JULY, 1961 THE ANALYST Vol. 86, No. 1024 PROCEEDINGS OF THE SOCIETY FOR ANALYTICAL CHEMISTRY DEATHS WE record with regret the deaths of Edgar Percy Campbell Albert Edward Fletcher Harold Wright Hodgson Reginald Arthur McNicol. 429 The Toxicology of Nitrates and Nitrites with Particular Reference to the Potability of Water Supplies A Review* BY E. H. W. J. BURDEN? (Firthorfie, South Ascot, B e h . ) INTRODUCTION THERE have been many reports in recent years that high concentrations of nitrates in drinking supplies have caused poisoning and death among infants and also among animals. A review has been made of the evidence available in order to suggest suitable limits for this constituent in drinking water supplies. THE TOXICOLOGY OF NITRATES AND NITRITES MODE OF ACTION- After ingestion, nitrates are converted to nitrites by bacterial reduction.According to Cornblath and Hartrnann,l this reduction occurs in the lower intestine of the adult. However, in very young babies, which have a physiological gastric achlorhydria, reduction occurs in the stomach and duodenum from which the nitrites are more readily absorbed into the blood stream. After absorption, nitrites convert oxyhaemoglobin into methaemoglobin and thus interfere with the oxygen transport in the blood. This effect may be cumulative, since the methaemoglobin is reduced only at a slow rate. * Reprints of this paper will be available shortly. t Formerly The Government Analyst, Ministry of Health, Khartoum, Sudan. For details, please see p. 492.430 BURDEN : TOXICOLOGY OF NITRATES AND NITRITES WITH vol.86 They occur in the order: (a) pain in the epigastrium; (b) purging, often with blood; (c) convulsions of the face muscles; (d) weak irregular pulse; (e) difficult respiration; (f) cold extremities followed by collapse. SYMPTOMS OF POISONING- Luff2 describes the symptoms of acute potassium nitrate poisoning. Cyanosis is usually observed and t:he blood becomes chocolate coloured. ANTIDOTE- to haemoglobin, and the patient makes a dra.matic recovery.3 Intravenous injection of methylene blue results in a rapid reduction of methaemoglobin LETHAL DOSE: HUMAN- Nitrates-Smith and Simpson4 consider the normal fatal dose of potassium nitrate to be 15 to 30 g for an adult, although they quote a report by Peterson, Haines and Webstefl of the death of an adult after taking 8 g.Luff2 records the death of an adult after taking 3.5 g of potassium nitrate, although Witthaws reports a recovery after taking 125 g. C0mly7 quotes the observation of Eustennan and Keiths that certain adults are extremely allergic to nitrates. These authors record that one adult suffered from extreme cyanosis after taking 7 g of ammonium nitrate daily for 8 successive days. Wood3 also states that nitrates are a cumulative poison. On the other han.d, Ambergg reports that a 13-year-old boy (110 lb) with glomerulonephritis took 6 g of potassium nitrate daily for a year without injury. Windmuellerlo states that an adult who took 4 teaspoonsful daily of an equal mixture of sulphur and potassium nitrate for 26 days died 17 days later.Nitrites-Smith and Simpson4 consider that the normal fatal dose of sodium nitrite is about log. Manicatidell records the death of a 3-month-old child after taking 0.35g of sodium nitrite, but the recovery of a 2-month-old child after taking 0-4 g. Tankard12 reports the death of an adult who probably took about 7 g of sodium nitrite. Scholes13 records the accidental death of three adults, two of them within an hour, after eating a meal seasoned with sodium nitrite instead of sodium chloride. Several other deaths from nitrite poisoning have been r e c ~ r d e d . ~ * J ~ J ~ J ~ J ~ However, Kobert, quoted by Autenrieth,lg mentions the case of a patient who took “several doses” of 0.5 g of sodium nitrite at hourly intervals “without dangerous symptoms,” although the srymptoms included nausea, diarrhoea, cyanosis and increased diuresis.Naidu and Venkatrao20 consider 2.0 g of sodium nitrite to be a fatal dose for a 10-stone man after examining results obtained on dogs. This is equivalent to 32 mg per kg. FATAL DOSE : ANIMALS- Crawford21 reports a case of sodium nitrate poisoning in cattle; he considers that & lb of sodium nitrate is a fatal dose. For an animal weighing 500 kg this is equivalent to 450 mg per kg. Bradley, Eppson and Beath22,23 have shown that “oat hay poisoning’’ in cattle is caused by abnormally high concentrations of nitrate in the affected hay. They havealso found that 1 g of potassium nitrate per kg is a lethal dose for cattle. Death is caused by methaemoglobinaemia, 70 to 80 per cent. of the haemoglobin being converted to methaemo- globin in a few hours.Sapiro, Hoflund, Clark and QuinN have demonstrated that nitrate is reduced to nitrite by the rumen contents of cattle. has shown that, in the rumen of sheep, nitrate is reduced successively to nitrite and ammonia. With large doses of nitrate, the conversion of nitrite to ammonia is limited and the nitrite concentration in the rumen increases rapidly. After giving large doses of nitrate, the methaemoglobin in the blood is found to reach a maximum about 7 hours later. Lewis finds that the doses of nitrate and nitrite required to give 60 per cent. conversionof haemoglobin to methaemoglobin are- Sodium nitrate (added to rumen) . . . . 