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Nitrate
Nitrate AM Fan, California Environmental Protection Agency, Oakland, CA, USA Ó 2014 Elsevier Inc. All rights reserved. l
Name: Nitrate Chemical Abstracts Service Registry Number: 14797-55-8 l Molecular Formula: NO3 l Chemical Structure: l
O N+ –O
O–
compounds or oxidized to nitrate by chemical and biological processes. Nitrates occur naturally in soil from microbial oxidation of ammonia derived from organic nitrogenous materials such as plant proteins, animals, and animal excreta. Other source contributions are wastewater, septic tank runoffs, airborne nitrogen compounds emitted by industry and automobiles, nitrogen fertilizer, and manure from animal feeding. Nitrate in groundwater is generally found below 10 ppm, with higher levels in areas of high agricultural activities.
Background
Exposure and Exposure Monitoring
Nitrate is commonly found in drinking water sources especially in agricultural areas where nitrogen fertilizer is used, and where unregulated shallow private wells are more at the risk of contamination. The World Health Organization (WHO) guideline of 50 ppm and the US maximum contaminant level (MCL) of 45 ppm for nitrate in drinking water have been established for protecting infants from methemoglobinemia, commonly known as blue baby syndrome. The health protective value continues to be a subject of public health interest for many years, with varying opinion on whether it is too high or too low. Evaluation of nitrate will need to include consideration of nitrite because both are closely related in the nitrogen cycle in the environment and the body, and nitrite plays a major role in inducing toxicity after its formation from nitrate. More recently, reports of nitrate in drinking water, especially at levels higher than 50 ppm, have been associated with other health effects other than methemoglobinemia. This toxicological review provides an update on the health effects of nitrate with a focus on methemoglobinemia, reproductive and developmental effects, potential carcinogenicity, and especially endocrine/thyroid effects.
The contribution of drinking water to nitrate intake is usually less than 14%, food and drinking water are the major sources of exposure, especially food (green vegetables and cured meat) unless the water level is higher than 45 ppm. Overall, for an average adult consumer who lives in an area with high water contamination, total exposure to nitrate from food and water is estimated to be about 60–90 mg per person per day, of which at least 90% is from food. The intake of nitrate may reach 200 mg per person per day for a high vegetable consumer or when the water is higher than the MCL. For infants, an average daily intake of nitrates from consumption of vegetable-based foods was reported to be 7.8 mg. For bottle-fed infants, intake from milk formula made with water containing 50 mg l1 nitrate would average about 8.3–8.5 mg nitrate kg1 day1.
Uses Nitrate is used in fertilizers; in the manufacture of nitrites, nitrous oxide, explosives, pyrotechnics, matches, freezing mixtures, and special cements; as a coloring agent and preserving additive in food; for coagulation of latexes; in the nuclear industry; and for odor (sulfide) and corrosion control in aqueous systems.
Environmental Behavior, Fate, Routes, and Pathways Nitrate (NO3), a product of nitrogen oxidation, is a naturally occurring ion in the environment and integrated into complex organic molecules such as proteins and enzymes required by living systems. Nitrate is a more stable form of oxidized nitrogen than nitrite; however, it can be reduced by microbial action to nitrite, which, in turn, can be reduced to various
Encyclopedia of Toxicology, Volume 3
Toxicokinetics Nitrate is widely distributed in the body via the nitrogen cycle. Nitrate ingestion is followed by endogenous nitrate synthesis, release from blood to saliva, conversion of nitrate to nitrite by bacteria in saliva, conversion of nitrite to nitrate in blood, and ultimate excretion mainly as nitrate in the urine. Bioavailability from food or drinking water is at least 92%. Absorbed nitrate is rapidly transported via the blood. Plasma nitrate is dosedependently secreted by the salivary gland via an active transport mechanism shared with iodide and thiocyanate. This amounts to approximately 25% of the ingested nitrate in saliva, or 10 times the concentration in plasma. Nitrate is secreted by passive diffusion into breast milk. Of toxicological concern is the metabolism of nitrate to nitrite and N-nitroso compounds. Approximately 5–20% of the ingested nitrate is reduced by oral bacteria to nitrite, and the in vivo nitrite formed represents 80% of total nitrite exposure. Nitrate biosynthesis appears to be from nitric oxide to N2O3 and the reaction of N2O3 with water to yield nitrite. Nitrite is rapidly oxidized to nitrate through reaction with hemoglobin. Other cell types can also form nitric oxide, generally from arginine. Ingested nitrate can result in the formation of Nnitroso compounds with concomitant ingestion of nitrosation cofactors and precursors (e.g., protein). Such endogenous
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nitrosation can occur in the stomach, small intestine, and other parts of the body where the bacterial flora reduce nitrate to nitrite. Active secretion of nitrate and conversion of nitrate to nitrite in saliva occur in humans and most laboratory species except rats. The endogenous production of nitrate and its role in the nitric oxide pathway is beneficial for protecting against oral and gastrointestinal diseases and also for its role in vascular fitness and exerting antihypertensive effects.
