Qualitative estimation of pesticide residues.
Steps in TLC Analysis
1. Preparation of TLC plates
2. Extraction of the sample (meat, milk, eggs, stomach contents etc.,)
1. Spotting of the substance
2. Development
6. Visualisation
7. Identification
8. Quantification
1. Preparation of Plates
Wash 3 glass plates of 10 cm x 20 cm x 3 mm. Arrange on a mounting board. Clean glass plates with water, acetone and finally with cotton. Clean the applicator of 250µ thickness. Prepare slurry of silica gel G 7 g in 14 ml water. Shake for one minute. Pour the slurry into the applicator. Draw the applicator uniformly in one motion. Dry at room temperature followed by drying at 110C/10 minutes. Cool in a dessicator.
Preparation of AgNO3 TLC plate (for detection of OC and synthetic pyrethroids)
Arrange 3 plates on mounting board. Clean with water, acetone and cotton. Weigh 7 g Silicagel G. Wash with 200 ml distilled water to remove chlorides in water. (If not AgNO3 forms a precipitate with chloride).
Filter under suction using buchner funnel. Transfer to a stoppered conical flask. Add 6 ml distilled water and 1 ml 0.1 N AgNO3. Shake for 1 minute and pour into applicator. Draw the applicator uniformly quickly. Dry at 1100C/10 minutes (activation). Cool in a dessicator.
2. Extraction of the sample (Eggs/tissues – meat)
The sample is homogenised in a tissue homogeniser. A 10 g of homogenate is taken, to which 60-75 g of sodium sulphate (anhydrous) is added .Mix it thoroughly in pestle & mortar till a clear powder is obtained. Take the powder in a conical flask and add 50ml of acetonitrile. Shake for few min allow it to stand for few more min and collect the supernatant. Repeat this procedure for 3 times. Take this 150ml of acetonitrile in a separatory funnel and add salt solution and distilled hexane 100-150ml, repeat for 3 times. The collected hexane is subjected to florisil “column clean up”. Elute with 6% diethyl ether in petroleum ether 200ml.Ether is removed by evaporating the eluted liquid on a water bath at 50 ºC. the residue is taken in acetone and spotted.
3. Spotting of Sample on TLC plates
Dress the plate by removing silica gel layer (2mm) from the edges. Place the plate on a spotting guide. Clean a microlitre pipette (5 µl) using acetone solvent. Spot the test solution (if serum, apply directly, if other materials like rumen liquor, unlabelled bottle etc., it has to be first extracted – refer above). 2 cm above the bottom edge of the plate and 2 cm inside from the left side edge.
Spot 5 µl each of the standard solution. The standard solution of the pesticide or other substance in question is prepared as follows: Take pure sample, weigh 10g and add 10 ml acetone (1ml=1g). Make it to1ug/ul (1ml=1000µl; 1mg= 1000µg). Leave 1 cm gap between the spots. Clean the pipette after each spotting. Mark at 10 cm from the origin of spotting.
4. Development of TLC plate: Pour a suitable solvent (50-60 ml) into an aluminium tray. Place it in an airtight developing chamber. Allow saturating with solvent vapours for 30 min. Keep the plate vertically in the tray and close with a lid. Allow the solvent to come up to 10cm mark. Remove the plate and allow the vapour to dry for a few seconds.
Developing Solvents
Organo Chlorine - Cyclohexane
Organophosphates - Toluene
Carbamates -Chloroform
Synthetic Pyrethroids - Hexane + Acetone (95+5)
For unknown pesticides - Hexane + Chloroform+Acetone(6.5 + 3.5 + drops)
5. Visualisation of Chromatogram
Different chromatogenic reagents are used for different classes of pesticides.
Organo Chlorine : AgNO3 - Silica gel plate. Expose to UV radiation (254 nm) for 10 min. Steam the plate and re expose to UV for 5 min for better visibility. Brown spots on white background are observed in positive cases. Or
Plain silica gel plate is developed and is sprayed with 2- phenoxyethanol. The advantage is increased sensitivity; 0.01- 0.1µg of the compound can be detected. AgNO30.1g is dissolved in 1ml of water. To this add 10ml of 2- phenoxy ethanol. Dilute this solution with 200ml acetone. Spray on the TLC plate. Dry for 5min at room temperature and for 15 min at 75°C and expose TLC plate to UV light. Development of dark spots indicates the incidence of OC compounds.
Organo Phosphate: Plain silica gel plate is developed and exposed to bromine vapours for 30 sec (Apply a facemask and take utmost precaution while exposing the developed plate to bromine vapours). Spray with Bromocresol green solution (0.5% in methanol). Yellow spots on greenish blue background are observed in positive cases.
Carbamates: Plain Silica gel plate is sprayed with 1 N methanolic NaOH solution followed with a solution of p-Nitrobenzenediazoniumtetrafluroborate (NDFB). (Mix 50 mg of NDFB, 50 ml of Diethyleneglycol, and 50 ml of Acetone).