420 mg per kg Sodium nitrite (added to rumen) . . . . 170 mg per kg Sodium nitrite (intravenously) .. . . . . 34mgperkg Sheep are similarly affected. Oltmann and Crandal126 find that the lethal dose of sodium nitrite for rabbits is 80 to Winks, Sutherland and Salisbury2’ report that the lethal dose of sodium nitrite 90 mg per kg. for pigs is 90 mg per kg, and that doses of 50 to SO mg per kg give toxic symptoms.July, 19611 PARTICULAR REFERENCE TO POTABILITY OF WATER SUPPLIES. A REVIEW 431 The lethal doses of sodium nitrite, calculated as nitrogen, quoted above for adults and various animals can be summarised as shown below- (1) For 10-stone adult . . .. .. 33 mg per kgQ (2) For 10-stone adult . . .. .. 22 mg per kgI2 (3) Rabbits . . . . . . . . 16 to 18 mg per kgZ6 (4) Pigs . . . . .. . . .. 18 mg per kg2' (5) Sheep . . . . . ... 34 mg per kg25 (6) Dogs . . . . . . . . .. 6.4 mg per kg20 A summary of the reported lethal doses of nitrate, calculated as nitrogen, is shown below- (1) For 10-stone adult . . .. . . 34 to 68 mg per kg4 (2) For 10-stone adult . . .. .. 18 mg per kg5 (3) Cattle. . .. . . . . . . 75 mg per kg21 (4) Cattle. . . . .. . . . . 140 mg per kg23 (5) Sheep . . . . .. .. 70 mg per kg25 NITRITES AND NITRATES IN WATER SUPPLIES- In most natural waters, nitrate is the final product of the aerobic decomposition of nitrogenous organic matter. Its presence in a water supply is usually interpreted as an indication of contamination by organic matter, although this may have happened in the remote past and may not necessarily be serious. Nitrite indicates less complete oxidation and may show more recent and possibly dangerous contamination. Traces of nitrate are present in most natural waters, and it is only rarely that the concentration rises to a dangerous level.The recorded cases of poisoning caused by high concentrations in water supplies come under two headings- (a) infantile cyanosis and (b) animal poisoning. (a) INFANTILE CYANOSIS- Comly7 cites from personal experience a number of cases of infantile cyanosis where symptoms were observed after ingestion of feeds made from waters containing 64 to 140 p.p.m. of nitrate-nitrogen. Taylor28 records a large number of cases of infantile cyanosis caused by nitrates in feed ~ater.2~JOv~l s32933934 Betke and Kleiha~eI3~ also refer to a case of infantile cyanosis occurring under similar circumstances.The reasons that make artificially fed infants particularly susceptible appear to be- He quotes a number of other cases including one fatality. (i) the normal gastric achlorhydria of infants, already referred to,l and (ii) the high relative fluid intake of an infant compared with that of an adult.3 In England, a baby under 15 lb requires about 2 i fluid 02. of water per pound body weight per diem.3 In the tropics, this figure is much higher and may rise to about 10 fluid 02. per pound body weight per diem.36 In comparison, a normal adult in England requires about 1.8 pints of liquid a day.37 For a 10-stone man, this is equivalent to 6 fluid oz. per pound body weight per diem. In the tropics, the requirement would be about 1Q fluid 02. per pound body weight per diem.Waring38 suggests that waters used for preparing infant feeds should not contain more than 20 p.p.m. of nitrate-nitrogen. Ewing and Mayon-White= concur with the suggestion. The Committee of the Division of Medical Science of the United States National Research Council3g regard any well water containing more than 10 p.p.m. of nitrate-nitrogen as unfit for consumption by infants. Wood3 regards supplies containing less than 20 p.p.m. of nitrate-nitrogen as satisfactory €or infant feeding, those containing 20 to 39 p.p.m. as undesirable, those containing 40 to 79 p.p.m. as unsafe and those containing more than 80 p.p.m. as dangerous, Any apparent discrepancy between these authors' findings may be explained by possible differences in climate. (b) ANIMAL POISONING- 1000 p.p.m.of nitrate-nitrogen. Cattle-Fincheflo reports the death of 4 cattle after drinking from a supply containing Successive Sudan Government Analysts have recorded132 BURDEN : TOXICOLOGY OF NITRATES AND NITRITES WITH [Vol. 86 deaths of cattle and camels after drinking from water supplies containing a high nitrate con~entration.41,~2,~3’~~,~~,~~ In the majority of cases, the nitrate-nitrogen concentration was found to lie in the range 70 to 150 p.p.m., although three supplies containing as much as 870 p.p.m. have been re~orded.4~,*~ In a recent case, a supply found to contain 320 p.p.m. was fatal to cattle in about 14 hours after wa~tering.