Mechanism of Toxicity The acute oral LD50 values for sodium nitrate range from 2480 to 9000 mg kg1 in rats, mice, and rabbits. Acute, subchronic, and chronic animal toxicity studies showed low toxicity for nitrate as sodium or potassium nitrate. A long-term study showed a slight depression in growth rate. Nitrite, but not nitrate, is capable of inducing methemoglobinemia (see Nitrites, for more details). Nitrate has been reported to be associated with thyroid effects in experimental animals and humans. Possible mode of action includes inhibition of iodine uptake to thyroid, serum T3 and T4 changes, and tissue T3 changes. However, there is a lack of knowledge on the differences in the mode of action to permit animal-to-human extrapolation. While the data indicate humans and rats exhibit similar dose–response relationships in acute inhibition of thyroidal iodide uptake, they show differences in thyroid hormone response following iodide uptake inhibition. Comparative data are needed for serum and brain tissue levels of thyroid hormones and characterization of the dose–response relationship between changes of thyroid hormone levels and adverse effects. Early experimental and field studies in mammals have found inorganic nitrate to be goitrogenic. The effects were observed in rats following oral and parenteral administration of potassium and sodium nitrate, whereas antithyroid effects were also reported in sheep and pigs administered potassium nitrate. Nitrate exposure through diet or drinking water caused functional and histological changes to the thyroid gland in rats and pigs. More recent investigations between 2000 and 2010 reported changes in thyroid and thyroid activity following exposure to nitrate. In these more recent studies, nitrate exposure has consistently resulted in increases in thyroid weight and/or changes to the follicle cell; however, the reported thyroidal hormone changes have not been as consistent. The studies reported increased thyroid weights with a decrease in thyroid hormones (i.e., T3 and T4) and/or decrease in thyroid stimulating hormone. However, not all the results are consistent with the expected outcome of a sodium–iodide symporter (NIS) inhibitor, which can be seen as supplementation of iodine in the diet that did not result in thyroid changes. Overall, the data support that nitrate impairs thyroid function involving the hypothalamic–pituitary–adrenal axis.
Acute and Short-Term Toxicity The major acute toxicity concern is methemoglobinemia after oral ingestion. Ingested nitrate is reduced to nitrite in the
gastrointestinal tract and binds to hemoglobin to form methemoglobin. Nitrite absorbed in the blood stream is involved in the oxidation of hemoglobin to methemoglobin in which the iron has been oxidized from the ferrous to the ferric state. NO2 þ oxyhemoglobin Fe2þ / methemoglobin Fe3þ þ NO 3 Nitrate is a competitive inhibitor of iodide uptake to thyroid at the NIS. It binds to NIS on the surface of thyroid follicular cells that can result in depression of thyroid hormone depression. It is believed to have a common mode of action with some other contaminants. In rats tested for radioactive iodine uptake, nitrate was found to be the least potent when compared with perchlorate and thiocyanate on a molar basis. Perchlorate was 10 times more potent than thiocyanate and 300 times more potent than nitrate. When the relative potencies of the three anions were determined in Chinese hamster ovary cells, perchlorate inhibited 125I uptake at the NIS at 15, 30, and 240 times that of thiocyanate, iodide, and nitrate, respectively. It was noted that the results are consistent with a common mode of action by these anions of simple competitive interaction, in which any one of the anions, either individually or in a mixture of the three, is indistinguishable from a concentration or dilution of either one of the remaining two ions in inhibiting iodine uptake at the NIS. The available data indicate that humans and rats exhibit similar dose–response relationships in terms of acute inhibition of thyroidal iodide uptake, but there are notable differences in terms of thyroid hormone response, which lead to iodide uptake inhibition. When dose–response data for changes in serum T3, T4, and thyroid stimulating hormone (TSH) levels from studies in humans, rats, mice, and rabbits were analyzed, thyroid homeostasis in the rat appears to be much more sensitive to perchlorate than the other species. Rats showed an increase in serum TSH at 0.1 mg kg1 day1, whereas other species were still unresponsive at 10 mg kg1 day1. Less pronounced but consistent effects were seen with serum T3 and T4. These cross-species comparisons provide a basis for special consideration in evaluating rat studies for their relevance to humans.
Chronic Toxicity Associations of nitrate exposure and thyroid effects in humans have been reported in the literature since 1994, and are the subject of ongoing investigations and research. The consumption of drinking water containing nitrate at levels higher than 50 ppm has been associated with: (1) increased thyroid volume and subclinical thyroid disorders (thyroid hypoechogenicity by ultrasound, increased serum thyrotropin level, and positive thyroperoxidase antibodies in school children in Eastern Slovakia); (2) increased thyroid volume in healthy women in the Netherlands; (3) increased incidence of goiter in children in villages of Bulgaria; and (4) increased relative risk of thyroid disorder and goiter rates in pregnant women in Bulgaria. In a cohort study of 21 977 women in Iowa, the authors
Nitrate
reported an increased risk of thyroid cancer with higher average nitrate levels in water and with longer consumption of water exceeding 22 ppm nitrate, but no association between nitrate in drinking water and prevalence of hypothyroidism or hyperthyroidism. Increased dietary nitrate was also associated with an increased risk of thyroid cancer and with the prevalence of hypothyroidism. Studies that did not report a relationship between nitrate and thyroid changes included: (1) a study in 10 adult volunteers given sodium nitrate in drinking water that found no effect on thyroidal I131 uptake and plasma concentrations of thyroid hormones (T3, rT3, T4, and TSH); (2) a study in 3059 clinically healthy people aged 18–70 from different regions of Germany that found no influence of urinary nitrate excretion on the prevalence of goiter; (3) a study of 3772 subjects aged 20–79 from Pomerania/Germany that showed no diagnosed thyroid disorder; (4) a study of infants that found no association between cord blood levels of nitrate, thiocyanate, and perchlorate anions and newborn weight, length, and head circumferences; and (5) an analysis of the urinary levels of nitrate, perchlorate, and thiocyanate; urinary iodide concentration; serum levels of thyroid hormones (T4, TSH), albumin, cotinine, and c-reactive protein in 2299 men and women (aged 12 years and higher, noninstitutionalized) in the 2001–02 US National Health and Nutrition Examination Survey.