For carbaryl, blue spots on white background are detected.
In case of carbofuran, purple / violet spots are observed.
Synthetic pyrethroids: Silica gel AgNO3 plate is exposed to UV radiation (254 nm) for 10 min. Steam the plate and re- expose to UV radiation for 5 min for better visibility.
Brown spots on a white background are conclusive for pyrethroids.
6. Identification of Pesticides
Measure the distance of the spot from the origin.
Calculate the Rf.
Rf = Distance Travelled by pesticide / Distance travelled by solvent (10cm)
Spots with same Rf as that of technical are said to be certain.
7. Quantification
By measuring the area of standard and unknown
Wt of std./technical x area of sample x purity of technical
Wt of sample x area of technical/std
Or
Scrap the spot and put it in solvent and elute in GLC or HPLC -especially for quinalphos and fenvalerate.
Qualitative Detection of Thiram in grains
Grains meant for germinating purpose are treated with fungicides like thiram.
Procedure: 100 g grain sample (suspected) and 100 ml chloroform are taken in a conical flask. Shake for 5 min. Filter through whatman No.1 filter paper. Add about 5g charcoal to filtrate and shake for 1-2 min. Filter again through whatman No.1 filter paper. Add few crystals of cuprous iodide to the filtrate (10 ml). Development of amber to brown color indicates the presence of thiram. (Ramasubba Reddy, V. and Rajashekar Reddy, A. 1995).
Fujiwara's test for Halogenated hydrocarbons (DDT)
A small aliquot of the extract (test sample) is heated in a boiling water bath with 1 ml of 20 % aqueous NaOH and 1 ml pyridine. Red colour in the pyridine layer indicates a positive reaction. (NB: do not dissolve the extract in chloroform).
Or
The test sample is dissolved in n-hexane and ethanolic NaOH is added. Mixture is evaporated to dryness in a water bath. After cooling, 4 drops of carbon tetrachloride are added to redissolve the compound. On shaking vigorously with a mixture of sulphuric acid and nitric acid, a green colour develops which lasts for 1 min.
(Toxicity, Mechanism and Methods - Stewart and Stolman Vol.II)
Monday, June 9, 2008
Detection of Fluorides
Qualitative Test for Fluorides (Gettler Test)
Aim
To detect the presence of fluorides in the given sample.
Materials and chemicals
Sodium hydroxide solution, conc. sulphuric acid, glass beaker, heater, 5% sodium chloride, glass plate, microscope.
Procedure
1. To a small quantity (1-3 ml) of urine or stomach contents, add 3 ml of sodium hydroxide solution in a glass or porcelain dish.
2. Add little quantity of powdered glass and mix thoroughly with the residue.
3. Apply moderate heat till dry.
4. Add 10 ml of conc. sulphuric acid and cover the dish with a small glass plate from the under surface of which is suspended a drop of 5 per cent sodium chloride solution. Place a small piece of ice on top of the plate to prevent evaporation of the drop.
5. Heat gently for 3-5 min carefully remove the drop and examine under low power of microscope.
Inference
If the given specimen contains fluorides, silicon fluoride is formed which appears as small, light pink, hexagonal crystals along the rim of the drop, whereas sodium chloride crystals are large and square shaped.
Aim
To detect the presence of fluorides in the given sample.
Materials and chemicals
Sodium hydroxide solution, conc. sulphuric acid, glass beaker, heater, 5% sodium chloride, glass plate, microscope.
Procedure
1. To a small quantity (1-3 ml) of urine or stomach contents, add 3 ml of sodium hydroxide solution in a glass or porcelain dish.
2. Add little quantity of powdered glass and mix thoroughly with the residue.
3. Apply moderate heat till dry.
4. Add 10 ml of conc. sulphuric acid and cover the dish with a small glass plate from the under surface of which is suspended a drop of 5 per cent sodium chloride solution. Place a small piece of ice on top of the plate to prevent evaporation of the drop.
5. Heat gently for 3-5 min carefully remove the drop and examine under low power of microscope.
Inference
If the given specimen contains fluorides, silicon fluoride is formed which appears as small, light pink, hexagonal crystals along the rim of the drop, whereas sodium chloride crystals are large and square shaped.
Detection of cyanide
Qualitative Estimation of Cyanide in Biological and Plant Material
Aim
To detect cyanide in biological material (copper-guaic test)
Principle
Acidification of biological fluids liberates cyanide, which interacts with suitable reagents on the filter paper to form chromophores.
Materials and chemicals
Tartaric acid, copper sulphate, alcohol, guaiacol, filter paper, 100 ml flask, heater.
Procedure
1.Take 50 g of finely ground tissue or stomach contents in a 100 ml flask and acidify contents with tartaric acid.
2. A filter paper previously moistened with 10 per cent of guaiacol in alcohol and 0.1 per cent of aqueous copper sulphate solution.
