschaft fur Technische Zusammenaabeit (GTZ) and the European Union.

Case background
The Plantation Corporation of Kerala (PCK), a Kerala government undertaking, has its cashew plantations on the upper reaches of western ghat hills around Padre village in Kasaragod district. PCK has resorted to aerial spraying of Endosulfan in cashew plantations to control Tea mosquito, which causes great havoc to the plantation. The spraying began in 1976 and is done thrice a year, one in December, second in January and third in March when the cashew plants start flowering. The pesticide used by PCK, Endosulfan, is a deadly organochlorine pesticide. It has already been banned in most of the developing and developed countries. The international negotiation by UNEP to ban such Persistent Organic Pollutants (POP's) has already short-listed 12 chemicals and endosulfan is expected to be the 13^th name in the list. The irreversible damages to the environment and human health by the pesticide already used in the last 18 years is beyond any assessment. The damages by the persistent pesticides are long lasting and will create problems for the generations to come.

Mehodology of Sample Collection
Sample collection: A researcher from CSE went to Padre to organize the collection of samples. Samples were collected on 28.1.01 and 29.1.01 under the technical guidance of M K Prasad, coordinator of the Environment Centre of the Kerala Sashtra Sahitya Parishad in Kochchi, Kerala, and V R Raghunandanan, associate professor and veterinary toxicologist with the Integrated Rural Technology Centre, Palakkad. Sripati Kajampady, a doctor who runs a nursing home in the neighboring Perla village, helped in collecting samples.

Method used for sample collection:
Soil: Representative soil samples were collected from three sites

• Near the house of D Subba Moolya of Jeentadka area of Kumbdaje village, neighbouring Padre.
• A few metres inside the plantation near the house of Krishna Naik, resident of Kajampady area of Padre. Aerial spraying took place here on Dec 26, 2000
• From the heart of the plantation at the top of a hill in Periyal.

Each surface soil sample (0-10cm), comprised collection from a minimum of 12 cores drew an acre unit with the help of a U-shaped tube of 2.8-cm internal diameter. The cores were pooled and collected in double walled polyethylene bags, transported to the laboratory on the same day, stored in a deep freezer at-18 ⁰C until analysed. Before extraction, the samples were thawed to room temperature, thoroughly mixed; stones and plant materials were removed. Sub-samples, weighing 50g, were drawn from the representative sample, which were used for residue analysis.

Water: Water samples were collected from three different sites

• From a small stream in Jeentadka area of Kumbdaje village, neighboring Padre; aerial spraying took place here on Dec 26, 2000
• From a tank in the house of Krishna Naik, about 20 meters from the cashew plantations in Kajampady area of Padre. Aerial spraying took place here on Dec 26, 2000
• From a channel that brings water from the Kodenkiri stream to the farms of S Narayan Bhat, resident of Padre.

Samples (1litre) were collected in clean plastic bottles (previously rinsed by hexane). The bottles were tightly capped to prevent contamination of the sample. The samples were stored under refrigeration at 2-4⁰ C, until analyzed.

Bovine milk: Samples of bovine milk were collected from a cow that grazes in the plantation around Krishna Naik’s house in Kajampady area of Padre. Its two-month-old calf died after epileptic fits, three days after aerial spraying took place in the area.

Each sample (500 ml) of milk was collected in glass bottles and refrigerated until analyzed.

Blood: Blood samples were collected from different people.

• Prabhawati Shastri, 46, of Kollenkana, Padre, adjacent to Kodenkiri stream has skin allergies and asthma.
• Vishnu Prasad Kulkarni, 16, has epilepsy and mental retardation
• Mohana Kumar, doctor, living in Kumbdaje village and practicing medicine in and around Padre has chronic throat infections.
• Kittanna Shetty, 21, lives right next to the Kodenkiri stream in Padre, has cerebral palsy. Brother Sridhar 16, suffers from mental retardation
• Muthakka Shetty 50, mother of Kittanna Shetty.
• Lalitha, 35, of Jeentadka, Kumbdaje village, near Padre, has a one-year-old child. Parents of Lalitha died of neurological problems two years ago and her sister Girija died of cancer four years ago. First sister-in-law died of an unknown cause. Second sister-in-law had a miscarriage.

Blood samples (5 ml) were collected in glass vials. Caps of these vials were airtight. The samples were stored at 2-4 ⁰ C until analyzed.