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control study in Prince Edward Island, Canada investigated the dose–response relationship between nitrate level and intrauterine growth restriction and prematurity. A case–control study examined all deliveries in Sweden for neural tube defects. A retrospective cohort study in Ostergotland County, Sweden investigated the association between water nitrate at or greater than 2 mg l1 and cardiac malformation using a geographic information system to link periconceptional or early pregnancy address to water supplies. A case–control study of counties along the Texas–Mexico border tested the association between increased water nitrate and spina bifida and anencephaly. A cross-sectional study in South Africa investigated the association between water from high nitrate regions and prematurity or size of South West Africa/Namibian infants based on samples taken from wells used at the time of home visit. A study investigated clusters of spontaneous abortion reported in Le Grange County, Indiana, after which the water was tested for nitrate.
Genotoxicity Genotoxicity studies showed mostly negative responses for nitrate.
Carcinogenicity Reproductive Toxicity In four teratogenicity studies by the US Food and Drug Administration (FDA), sodium and potassium nitrate were given orally at four dose levels to pregnant hamsters, mice, rats, and rabbits. No teratogenicity, soft or skeletal tissue abnormalities, or other effects were observed on nidation, maternal or fetal survival, fetal toxicity, malformations, or maternal reproductive effects. Testicular toxicity has been reported in rats and mice following subchronic exposure to nitrate. Epidemiological studies have suggested an association between exposure to nitrate in drinking water and spontaneous abortions, intrauterine growth restrictions, and birth defects, but no clear exposure–response relationship can be established. A case–control study in New Brunswick, Canada examined the relationship between maternal exposure to nitrates in drinking water and risk of delivering an infant with a central nervous system malformation. A population-based case–control study in California investigated the association between maternal periconceptional exposure to nitrate from drinking water and diet and risk for neural tube defects. A study investigated the relationship between community drinking water quality and spontaneous abortion in patients who entered Boston Hospital for Women Division of Brigham and Women’s Hospital in Massachusetts. A case–control study in South Australia investigated malformation (mainly neural tube defects and defects affecting multiple systems) in women and association with drinking water consumption from specific sources differing in nitrate content. A case–control study in the Mount Gambier region of South Australia investigated the relationship between mothers’ maternal drinking water source and malformations in offspring as a follow-up study. A case–
Sodium and potassium nitrate have been tested for potential carcinogenicity, alone and in combination with nitrosatable compounds. Nitrosating agents can be ingested from food and drinking water, and synthesized from ingested nitrate and nitrite. Nitrosating agents can react under certain conditions with nitrosatable compounds to form N-nitrosamines and N-nitrosamides, some of which are animal carcinogens. Nitrosating agents (e.g., nitrous acid and nitrous anhydride) that arise from nitrite under acidic gastric conditions can react with amines or amides to form nitrosamines or nitrosamides, and the induction of tumors in animals via endogenous synthesis of N-nitroso compounds has been demonstrated. Nitrosamines need to be activated metabolically by cytochrome P450 enzymes to electrophilic intermediates to exert a carcinogenic effect, while nitrosamides are direct-acting carcinogens. Ascorbic acid is an inhibitor of nitrosation reactions. It has been shown to lower the incidence of tumors in animal experiments, and reduce the risk for cancer that is associated with ingested nitrite in epidemiological studies. NTP chronic bioassay studies in rats and male mice did not show carcinogenicity where nitrate was administered alone in drinking water or diet (three studies in mice and four studies in rats) or was coadministered with nitrosatable compounds, and when nitrite was given alone in the diet by gavage or in the drinking water to rats and mice. Various ecological studies, case–control studies, and cohort studies conducted worldwide on the relationship between human exposure to nitrate and the risk for various cancers reported inconsistent results. These were reviewed by the International Agency for Research in Cancer (IARC) and included studies of: (1) cancers of the colon, liver, pancreas, and rectum
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Nitrate