3. Plug the mouth of flask and warm gently.
4. Allow it to stand for 30 minutes.
5. Observe the colour of paper.
Inference
If cyanide is present, a blue colour is developed.
Sodium picrate paper test to detect cyanide in plant sample
Aim
To detect the presence of cyanide in the given plant sample.
Materials and chemicals
Sodium bicarbonate, picric acid, filter paper, distilled water, chloroform, test tube, spirit lamp.
Preparation of sodium picrate paper
Dissolve5 g sodium bicarbonate and 0.5 g picric acid in 100 ml distilled water, cut filter paper into strips of 2" x6" size. Dip the strips in the reagent and dry in cool place.
Test procedure
1.Take the given sample in a test tube and add few drops of water and chloroform.
2. Plug the test tube with cotton, hanging the dried picrate paper. The paper should not touch the fluid in the tube.
3. Heat the tube gently till fumes evolve.
4. Observe the colour of paper.
Inference
If the colour of paper changes from yellow to brown it indicates that the plant sample is positive for cyanide.
The leaves and stomach contents are to be frozen immediately after collection in a polythene bag as cyanide is likely to evaporate.
Scheerer's test for detecting cyanide.
Materials and chemicals:
10% tartaric acid, a strip of filter paper dipped in a saturated solution of picric acid, dried and soaked in 10% sodium carbonate or sodium hydroxide at a concentration of 2 mol/L.
Method.
Place a small amount of the suspected material into a wide test-tube or conical flask and add tartaric acid. Fix the filter paper in the neck of the tube or flask and seal the neck.
Inference: If hydrogen cyanide is given off, the paper turns pink or red
Aim
To detect cyanide in biological material (copper-guaic test)
Principle
Acidification of biological fluids liberates cyanide, which interacts with suitable reagents on the filter paper to form chromophores.
Materials and chemicals
Tartaric acid, copper sulphate, alcohol, guaiacol, filter paper, 100 ml flask, heater.
Procedure
1.Take 50 g of finely ground tissue or stomach contents in a 100 ml flask and acidify contents with tartaric acid.
2. A filter paper previously moistened with 10 per cent of guaiacol in alcohol and 0.1 per cent of aqueous copper sulphate solution.
3. Plug the mouth of flask and warm gently.
4. Allow it to stand for 30 minutes.
5. Observe the colour of paper.
Inference
If cyanide is present, a blue colour is developed.
Sodium picrate paper test to detect cyanide in plant sample
Aim
To detect the presence of cyanide in the given plant sample.
Materials and chemicals
Sodium bicarbonate, picric acid, filter paper, distilled water, chloroform, test tube, spirit lamp.
Preparation of sodium picrate paper
Dissolve5 g sodium bicarbonate and 0.5 g picric acid in 100 ml distilled water, cut filter paper into strips of 2" x6" size. Dip the strips in the reagent and dry in cool place.
Test procedure
1.Take the given sample in a test tube and add few drops of water and chloroform.
2. Plug the test tube with cotton, hanging the dried picrate paper. The paper should not touch the fluid in the tube.
3. Heat the tube gently till fumes evolve.
4. Observe the colour of paper.
Inference
If the colour of paper changes from yellow to brown it indicates that the plant sample is positive for cyanide.
The leaves and stomach contents are to be frozen immediately after collection in a polythene bag as cyanide is likely to evaporate.
Scheerer's test for detecting cyanide.
Materials and chemicals:
10% tartaric acid, a strip of filter paper dipped in a saturated solution of picric acid, dried and soaked in 10% sodium carbonate or sodium hydroxide at a concentration of 2 mol/L.
Method.
Place a small amount of the suspected material into a wide test-tube or conical flask and add tartaric acid. Fix the filter paper in the neck of the tube or flask and seal the neck.
Inference: If hydrogen cyanide is given off, the paper turns pink or red
Detection of nitrate and nitrite
Qualitative test for Nitrite
Nitrate is less toxic but when converted into nitrite in the rumen or in the forage/silage as an intermediary product before being converted into ammonia it is more toxic (6-10 times more than nitrate). A simple field test for detection of nitrite is cooking test.
Aim
To detect the presence of nitrite in the blood.
Procedure
1. Draw blood (10 ml) without anticoagulant from an affected and a known normal animal in 20 ml capacity test tubes.
2. Place them in boiling water bath for 45 minutes. Cool them.
3. Observe the colour of the blood and the surface.
Inference
Nitrite containing blood sample is salmon pink in colour does not pull away from the side and the surface is level or concave. Normal blood sample is chocolate brown, pulls away from the side of tube and the surface is convex.
Second test for Nitrite.
Materials and chemicals
Sulphanilic acid, glacial acetic acid, α-naphthyl amine hydrochloride.