Butter: Butter sample (10 g), churned from the milk of a 4-year-old cow that grazes around the cashew plantations adjacent to Saletadka Area of Vaninagar, Padre, about 30 metres from the plantation, was collected in air tight vial and refrigerated until analyzed.

Coconut oil: Sample (10 ml) of coconut oil was collected from produce of coconut trees about 50 meters from the plantations near the house of Vishnu Bhat in Saletadka Area of Vaninagar, Padre. Aerial spraying took place here on Dec 26, 2000

Vegetables: Samples of vegetables, each weighing approximately 500g were collected

• Basale, a leafy vegetable eaten likes spinach, from the house of Krishna Naik of Kajampady.
• From just inside the plantation near the house of Krishna Naik in Kajampady area of Padre.
• Pepper bunch from tree close to the house of Krishna Naik in Kajampady area of Padre, about 20 meters from the plantation.

The samples were wrapped in polyethylene bags and immediately taken to the laboratory for analysis. The samples were chopped to small pieces and thoroughly mixed. From the well-mixed samples, sub-sample of 50 g was drawn. These sub-samples were analyzed separately for Endosulfan residues.

Fish: Fish samples were collected from a tank in the house of Krishna Naik, about 20 metres from the cashew plantations in Kajampady area of Padre. The samples were packed in polyethylene bags and transferred immediately to the laboratory and stored at -20⁰C until analysed.

The sample was thawed in laboratory and inedible portion like head, scales, etc., were removed, then sample was homogenised in a Waring blender. Sub-sample weighing 50 g was taken from the homogenised sample for analysis.

Human milk: Human milk samples were obtained from a nursing mother, Lalitha, 35, of Jeentadka, Kumbdaje village, near Padre. She has a one-year-old child. Approximately 20 ml of milk was collected with hands in a stoppered glass vial and was stored at -20⁰ C until analysed.

Extraction, Clean up and Analysis

Soil: The samples were extracted for residues following the extraction and cleanup procedure of Drager (1969) with suitable modifications. A sub-sample (50 g. of soil) was extracted twice by dipping in a 100 ml of methanol-water solvent mixture (2:1, v/v) for a day with occasional shaking. After filtration, the extract was partitioned with 100 and 50-ml portions of n-hexane. The aqueous layer was then partitioned with 50-ml dichloromethane. The combined organic phase of n-hexane and dichloromethane was concentrated to about 5 ml. The concentrated extract normally did not require any further cleanup. However, certain samples, which needed further cleanup, were chromatographed on silica gel using n-hexane and n-hexane-acetone (4:1, v/v) as eluants.

Water: Water samples (500 ml) were shaken well and filtered. After filtration, the extract was partitioned with 100 and 50-ml portions of n-hexane (twice). The aqueous layer was then discarded. The combined organic phase of n-hexane was concentrated to about 5 ml. The concentrated extract normally did not require any further cleanup. However, certain samples, which needed further cleanup, were chromatographed on Silica gel using n-hexane and n-hexane-acetone (4:1, v/v) as eluants.

Bovine milk: Extraction and cleanup of the milk samples was accomplished by suitable combination of the extraction method of de Faubert Maunder et al, (1964) and cleanup technique of Veirov and Aharonson (1978). A sub sample of milk (5 ml) was homogenised with 40 ml of acetone-hexane (1:1, v/v) mixture. The homogenate was allowed to stand till a clear separation into two layers occurred. After the removal of the upper organic phase, the lower aqueous phase was re-extracted twice with n-hexane (40 ml). The combined organic phase was evaporated till almost free of the solvent. The residue was dissolved in 40 ml of petroleum ether (B.P. 60-80 ⁰ C) and was cleaned up by drop-wise addition of 10 ml of concentrated sulphuric acid (sp. gravity 1.84). The petroleum ether fraction was washed with distilled water till neutral to litmus and concentrated to a suitable volume.