in Canada, China, Slovakia, and Thailand; (2) leukemia and lymphoma in Canada, China, Egypt, Finland, Italy, Slovakia, the United Kingdom, and the USA; (3) gastric and esophageal tumors in China, Columbia, Costa Rica, Denmark, Japan, Netherlands, Poland, Scotland, Spain, Sweden, the United Kingdom, and the USA; (4) tumors of the nervous system, mainly brain, in Australia, Canada, England, France, Germany, Israel, and the USA; and (5) genital and urinary tract tumors in Denmark, Egypt, Germany, Slovakia, Spain, and the USA. An increased risk of non-Hodgkin’s lymphoma and urinary bladder was reported in some studies but not others at similar exposure levels of nitrate in drinking water. Meta-analysis of prospective, case–control, and cohort studies reported greater risks for colorectal cancer associated with consumption of processed meat. IARC concluded that ingested nitrate under conditions that result in endogenous nitrosation is probably carcinogenic to humans (Group 2A). The underlying mechanism for the carcinogenicity determination is endogenous nitrosation that results in the formation of N-nitroso compounds, some of which are known carcinogens. There is an active endogenous nitrogen cycle in humans wherein nitrosating agents that arise from nitrite under acidic gastric conditions react readily with nitrosatable compounds, especially secondary amines and amides, to generate N-nitroso compounds. These nitrosating conditions are enhanced following ingestion of additional nitrate, nitrite, or nitrosatable compounds. Specifically, for nitrate alone, IARC found that there is inadequate evidence in humans for the carcinogenicity of nitrate in food. There is inadequate evidence in humans for the carcinogenicity of nitrate in drinking water. There is inadequate evidence in experimental animals for the carcinogenicity of nitrate. For nitrite, in food, IARC found that it is associated with an increased incidence of stomach cancer. There is sufficient evidence in experimental animals for the carcinogenicity of nitrite in combination with amines or amides. There is limited evidence in experimental animals for the carcinogenicity of nitrite per se. More recent studies after the IARC evaluation did not report an association between nitrate in water and nonHodgkin lymphoma, breast, bladder, colon, urinary, and pancreatic cancer. Studies on dietary intake of nitrate/nitrite reported some associations with increased risk of bladder cancer, esophageal squamous cell carcinomas, colorectal cancer, and thyroid cancer. Studies in Iowa reported no association to renal cell carcinoma (>5 and >10 ppm nitrateN); no association with non-Hodgkin lymphoma (below 3 ppm); and increased risk of thyroid cancer in older women (>5 ppm nitrate for 5 years or longer, relative risk ¼ 2.6, 95% confidence interval ¼ 1.6–6.2) (no association with prevalence of hypothyroidism or hyperthyroidism). Study on dietary intake of nitrate and nitrite (National Institutes of Health–American Association of Retired Persons Diet and Health Study) suggested a role and further studies on ovarian and thyroid cancer risk and pancreatic cancer in men. Overall, interpretation of the data is complicated by various factors such as the amount of nitrate/nitrite ingested, the concomitant ingestion of nitrosation cofactors and precursors, specific factors that increase nitrosation, and some study limitations.
Carcinogenicity Carcinogenicity is a possible endpoint of concern because of the biological plausibility of endogenous nitrosation of ingested nitrate and nitrite, with the conversion of nitrate to nitrite, and formation of genotoxic/carcinogenic N-nitroso compounds, such as N-nitrosamines and N-nitrosamides, some of which are known carcinogens. The mechanism may involve acid-catalyzed (e.g., in acidic stomach) or cell-mediated (such as bacteria and immune cell, neutral pH) formation. In humans, nitrite swallowed in saliva is usually first converted in the stomach to nitrous acid (HNO2) which is spontaneously converted to the active nitrosating species nitrous anhydride (N2O3). Nitrous anhydride is a strong nitrosating agent, which donates NOþ to secondary and tertiary amines to form nitrosamines. Nitrous acid can also be protonated to nitrous acidium ion, which reacts directly with neutral amides to form nitrosamides. About 40–70% of total human exposure to Nnitroso compounds is from endogenous formation.
Clinical Management of Methemoglobinemia People may have lifelong methemoglobinemia and can be asymptomatic at relatively high levels. Methemoglobinemia is a side effect of nitric oxide therapy for acute respiratory stress syndrome and persistent hypertension in newborns. Rapidity of methemoglobin formation may lead to severe symptoms. Acquired methemoglobinemia can also result from exposure to other chemicals and pharmaceuticals. People with glucose-6phoshate dehydrogenase deficiency and infants aged 6 months and less are at increased risk. Methemoglobinemia should be suspected in patients with central cyanosis and low oxygen saturations, which are unresponsive to oxygen therapy. Treatment should be guided by the severity of the symptoms initially, aimed at decreasing the level of methemoglobin found in the blood, and accompanied by removal from exposure. Methylene blue is used when significant symptoms are present (dizziness, confusion, seizure, somnolence, and headache), with clinical observation of lowering methemoglobin levels. However, methylene blue will not be responsive in patients with G6PD deficiency, and it has not been FDA approved for the pediatric population.
Other Health Effects Associations between nitrate in drinking water and hypertension, immunologic effects, recurrent respiratory tract infection, diarrhea, and childhood onset of diabetes mellitus have been reported. The data are very limited and not convincing, and results were conflicting in studies of childhood diabetes mellitus.
Exposure Standards and Guidelines The WHO guideline for nitrate in drinking water is 50 ppm. The US MCL for nitrate in drinking water is 45 ppm as nitrate, or 10 ppm as nitrate-N, and 1 ppm for nitrite as nitrite-N. The combined MCL is 10 ppm nitrate/nitrite-N.
Nitrate
See also: Ammonium Nitrate; Butyl Nitrite; Nitrites; Amyl Nitrite; Nitrite Inhalants.