Procedure
1. Dissolve 0.5 g of sulphanilic acid in 150 ml of 20% glacial acetic acid-solution A.
2. Dissolve 0.2 gof α-naphthylamine hydrochloride in 150 ml of 20% glacial acetic acid by gently heating - solution B.
3. In a small test tube the given sample is taken (solution of feed material or ruminal contents) and 2 ml of solution A and B are added. Observe the colour.
Inference
A pink to red colour is positive for the presence of nitrites.
v Nitrite is easily decomposed and destroyed hence it may not be present in the sample preserved for chemical analysis even in cases of definite nitrite poisoning.
Qualitative test for Nitrate in a plant sample
Materials and chemicals
Sulphuric acid, diphenylamine and glass slide.
Procedure
1. Dissolve 0.5 g of diphenylamine in 20 ml of distilled water and make the volume to 100 ml with sulphuric acid.
2. Store it in amber coloured bottle. This is full strength solution and can be made into half strength by diluting with equal parts of 80% sulphuric acid.
3. To test a plant place a drop of the test reagent on the cut surface of plant. Observe the colour.
Inference
o A green to blue colour indicates the presence of nitrate.
A green to blue colour with a half strength solution indicates + + for nitrate which could be toxic to animal.
Nitrate is less toxic but when converted into nitrite in the rumen or in the forage/silage as an intermediary product before being converted into ammonia it is more toxic (6-10 times more than nitrate). A simple field test for detection of nitrite is cooking test.
Aim
To detect the presence of nitrite in the blood.
Procedure
1. Draw blood (10 ml) without anticoagulant from an affected and a known normal animal in 20 ml capacity test tubes.
2. Place them in boiling water bath for 45 minutes. Cool them.
3. Observe the colour of the blood and the surface.
Inference
Nitrite containing blood sample is salmon pink in colour does not pull away from the side and the surface is level or concave. Normal blood sample is chocolate brown, pulls away from the side of tube and the surface is convex.
Second test for Nitrite.
Materials and chemicals
Sulphanilic acid, glacial acetic acid, α-naphthyl amine hydrochloride.
Procedure
1. Dissolve 0.5 g of sulphanilic acid in 150 ml of 20% glacial acetic acid-solution A.
2. Dissolve 0.2 gof α-naphthylamine hydrochloride in 150 ml of 20% glacial acetic acid by gently heating - solution B.
3. In a small test tube the given sample is taken (solution of feed material or ruminal contents) and 2 ml of solution A and B are added. Observe the colour.
Inference
A pink to red colour is positive for the presence of nitrites.
v Nitrite is easily decomposed and destroyed hence it may not be present in the sample preserved for chemical analysis even in cases of definite nitrite poisoning.
Qualitative test for Nitrate in a plant sample
Materials and chemicals
Sulphuric acid, diphenylamine and glass slide.
Procedure
1. Dissolve 0.5 g of diphenylamine in 20 ml of distilled water and make the volume to 100 ml with sulphuric acid.
2. Store it in amber coloured bottle. This is full strength solution and can be made into half strength by diluting with equal parts of 80% sulphuric acid.
3. To test a plant place a drop of the test reagent on the cut surface of plant. Observe the colour.
Inference
o A green to blue colour indicates the presence of nitrate.
A green to blue colour with a half strength solution indicates + + for nitrate which could be toxic to animal.
Detection of heavy metals
Qualitative estimation of heavy metals (Reinsch's Test)
Aim
To detect the presence of heavy metals in the given sample.
Principle
Copper displaces from the solution elements below it in the electrochemical series. The displaced element coats the copper giving a characteristic colour, which indicates a positive result.
Materials and chemicals
Beaker, conc. HCl, copper foil or wire, test tube and spirit lamp.
Test procedure
1. Place 10 g of tissue (finely minced) in a beaker or test tube.
2. Add 10 ml of 10% HCl.
3. Introduce copper wire into the test tube.
4. Boil for 10-15 min (never inhale fumes).
5. Remove the copper wire and place it on a filter paper.
6. Observe the colour of the copper wire.
Inference:
Black colouration - Arsenic or Bismuth
Shining silver deposit - Mercury
Dull white deposit - Silver
Dark colour with purple to blue-violet sheen - Antimony.
Depending on the colour of copper wire the confirmatory test is conducted.
If the deposit is black it can be confirmed whether it is due to arsenic or bismuth by placing the copper strip in 1-2 ml of 10 per cent potassium cyanide. If the deposit is due to arsenic, it will dissolve, but if it is due to bismuth or antimony, it will persist.
In mercury, colour of the deposit ranges from greyish (50 mg) to shiny silver (100 mg)
Confirmation test for mercury (Cuprous Iodide test)
Procedure
1. Dissolve 5 g of cupric sulphate and 3 g of ferrous sulphate in 10 ml of water.
2. Slowly add 7 g of KI in 50 ml of water while stirring. A precipitate is formed. Filter and wash the precipitate with water (by decantation) to remove the excess iodide. Mix 1 and 2. The cuprous iodide so formed is stored in a brown bottle in 10 ml of water and may be stirred and used in the form of suspension.