~~ Unfortunately, as far as the records show, the bodies of poisoned animals have not been examined by veterinary inspectors because the supplies have come from wells in remote areas, and any evidence has been lost before a post-mortem examination could be made. On the basis of available evidence, I introduced a tentative limit of 50p.p.m. of nitrate-nitrogen, which has been applied in recent years in Khartoum when making recommendations on the suitability of a particular supply.However, Ibrahim has pointed that the nitrate concentration of a water supply may fluctuate within a wide range, and he re-examines regularly any supply with a nitrate-nitrogen concentration in the range 30 to 50 p.p.m. W i l l i m ~ t t ~ ~ has also had experience of cattle poisoning in Cyprus caused by well waters containing high concentrations of nitrate. Pigs-Winks, Sutherland and Sa1isbu1-y~~ record heavy mortality among pigs fed on a soup made by cooking beef and offal in a well water containing 290 to 495 p.p.m.of nitrate- nitrogen and less than 1.5 p.p.m. of nitrite-nitrogen. The soup itself contained 80 to 285 p. p .m. of ni trit e-ni trogen . Fish-Sanborn48 states that water containing 2000 p.p.m. of sodium nitrate (330 p.p.m. of nitrate-nitrogen) is fatal to fish, whereas a concentration of 1000 p.p.m. of sodium nitrate (165 p.p.m. of nitrate-nitrogen) is without apparent effect. DISCUSSION ANI) CONCLUSIONS and animals, the following assumptions have been made- In deducing a suitable limit for nitrates in water to be consumed by infants, adults (i) that the nitrate is completely reduced to, and absorbed as, nitrite; (ii) that the lethal dose of nitrite-nitrogen for men and cattle is about 20 mg per kg; (iii) that one fifth of the lethal dose, i.e., 4- mg per kg, is the maximum daily amount of nitrite-nitrogen that can be tolerated without giving rise to toxic symptoms.When computing limits for cattle in tropical climates it should be remembered that in many places cattle are watered only once or twice a day, and that, as a consequence, large volumes are taken at a time. On the above basis the limits shown in Table I for the permissible concentrations of nitrate-nitrogen in drinking water for infants, adults and cattle in England and in the tropics have been calculated. TABLE I PERMISSIBLE LIMITS FOR NITRATE-NITROGEN I N DRINKING WATER Assumed Consumer weight, kg Infant . . 3 Infant . . 3 Adult .. 60 Adult . . 60 cow .. 500 Maximum permissible Approximate daily Region dose of nitrate-nitrogen, water intake, mg litres England 12 0.5 Tropics 12 2 England 240 1 Tropics 240 5.3 Tropics 2000 45 Permissible limit of nitrate-nitrogen in water, p.p.m. 24 6 240 45 45 These figures can only be approximate, but they give general support to the limits already suggested. They also show how very susceptible an infant artificially reared in the tropics can be to relatively small amounts of nitrates in drinking water. In areas where the water supplies have a high nitrate concentration special arrangements must be made for supplying infants with low- nitrate water. I t may also be concluded that, since the lethal doses of nitrate-nitrogen and nitrite- nitrogen for adults are not very different, most of the nitrate consumed is reduced to nitrite and absorbed into the blood stream.Thus it .would appear that the significant factor that makes infants susceptible to nitrates is the high relative fluid intake and not the gastric achlorhydria. They emphasise the variations caused by differences in climate. This is already being done in Norfolk and East S~ffolk.~July, 19611 PARTICULAR REFERENCE TO POTABILITY OF WATER SUPPLIES. A REVIEW 433 REFERENCES 1. 2. 3. 4. 3 . ti. - 8. 9. 10. 11. 12. 13. 14. 16. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 39. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. Cornblath, M., and Hartmann, A. F., J . Pediat., 1948, 33, 421. Luff, A. P., “Text Book of Forensic Medicine and Toxicology,” Longmans, Green and Co., London, Wood, E.C., personal communication. 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Edition, Saunders, Philadelphia. 1911, p. 305. Edition, J. & A. Churchill, London, 1928, p. 321. , , , Ibid., 1940, 96, 41. - ~ - Chem. Abstr., 1951, 45, 3507. Seventh Edition, J . & A. Churchill, London, 1958, p. 548. 32, 979. Editon, Edward Arnold (Publishers) Ltd., London, 1956, p. 399. -, Ibid., 1957/58, 8. Received Janziary 16th, 1961