Further Reading Aly, H.A., Mansour, A.M., Abo-Salem, O.M., Abd-Ellah, H.F., Abdel-Naim, A.B., 2010. Potential testicular toxicity of sodium nitrate in adult rats. Food Chem. Toxicol. 48 (2), 572–578. Aschebrook-Kilfoy, B., Cross, A., Stolzenberg-Solomon, R.Z., Schatzkin, A., Hollenbeck, A.R., Sinha, R., Ward, M.H., August 1, 2011. Pancreatic cancer and exposure to dietary nitrate and nitrite in the NIH-AARP diet and health study. Am. J. Epidemiol. 174 (3), 305–315. Aschebrook-Kilfoy, B., Ward, M.H., Gierach, G.L., Schatzkin, A., Hollenbeck, A.R., Sinha, R., Cross, A.J., 2012. Epithelial ovarian cancer and exposure to dietary nitrate and nitrite in the NIH-AARP Diet and Health Study. Eur. J. Cancer Prev. 21 (1), 65–72. Eskiocak, S., Dundar, C., Basoglu, T., Altaner, S., 2005. The effects of taking chronic nitrate by drinking water on thyroid functions and morphology. Clin. Exp. Med. 5, 66–71. Fan, A.M., 2011. Nitrate and nitrite in drinking water: a toxicological review. In: Nriagu, J.O. (Ed.), Encyclopedia of Environmental Health, vol. 4. Elsevier, Burlington, pp. 137–145.
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Fan, A.M., Steinberg, V.E., 1996. Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. Reg. Toxicol. Pharmacol. 23, 35–43. Kilfoy, B.A., Zhang, Y., Park, Y., Holford, T.R., Schatzkin, A., Hollenbeck, A., Ward, M.H., 2011. Dietary nitrate and nitrite and the risk of thyroid cancer in the NIH-AARP Diet and Health Study. Int. J. Cancer 129 (1), 160–172. L’hirondel, J., L’hirondel, J.L., 2001. Nitrate and Man, Toxic, Harmless or Beneficial? CABI Publishing, Wallingford, UK. Ward, M.H., Kilfoy, B.A., Weyer, P.J., Anderson, K.E., Folsom, A.R., Cerhan, J.R., 2010. Nitrate intake and the risk of thyroid cancer and thyroid disease. Epidemiology 21 (3), 389–395. Zaki, A., Ait Chaoui, A., Talibi, A., Derouiche, A.F., Aboussaouira, T., Zarrouck, K., Chait, A., Himmia, T., 2004. Impact of nitrate intake in drinking water on the thyroid gland activity in male rat. Toxicol. Lett. 147, 27–33.
Relevant Website http://www.who.int/water_sanitation_health/dwq/chemicals/nitratenitrite2ndadd.pdf – Background Document for Development of WHO Guidelines for Drinking-water Quality. World Health Organization, Geneva, Switzerland.
Name: Nitrate Chemical Abstracts Service Registry Number: 14797-55-8 l Molecular Formula: NO3 l Chemical Structure: l
O N+ –O
O–
compounds or oxidized to nitrate by chemical and biological processes. Nitrates occur naturally in soil from microbial oxidation of ammonia derived from organic nitrogenous materials such as plant proteins, animals, and animal excreta. Other source contributions are wastewater, septic tank runoffs, airborne nitrogen compounds emitted by industry and automobiles, nitrogen fertilizer, and manure from animal feeding. Nitrate in groundwater is generally found below 10 ppm, with higher levels in areas of high agricultural activities.
Background
Exposure and Exposure Monitoring
Nitrate is commonly found in drinking water sources especially in agricultural areas where nitrogen fertilizer is used, and where unregulated shallow private wells are more at the risk of contamination. The World Health Organization (WHO) guideline of 50 ppm and the US maximum contaminant level (MCL) of 45 ppm for nitrate in drinking water have been established for protecting infants from methemoglobinemia, commonly known as blue baby syndrome. The health protective value continues to be a subject of public health interest for many years, with varying opinion on whether it is too high or too low. Evaluation of nitrate will need to include consideration of nitrite because both are closely related in the nitrogen cycle in the environment and the body, and nitrite plays a major role in inducing toxicity after its formation from nitrate. More recently, reports of nitrate in drinking water, especially at levels higher than 50 ppm, have been associated with other health effects other than methemoglobinemia. This toxicological review provides an update on the health effects of nitrate with a focus on methemoglobinemia, reproductive and developmental effects, potential carcinogenicity, and especially endocrine/thyroid effects.
The contribution of drinking water to nitrate intake is usually less than 14%, food and drinking water are the major sources of exposure, especially food (green vegetables and cured meat) unless the water level is higher than 45 ppm. Overall, for an average adult consumer who lives in an area with high water contamination, total exposure to nitrate from food and water is estimated to be about 60–90 mg per person per day, of which at least 90% is from food. The intake of nitrate may reach 200 mg per person per day for a high vegetable consumer or when the water is higher than the MCL. For infants, an average daily intake of nitrates from consumption of vegetable-based foods was reported to be 7.8 mg. For bottle-fed infants, intake from milk formula made with water containing 50 mg l1 nitrate would average about 8.3–8.5 mg nitrate kg1 day1.
Uses Nitrate is used in fertilizers; in the manufacture of nitrites, nitrous oxide, explosives, pyrotechnics, matches, freezing mixtures, and special cements; as a coloring agent and preserving additive in food; for coagulation of latexes; in the nuclear industry; and for odor (sulfide) and corrosion control in aqueous systems.