3. Place the copper wire used in Reinsch's test in few drops of cuprous iodide suspension. Cover with a watch glass and allow it to stand for about one hour. The copper wire turns to light pink.
Inference
Presence of mercury (30 μg) turns cuprous iodide to mercury iodide that is light pink; the intensity of colour is proportional to the concentration of mercury present.
Qualitative Test for Lead (Grunwald test)
Aim
To detect the presence of lead in the given sample.
Principle
The determination of lead in biological material is done by wet digestion. Nitric acid is used to digest the organic matter. Addition of potassium iodide imparts yellow colour due to formation of lead iodide.
Materials and chemicals
Conc. nitric acid, 10% potassium iodide solution, test tube and spirit lamp.
Procedure
1. Mince the liver/kidney piece or collect a small amount of scraping from stomach wall.
2. Add few drops of conc. nitric acid and heat gently till dry (never over heat the sample as it turns black, making reading difficult).
3. Add few drops of water and two drops of 10 % potassium iodide solution.
Inference
Development of yellow colour indicates the presence of lead.
Other methods for detection of lead
To the test solution/ sample add the following: -
With dilute sulphuric acid: Lead ions in dilute H2SO4 produce an insoluble white precipitate of PbSO4, which is insoluble in acids but soluble in hydroxides (alkalis).
Further, filter the precipitate and to the residue on filter paper add sodium sulphide solution, formation of black coloured lead sulphide is seen.
With potassium chromate: - To the test sample, add few drops of dilute potassium chromate solution. Lead chromate (yellow in colour) is formed in presence of lead. Lead chromate is insoluble in water, dilute acetic acid and ammonia, but soluble in dilute mineral acids and hydroxides.
Aim
To detect the presence of heavy metals in the given sample.
Principle
Copper displaces from the solution elements below it in the electrochemical series. The displaced element coats the copper giving a characteristic colour, which indicates a positive result.
Materials and chemicals
Beaker, conc. HCl, copper foil or wire, test tube and spirit lamp.
Test procedure
1. Place 10 g of tissue (finely minced) in a beaker or test tube.
2. Add 10 ml of 10% HCl.
3. Introduce copper wire into the test tube.
4. Boil for 10-15 min (never inhale fumes).
5. Remove the copper wire and place it on a filter paper.
6. Observe the colour of the copper wire.
Inference:
Black colouration - Arsenic or Bismuth
Shining silver deposit - Mercury
Dull white deposit - Silver
Dark colour with purple to blue-violet sheen - Antimony.
Depending on the colour of copper wire the confirmatory test is conducted.
If the deposit is black it can be confirmed whether it is due to arsenic or bismuth by placing the copper strip in 1-2 ml of 10 per cent potassium cyanide. If the deposit is due to arsenic, it will dissolve, but if it is due to bismuth or antimony, it will persist.
In mercury, colour of the deposit ranges from greyish (50 mg) to shiny silver (100 mg)
Confirmation test for mercury (Cuprous Iodide test)
Procedure
1. Dissolve 5 g of cupric sulphate and 3 g of ferrous sulphate in 10 ml of water.
2. Slowly add 7 g of KI in 50 ml of water while stirring. A precipitate is formed. Filter and wash the precipitate with water (by decantation) to remove the excess iodide. Mix 1 and 2. The cuprous iodide so formed is stored in a brown bottle in 10 ml of water and may be stirred and used in the form of suspension.
3. Place the copper wire used in Reinsch's test in few drops of cuprous iodide suspension. Cover with a watch glass and allow it to stand for about one hour. The copper wire turns to light pink.
Inference
Presence of mercury (30 μg) turns cuprous iodide to mercury iodide that is light pink; the intensity of colour is proportional to the concentration of mercury present.
Qualitative Test for Lead (Grunwald test)
Aim
To detect the presence of lead in the given sample.
Principle
The determination of lead in biological material is done by wet digestion. Nitric acid is used to digest the organic matter. Addition of potassium iodide imparts yellow colour due to formation of lead iodide.
Materials and chemicals
Conc. nitric acid, 10% potassium iodide solution, test tube and spirit lamp.
Procedure
1. Mince the liver/kidney piece or collect a small amount of scraping from stomach wall.
2. Add few drops of conc. nitric acid and heat gently till dry (never over heat the sample as it turns black, making reading difficult).
3. Add few drops of water and two drops of 10 % potassium iodide solution.
Inference
Development of yellow colour indicates the presence of lead.
Other methods for detection of lead
To the test solution/ sample add the following: -
With dilute sulphuric acid: Lead ions in dilute H2SO4 produce an insoluble white precipitate of PbSO4, which is insoluble in acids but soluble in hydroxides (alkalis).
Further, filter the precipitate and to the residue on filter paper add sodium sulphide solution, formation of black coloured lead sulphide is seen.