Butter fat: The method described by deFaubert Maunder et al. (1964) with slight modifications was used to extract and isolate residues from butter sample. Butter was warmed at about 50⁰C to separate the fat which was decanted through dry filter paper. A 5-g sample of the clarified fat was dissolved in 10 ml of hexane and transferred quantitatively to a 125-ml separatory funnel. The hexane extract was partitioned three times into dimethyl formamide (hexane saturated), using 10 ml of solvent each time. The dimethyl formamide after back washing with 10 ml of hexane (dimethyl formamide-saturated) was diluted with 250 ml of water and 50 ml of sodium chloride-saturated aqueous solution, and was extracted twice with 100 ml of hexane. The combined n-hexane extract was concentrated to about 5-10 ml and clean up was done on silica gel column.

Oil: The oil sample was extracted for the total pesticides residues according to method of Verion and Aharonson (1978) with slight modification. 10 ml of oil sample, dissolved in 40 ml of hexane. Partitioned thrice with equal volume of acetonitrile (3x40 ml). Combine the acetonitrile fraction and diluted with 650 ml distilled water and 20-30 ml of saturated. NaCl solution. Further partitioned with hexane (3x100 ml). The hexane fractions were collected and passed through sodium sulphate and concentrated.

Vegetables: The procedure of Mills et al. (1963) with slight modification was followed for extraction of residues. Sub-sample (50 g) was blended with 100 ml of acetonitrile for 2-3 minutes in the Waring blender. The macerate was filtered through a suction filter using mild vacuum. The filter-cake was blended again with 100 and 50 ml of acetonitrile and filtered. The blending jar and the filter were rinsed with additional 50 ml of acetonitrile. The filtrates and the washings were combined, diluted with volumes of water and 30 ml of sodium chloride saturated aqueous solution and then partitioned thrice using 100, 50 and 50 ml of petroleum ether (BP 60-80⁰ C). The petroleum ether fraction after concentration to a small volume was further cleaned up by column chromatography using silica gel as an adsorbent (Joia et al. 1987). The elutes were concentrated to small volume for analysis.

Fish: The procedure of Mills et al, (1963) with slight modification was followed for extraction of pesticide residue from fish. Sub-sample (50 g) was homogenised with 100-ml acetonitrile and 25 ml of distilled water in a Waring blender for 3 minutes at high speed. The homogenate was filtered and the residual material was re-extracted with 100 ml of methyl cyanide and 15 ml of distilled water. The filtrates were combined and the aliquot equivalent to 10 g of the sample was transferred to a 1 litre separatory funnel. To it, distilled water (250 ml), n-hexane (100 ml) and brine solution (50 ml) were added. The contents were shaken for few minutes and allowed to stand till there was complete separation of two layers. The upper organic layer was removed and the lower aqueous layer was re-extracted with 50-ml n-hexane. Both the n-hexane fractions were combined, washed with distilled water and then dried over anhydrous sodium sulphate. The n-hexane fraction was concentrated to 20 ml and further cleaned up by column chromatography using silica gel as an absorbent. The elute were concentrated to small volume for analysis.

Animal tissue: Samples weighing approximately 3-5 g were admixtures with about 20 g anhydrous sodium sulphate and were extracted thrice with acetonitrile after thorough maceration. The combined acetonitrile fraction, after dilution with 3 volumes of water and 10 ml of brine solution was partitioned thrice using 100 hexane:acetone (80:20,v/v). The combined hexane fraction was concentrated to about 5-10 ml and chromatographed on activated silica gel for further cleanup.

Milk: 5 ml of the sub-sample was extracted with 2 volumes of n-hexane-acetone (1:1, v/v) mixture The homogenate was allowed to stand till clear separation into two layers occurred. After the removal of organic phase, the lower phase was re-extracted twice with 15-ml portions of n-hexane. The combined n-hexane extract after concentration to 20 ml was transferred to a separatory funnel, to which 5-ml conc. Sulphuric acid (sp. Gr., 1.84) was added drop wise. The contents in the separatory funnel were shaken and allowed to stand. The lower sulphuric acid layer containing digested fat was discarded. The n-hexane phase was washed with distilled water till neutral to litmus and passed through sodium sulphate and concentrated to a small volume.

Blood: 2-ml blood sample was taken in a stoppered vial and 10-ml hexane was added and Centrifuged. Hexane fraction was collected and repeated twice. Combined hexane fraction was dried by using vacuum rotatory evaporator.

Method for Analysis
The residues in hexane were analyzed on Gas chromatography (GC-Trace, Thermoquest) equipped with electron capture detector by injecting 1ul sample. The operating parameters for Endosulfan were as follows:

METHOD FOR ANALYSIS