Environmental Behavior, Fate, Routes, and Pathways Nitrate (NO3), a product of nitrogen oxidation, is a naturally occurring ion in the environment and integrated into complex organic molecules such as proteins and enzymes required by living systems. Nitrate is a more stable form of oxidized nitrogen than nitrite; however, it can be reduced by microbial action to nitrite, which, in turn, can be reduced to various
Encyclopedia of Toxicology, Volume 3
Toxicokinetics Nitrate is widely distributed in the body via the nitrogen cycle. Nitrate ingestion is followed by endogenous nitrate synthesis, release from blood to saliva, conversion of nitrate to nitrite by bacteria in saliva, conversion of nitrite to nitrate in blood, and ultimate excretion mainly as nitrate in the urine. Bioavailability from food or drinking water is at least 92%. Absorbed nitrate is rapidly transported via the blood. Plasma nitrate is dosedependently secreted by the salivary gland via an active transport mechanism shared with iodide and thiocyanate. This amounts to approximately 25% of the ingested nitrate in saliva, or 10 times the concentration in plasma. Nitrate is secreted by passive diffusion into breast milk. Of toxicological concern is the metabolism of nitrate to nitrite and N-nitroso compounds. Approximately 5–20% of the ingested nitrate is reduced by oral bacteria to nitrite, and the in vivo nitrite formed represents 80% of total nitrite exposure. Nitrate biosynthesis appears to be from nitric oxide to N2O3 and the reaction of N2O3 with water to yield nitrite. Nitrite is rapidly oxidized to nitrate through reaction with hemoglobin. Other cell types can also form nitric oxide, generally from arginine. Ingested nitrate can result in the formation of Nnitroso compounds with concomitant ingestion of nitrosation cofactors and precursors (e.g., protein). Such endogenous
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nitrosation can occur in the stomach, small intestine, and other parts of the body where the bacterial flora reduce nitrate to nitrite. Active secretion of nitrate and conversion of nitrate to nitrite in saliva occur in humans and most laboratory species except rats. The endogenous production of nitrate and its role in the nitric oxide pathway is beneficial for protecting against oral and gastrointestinal diseases and also for its role in vascular fitness and exerting antihypertensive effects.
Mechanism of Toxicity The acute oral LD50 values for sodium nitrate range from 2480 to 9000 mg kg1 in rats, mice, and rabbits. Acute, subchronic, and chronic animal toxicity studies showed low toxicity for nitrate as sodium or potassium nitrate. A long-term study showed a slight depression in growth rate. Nitrite, but not nitrate, is capable of inducing methemoglobinemia (see Nitrites, for more details). Nitrate has been reported to be associated with thyroid effects in experimental animals and humans. Possible mode of action includes inhibition of iodine uptake to thyroid, serum T3 and T4 changes, and tissue T3 changes. However, there is a lack of knowledge on the differences in the mode of action to permit animal-to-human extrapolation. While the data indicate humans and rats exhibit similar dose–response relationships in acute inhibition of thyroidal iodide uptake, they show differences in thyroid hormone response following iodide uptake inhibition. Comparative data are needed for serum and brain tissue levels of thyroid hormones and characterization of the dose–response relationship between changes of thyroid hormone levels and adverse effects. Early experimental and field studies in mammals have found inorganic nitrate to be goitrogenic. The effects were observed in rats following oral and parenteral administration of potassium and sodium nitrate, whereas antithyroid effects were also reported in sheep and pigs administered potassium nitrate. Nitrate exposure through diet or drinking water caused functional and histological changes to the thyroid gland in rats and pigs. More recent investigations between 2000 and 2010 reported changes in thyroid and thyroid activity following exposure to nitrate. In these more recent studies, nitrate exposure has consistently resulted in increases in thyroid weight and/or changes to the follicle cell; however, the reported thyroidal hormone changes have not been as consistent. The studies reported increased thyroid weights with a decrease in thyroid hormones (i.e., T3 and T4) and/or decrease in thyroid stimulating hormone. However, not all the results are consistent with the expected outcome of a sodium–iodide symporter (NIS) inhibitor, which can be seen as supplementation of iodine in the diet that did not result in thyroid changes. Overall, the data support that nitrate impairs thyroid function involving the hypothalamic–pituitary–adrenal axis.
Acute and Short-Term Toxicity The major acute toxicity concern is methemoglobinemia after oral ingestion. Ingested nitrate is reduced to nitrite in the
gastrointestinal tract and binds to hemoglobin to form methemoglobin. Nitrite absorbed in the blood stream is involved in the oxidation of hemoglobin to methemoglobin in which the iron has been oxidized from the ferrous to the ferric state. NO2 þ oxyhemoglobin Fe2þ / methemoglobin Fe3þ þ NO 3 Nitrate is a competitive inhibitor of iodide uptake to thyroid at the NIS. It binds to NIS on the surface of thyroid follicular cells that can result in depression of thyroid hormone depression. It is believed to have a common mode of action with some other contaminants. In rats tested for radioactive iodine uptake, nitrate was found to be the least potent when compared with perchlorate and thiocyanate on a molar basis. Perchlorate was 10 times more potent than thiocyanate and 300 times more potent than nitrate. When the relative potencies of the three anions were determined in Chinese hamster ovary cells, perchlorate inhibited 125I uptake at the NIS at 15, 30, and 240 times that of thiocyanate, iodide, and nitrate, respectively. It was noted that the results are consistent with a common mode of action by these anions of simple competitive interaction, in which any one of the anions, either individually or in a mixture of the three, is indistinguishable from a concentration or dilution of either one of the remaining two ions in inhibiting iodine uptake at the NIS. The available data indicate that humans and rats exhibit similar dose–response relationships in terms of acute inhibition of thyroidal iodide uptake, but there are notable differences in terms of thyroid hormone response, which lead to iodide uptake inhibition. When dose–response data for changes in serum T3, T4, and thyroid stimulating hormone (TSH) levels from studies in humans, rats, mice, and rabbits were analyzed, thyroid homeostasis in the rat appears to be much more sensitive to perchlorate than the other species. Rats showed an increase in serum TSH at 0.1 mg kg1 day1, whereas other species were still unresponsive at 10 mg kg1 day1. Less pronounced but consistent effects were seen with serum T3 and T4. These cross-species comparisons provide a basis for special consideration in evaluating rat studies for their relevance to humans.