With potassium chromate: - To the test sample, add few drops of dilute potassium chromate solution. Lead chromate (yellow in colour) is formed in presence of lead. Lead chromate is insoluble in water, dilute acetic acid and ammonia, but soluble in dilute mineral acids and hydroxides.
Pathological effects of heavy metal water pollutants on Animals
Pathological effects of heavy metal water pollutants on Animals
1.MercuryAbdominal pain, headache, diarrhoea, hemolysis, chest pain.
2.Lead Anaemia, vomiting, loss of appetite, convulsions, damage of brain, liver and kidney.
3.Arsenic Disturbed peripheral circulation, mental disturbance, liver cirrhosis, hyperkeratosis, lung cancer, ulcers in gastro intestinal tract and kidney damage.
4.Cadmium Diarrhoea, growth retardation, bone deformation, kidney damage, testicular atrophy, anaemia, injury of central nervous system and liver, hypertension.
5.Copper Hypertension, uremia, coma, sporadic fever.
6.Barium Excessive salivation, vomiting, diarrhoea, paralysis, colic pain.
7.Zinc Vomiting, renal damage, cramps.
8.Selenium Damage of liver, kidney and spleen, fever, nervousness, vomiting, low blood pressure, blindness, and even death.
9.Hexavalent chromiumNephritis, gastro-intestinal ulceration, diseases in central nervous system, cancer.
10.Cobalt Diarrhoea, low blood pressure, lung irritation, bone deformities, paralysis.
1.MercuryAbdominal pain, headache, diarrhoea, hemolysis, chest pain.
2.Lead Anaemia, vomiting, loss of appetite, convulsions, damage of brain, liver and kidney.
3.Arsenic Disturbed peripheral circulation, mental disturbance, liver cirrhosis, hyperkeratosis, lung cancer, ulcers in gastro intestinal tract and kidney damage.
4.Cadmium Diarrhoea, growth retardation, bone deformation, kidney damage, testicular atrophy, anaemia, injury of central nervous system and liver, hypertension.
5.Copper Hypertension, uremia, coma, sporadic fever.
6.Barium Excessive salivation, vomiting, diarrhoea, paralysis, colic pain.
7.Zinc Vomiting, renal damage, cramps.
8.Selenium Damage of liver, kidney and spleen, fever, nervousness, vomiting, low blood pressure, blindness, and even death.
9.Hexavalent chromiumNephritis, gastro-intestinal ulceration, diseases in central nervous system, cancer.
10.Cobalt Diarrhoea, low blood pressure, lung irritation, bone deformities, paralysis.
Pm examination of veterolegal case
MODE OF COLLECTION AND DESPATCH OF TOXICOLOGICAL SPECIMENS FOR DIAGNOSIS OF POISONING
Generally every veterinarian is encountered with two types of cases in poisoning of farm animals. (1) Accidental poisoning and (2) Malicious poisoning. The necropsy of the animals and the rapid diagnosis is helpful in the treatment of other affected animals in case of accidental poisoning. In case of malicious poisoning, which may turn up into medico legal case, the identification of poison is a must to establish the cause of death. In all poisonous cases, chemical analysis of the biological specimens is essential to know the cause of death or illness. Therefore, every veterinarian should know the salient points in collection and despatch of toxicological specimens to a laboratory.
History of the case is of great importance in the diagnosis of poisoning. This includes the number of animals in the farm, number of affected, method of feeding, regularity of feeding, recent changes in the rations or attendants, whether pastures have been sprayed with pesticides or fertilizers, if rodenticides have been used and remnants of the bait removed and disposed properly, storage of poisonous substances etc.
Inspection of the surroundings for empty pesticides or paint containers that are not really empty, presence of poisonous plants in the farm environment. Also, the possibility of industrial effluents coming in contact with grazing/watering sources should be given thought of.
POST MORTEM PROPER
Necropsy by routine procedure is to be performed as soon as possible after the death of animal. Examine the animal externally and note incisions (for sui poisoning, snake bite etc.,) on the skin or mucous membranes
Examine the oral cavity for corrosive lesions (acids/alkali) or changes in colour of mucous membrane (nitrate, co, cyanide poisoning). As most of the toxins gain entry through gut, examination of gut mucosa, the contents, their smell, colour and pH (acids, alkali, urea) is a valuable guide in diagnosing toxicoses. Poisoning by salts of heavy metals results in significant post mortem lesions but poisoning by alkaloids like strychnine produces very feeble lesions.
The natural orifices, sub-cutaneous fat tissue, muscles, bones and teeth (in fluorine poisoning), body cavities, and internal organs should be examined. The stomach should be punctured rather than cut open for organoleptic examination to note the character of smell. Puncture ensures greater accuracy and a longer time smell. Some of the poisons which emit characteristic smell are: bitter almond -hydrogen cyanide poisoning, garlic odour - phosphorus poisoning, rotten garlic or horse radish smell - selenium, tobacco odour –nicotine, acetylene odour - zinc phosphide and ammoniacal odour- urea.