Chronic Toxicity Associations of nitrate exposure and thyroid effects in humans have been reported in the literature since 1994, and are the subject of ongoing investigations and research. The consumption of drinking water containing nitrate at levels higher than 50 ppm has been associated with: (1) increased thyroid volume and subclinical thyroid disorders (thyroid hypoechogenicity by ultrasound, increased serum thyrotropin level, and positive thyroperoxidase antibodies in school children in Eastern Slovakia); (2) increased thyroid volume in healthy women in the Netherlands; (3) increased incidence of goiter in children in villages of Bulgaria; and (4) increased relative risk of thyroid disorder and goiter rates in pregnant women in Bulgaria. In a cohort study of 21 977 women in Iowa, the authors
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reported an increased risk of thyroid cancer with higher average nitrate levels in water and with longer consumption of water exceeding 22 ppm nitrate, but no association between nitrate in drinking water and prevalence of hypothyroidism or hyperthyroidism. Increased dietary nitrate was also associated with an increased risk of thyroid cancer and with the prevalence of hypothyroidism. Studies that did not report a relationship between nitrate and thyroid changes included: (1) a study in 10 adult volunteers given sodium nitrate in drinking water that found no effect on thyroidal I131 uptake and plasma concentrations of thyroid hormones (T3, rT3, T4, and TSH); (2) a study in 3059 clinically healthy people aged 18–70 from different regions of Germany that found no influence of urinary nitrate excretion on the prevalence of goiter; (3) a study of 3772 subjects aged 20–79 from Pomerania/Germany that showed no diagnosed thyroid disorder; (4) a study of infants that found no association between cord blood levels of nitrate, thiocyanate, and perchlorate anions and newborn weight, length, and head circumferences; and (5) an analysis of the urinary levels of nitrate, perchlorate, and thiocyanate; urinary iodide concentration; serum levels of thyroid hormones (T4, TSH), albumin, cotinine, and c-reactive protein in 2299 men and women (aged 12 years and higher, noninstitutionalized) in the 2001–02 US National Health and Nutrition Examination Survey.
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control study in Prince Edward Island, Canada investigated the dose–response relationship between nitrate level and intrauterine growth restriction and prematurity. A case–control study examined all deliveries in Sweden for neural tube defects. A retrospective cohort study in Ostergotland County, Sweden investigated the association between water nitrate at or greater than 2 mg l1 and cardiac malformation using a geographic information system to link periconceptional or early pregnancy address to water supplies. A case–control study of counties along the Texas–Mexico border tested the association between increased water nitrate and spina bifida and anencephaly. A cross-sectional study in South Africa investigated the association between water from high nitrate regions and prematurity or size of South West Africa/Namibian infants based on samples taken from wells used at the time of home visit. A study investigated clusters of spontaneous abortion reported in Le Grange County, Indiana, after which the water was tested for nitrate.
Genotoxicity Genotoxicity studies showed mostly negative responses for nitrate.
Carcinogenicity Reproductive Toxicity In four teratogenicity studies by the US Food and Drug Administration (FDA), sodium and potassium nitrate were given orally at four dose levels to pregnant hamsters, mice, rats, and rabbits. No teratogenicity, soft or skeletal tissue abnormalities, or other effects were observed on nidation, maternal or fetal survival, fetal toxicity, malformations, or maternal reproductive effects. Testicular toxicity has been reported in rats and mice following subchronic exposure to nitrate. Epidemiological studies have suggested an association between exposure to nitrate in drinking water and spontaneous abortions, intrauterine growth restrictions, and birth defects, but no clear exposure–response relationship can be established. A case–control study in New Brunswick, Canada examined the relationship between maternal exposure to nitrates in drinking water and risk of delivering an infant with a central nervous system malformation. A population-based case–control study in California investigated the association between maternal periconceptional exposure to nitrate from drinking water and diet and risk for neural tube defects. A study investigated the relationship between community drinking water quality and spontaneous abortion in patients who entered Boston Hospital for Women Division of Brigham and Women’s Hospital in Massachusetts. A case–control study in South Australia investigated malformation (mainly neural tube defects and defects affecting multiple systems) in women and association with drinking water consumption from specific sources differing in nitrate content. A case–control study in the Mount Gambier region of South Australia investigated the relationship between mothers’ maternal drinking water source and malformations in offspring as a follow-up study. A case–