Check the pH of the stomach contents by pH paper. Any variation in the normal pH of the sps. being examined indicates abnormality. (In urea poisoning-alkaline pH is observed in rumen liquor due to release of ammonia).
The colour of stomach contents also indicates the cause of poisoning. Copper salts impart a greenish blue colour whereas picric and nitric acid impart yellow colour to the contents.
The contents of the stomach vary from traces to flakes of paints or lead objects, grains or baits, seeds etc., like wise small and large intestine should be examined. Blood should be examined for its colour and clotting characters. Cyanide poisoning imparts cherry red colour, arsenic imparts rose red colour and nitrate poisoning turns blood brown in colour. In abrus and cyanide poisoning-blood remains fluid after death.
Examination of other visceral organs should be done in relation to their size, colour etc. eg: - spleen size is decreased and colour is changed to dark brown or black in copper poisoning and spleen size is increased in T-2 mycotoxicoses.
Lymph nodes are swollen, haemorrhagic, oedematous and dark upon exposure to radiation. Bone marrow becomes pale and gelatinous with yellowish tint.
The description of morphological changes should be noted clearly and absence of changes should be notified. The most important lesions found should be underlined.
COLLECTION OF SAMPLES FOR ANALYSIS
A sample feed/suspected bait to establish source of poisoning.
Rumen contents/vomitus to establish that the toxin has been ingested.
Liver, spleen and blood - to establish absorption.
Kidney, urine, milk - to establish excretion.
In case of small animals (poultry, small dogs, lab animals) the cadavers are sent as it is, in case of large animals the stomach contents are collected from the vicinity of patho anatomic changes in the gastric mucosa. If there are no changes a representative sample is collected, but in medium sized animals the stomach tied at oesophageal and duodenal end, intestine tied at both ends and bladder with tied ends is sent separately.
All the specimens are to be taken in separate containers (polythene jars/covers), securely tied, properly labelled with particulars of date, case No., organs collected, species, name of preservative used etc. A sample of the preservative used, brief history of the case along with treatment given particulars should be sent.
------------------------------------------------------------------------------------------------
Kind of sample Quantity
-------------------------------------------------------------------------------------------------
Whole blood 10-20 ml
Serum 10-20 ml
Milk 50-100 ml
Urine 50-100 ml
Water 200 ml
Faeces 50 g
Feed 0.5 - 1kg.
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It is always preferable to send the specimens through a special messenger. In medico legal cases, the specimens should be sealed in the presence of a witness.
Mode of preservation
1. Ice/72 hrs.
2. Alcohol (95% ethyl alcohol) 1 ml/g of tissue is the ideal preservative for toxicological specimens. Formaline should never be used as it hardens the tissue without giving scope for scraping and interferes in the analysis. Blood and serum should be refrigerated and never frozen. A sample of the preservative used should be sent. It is always better to have a duplicate sample stored properly in a refrigerator for future reference.
Type of poison Organs to be collected
Arsenic - Liver, kidney, feed, stomach contents, urine.
Lead - Bone, blood, and stomach contents
Fluoride - Urine, stomach contents, forages and water sample
Nitrate - Water, forage, silage, whole blood, plant material, frozen and sent without chopping in a ploythene cover
Copper - Kidney, liver, whole blood, faeces
Cyanide -Blood, liver, forage, stomach contents in1% mercuricchloride and refrigerated
Organo phosphorus pesticides - Body fat, stomach contents, blood (heparinised), urine, feed and half brain.
Organochlorine pesticides - Fat, stomach contents, liver, kidney, whole blood, serum.
Carbon monoxide - Blood (refrigerated).
Urea - Blood (refrigerated), rumen liquor (frozen).
Generally every veterinarian is encountered with two types of cases in poisoning of farm animals. (1) Accidental poisoning and (2) Malicious poisoning. The necropsy of the animals and the rapid diagnosis is helpful in the treatment of other affected animals in case of accidental poisoning. In case of malicious poisoning, which may turn up into medico legal case, the identification of poison is a must to establish the cause of death. In all poisonous cases, chemical analysis of the biological specimens is essential to know the cause of death or illness. Therefore, every veterinarian should know the salient points in collection and despatch of toxicological specimens to a laboratory.
History of the case is of great importance in the diagnosis of poisoning. This includes the number of animals in the farm, number of affected, method of feeding, regularity of feeding, recent changes in the rations or attendants, whether pastures have been sprayed with pesticides or fertilizers, if rodenticides have been used and remnants of the bait removed and disposed properly, storage of poisonous substances etc.
Inspection of the surroundings for empty pesticides or paint containers that are not really empty, presence of poisonous plants in the farm environment. Also, the possibility of industrial effluents coming in contact with grazing/watering sources should be given thought of.
POST MORTEM PROPER
Necropsy by routine procedure is to be performed as soon as possible after the death of animal. Examine the animal externally and note incisions (for sui poisoning, snake bite etc.,) on the skin or mucous membranes
Examine the oral cavity for corrosive lesions (acids/alkali) or changes in colour of mucous membrane (nitrate, co, cyanide poisoning). As most of the toxins gain entry through gut, examination of gut mucosa, the contents, their smell, colour and pH (acids, alkali, urea) is a valuable guide in diagnosing toxicoses. Poisoning by salts of heavy metals results in significant post mortem lesions but poisoning by alkaloids like strychnine produces very feeble lesions.
The natural orifices, sub-cutaneous fat tissue, muscles, bones and teeth (in fluorine poisoning), body cavities, and internal organs should be examined. The stomach should be punctured rather than cut open for organoleptic examination to note the character of smell. Puncture ensures greater accuracy and a longer time smell. Some of the poisons which emit characteristic smell are: bitter almond -hydrogen cyanide poisoning, garlic odour - phosphorus poisoning, rotten garlic or horse radish smell - selenium, tobacco odour –nicotine, acetylene odour - zinc phosphide and ammoniacal odour- urea.
Check the pH of the stomach contents by pH paper. Any variation in the normal pH of the sps. being examined indicates abnormality. (In urea poisoning-alkaline pH is observed in rumen liquor due to release of ammonia).
The colour of stomach contents also indicates the cause of poisoning. Copper salts impart a greenish blue colour whereas picric and nitric acid impart yellow colour to the contents.
The contents of the stomach vary from traces to flakes of paints or lead objects, grains or baits, seeds etc., like wise small and large intestine should be examined. Blood should be examined for its colour and clotting characters. Cyanide poisoning imparts cherry red colour, arsenic imparts rose red colour and nitrate poisoning turns blood brown in colour. In abrus and cyanide poisoning-blood remains fluid after death.
Examination of other visceral organs should be done in relation to their size, colour etc. eg: - spleen size is decreased and colour is changed to dark brown or black in copper poisoning and spleen size is increased in T-2 mycotoxicoses.
Lymph nodes are swollen, haemorrhagic, oedematous and dark upon exposure to radiation. Bone marrow becomes pale and gelatinous with yellowish tint.
The description of morphological changes should be noted clearly and absence of changes should be notified. The most important lesions found should be underlined.
COLLECTION OF SAMPLES FOR ANALYSIS
A sample feed/suspected bait to establish source of poisoning.
Rumen contents/vomitus to establish that the toxin has been ingested.
Liver, spleen and blood - to establish absorption.
Kidney, urine, milk - to establish excretion.
In case of small animals (poultry, small dogs, lab animals) the cadavers are sent as it is, in case of large animals the stomach contents are collected from the vicinity of patho anatomic changes in the gastric mucosa. If there are no changes a representative sample is collected, but in medium sized animals the stomach tied at oesophageal and duodenal end, intestine tied at both ends and bladder with tied ends is sent separately.
All the specimens are to be taken in separate containers (polythene jars/covers), securely tied, properly labelled with particulars of date, case No., organs collected, species, name of preservative used etc. A sample of the preservative used, brief history of the case along with treatment given particulars should be sent.
------------------------------------------------------------------------------------------------
Kind of sample Quantity
-------------------------------------------------------------------------------------------------
Whole blood 10-20 ml
Serum 10-20 ml
Milk 50-100 ml
Urine 50-100 ml
Water 200 ml
Faeces 50 g
Feed 0.5 - 1kg.
-------------------------------------------------------------------------------------------------
It is always preferable to send the specimens through a special messenger. In medico legal cases, the specimens should be sealed in the presence of a witness.
Mode of preservation
1. Ice/72 hrs.
2. Alcohol (95% ethyl alcohol) 1 ml/g of tissue is the ideal preservative for toxicological specimens. Formaline should never be used as it hardens the tissue without giving scope for scraping and interferes in the analysis. Blood and serum should be refrigerated and never frozen. A sample of the preservative used should be sent. It is always better to have a duplicate sample stored properly in a refrigerator for future reference.
Type of poison Organs to be collected
Arsenic - Liver, kidney, feed, stomach contents, urine.
Lead - Bone, blood, and stomach contents
Fluoride - Urine, stomach contents, forages and water sample
Nitrate - Water, forage, silage, whole blood, plant material, frozen and sent without chopping in a ploythene cover
Copper - Kidney, liver, whole blood, faeces
Cyanide -Blood, liver, forage, stomach contents in1% mercuricchloride and refrigerated
Organo phosphorus pesticides - Body fat, stomach contents, blood (heparinised), urine, feed and half brain.
Organochlorine pesticides - Fat, stomach contents, liver, kidney, whole blood, serum.
Carbon monoxide - Blood (refrigerated).
Urea - Blood (refrigerated), rumen liquor (frozen).
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