Sodium and potassium nitrate have been tested for potential carcinogenicity, alone and in combination with nitrosatable compounds. Nitrosating agents can be ingested from food and drinking water, and synthesized from ingested nitrate and nitrite. Nitrosating agents can react under certain conditions with nitrosatable compounds to form N-nitrosamines and N-nitrosamides, some of which are animal carcinogens. Nitrosating agents (e.g., nitrous acid and nitrous anhydride) that arise from nitrite under acidic gastric conditions can react with amines or amides to form nitrosamines or nitrosamides, and the induction of tumors in animals via endogenous synthesis of N-nitroso compounds has been demonstrated. Nitrosamines need to be activated metabolically by cytochrome P450 enzymes to electrophilic intermediates to exert a carcinogenic effect, while nitrosamides are direct-acting carcinogens. Ascorbic acid is an inhibitor of nitrosation reactions. It has been shown to lower the incidence of tumors in animal experiments, and reduce the risk for cancer that is associated with ingested nitrite in epidemiological studies. NTP chronic bioassay studies in rats and male mice did not show carcinogenicity where nitrate was administered alone in drinking water or diet (three studies in mice and four studies in rats) or was coadministered with nitrosatable compounds, and when nitrite was given alone in the diet by gavage or in the drinking water to rats and mice. Various ecological studies, case–control studies, and cohort studies conducted worldwide on the relationship between human exposure to nitrate and the risk for various cancers reported inconsistent results. These were reviewed by the International Agency for Research in Cancer (IARC) and included studies of: (1) cancers of the colon, liver, pancreas, and rectum
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in Canada, China, Slovakia, and Thailand; (2) leukemia and lymphoma in Canada, China, Egypt, Finland, Italy, Slovakia, the United Kingdom, and the USA; (3) gastric and esophageal tumors in China, Columbia, Costa Rica, Denmark, Japan, Netherlands, Poland, Scotland, Spain, Sweden, the United Kingdom, and the USA; (4) tumors of the nervous system, mainly brain, in Australia, Canada, England, France, Germany, Israel, and the USA; and (5) genital and urinary tract tumors in Denmark, Egypt, Germany, Slovakia, Spain, and the USA. An increased risk of non-Hodgkin’s lymphoma and urinary bladder was reported in some studies but not others at similar exposure levels of nitrate in drinking water. Meta-analysis of prospective, case–control, and cohort studies reported greater risks for colorectal cancer associated with consumption of processed meat. IARC concluded that ingested nitrate under conditions that result in endogenous nitrosation is probably carcinogenic to humans (Group 2A). The underlying mechanism for the carcinogenicity determination is endogenous nitrosation that results in the formation of N-nitroso compounds, some of which are known carcinogens. There is an active endogenous nitrogen cycle in humans wherein nitrosating agents that arise from nitrite under acidic gastric conditions react readily with nitrosatable compounds, especially secondary amines and amides, to generate N-nitroso compounds. These nitrosating conditions are enhanced following ingestion of additional nitrate, nitrite, or nitrosatable compounds. Specifically, for nitrate alone, IARC found that there is inadequate evidence in humans for the carcinogenicity of nitrate in food. There is inadequate evidence in humans for the carcinogenicity of nitrate in drinking water. There is inadequate evidence in experimental animals for the carcinogenicity of nitrate. For nitrite, in food, IARC found that it is associated with an increased incidence of stomach cancer. There is sufficient evidence in experimental animals for the carcinogenicity of nitrite in combination with amines or amides. There is limited evidence in experimental animals for the carcinogenicity of nitrite per se. More recent studies after the IARC evaluation did not report an association between nitrate in water and nonHodgkin lymphoma, breast, bladder, colon, urinary, and pancreatic cancer. Studies on dietary intake of nitrate/nitrite reported some associations with increased risk of bladder cancer, esophageal squamous cell carcinomas, colorectal cancer, and thyroid cancer. Studies in Iowa reported no association to renal cell carcinoma (>5 and >10 ppm nitrateN); no association with non-Hodgkin lymphoma (below 3 ppm); and increased risk of thyroid cancer in older women (>5 ppm nitrate for 5 years or longer, relative risk ¼ 2.6, 95% confidence interval ¼ 1.6–6.2) (no association with prevalence of hypothyroidism or hyperthyroidism). Study on dietary intake of nitrate and nitrite (National Institutes of Health–American Association of Retired Persons Diet and Health Study) suggested a role and further studies on ovarian and thyroid cancer risk and pancreatic cancer in men. Overall, interpretation of the data is complicated by various factors such as the amount of nitrate/nitrite ingested, the concomitant ingestion of nitrosation cofactors and precursors, specific factors that increase nitrosation, and some study limitations.
Carcinogenicity Carcinogenicity is a possible endpoint of concern because of the biological plausibility of endogenous nitrosation of ingested nitrate and nitrite, with the conversion of nitrate to nitrite, and formation of genotoxic/carcinogenic N-nitroso compounds, such as N-nitrosamines and N-nitrosamides, some of which are known carcinogens. The mechanism may involve acid-catalyzed (e.g., in acidic stomach) or cell-mediated (such as bacteria and immune cell, neutral pH) formation. In humans, nitrite swallowed in saliva is usually first converted in the stomach to nitrous acid (HNO2) which is spontaneously converted to the active nitrosating species nitrous anhydride (N2O3). Nitrous anhydride is a strong nitrosating agent, which donates NOþ to secondary and tertiary amines to form nitrosamines. Nitrous acid can also be protonated to nitrous acidium ion, which reacts directly with neutral amides to form nitrosamides. About 40–70% of total human exposure to Nnitroso compounds is from endogenous formation.
Clinical Management of Methemoglobinemia People may have lifelong methemoglobinemia and can be asymptomatic at relatively high levels. Methemoglobinemia is a side effect of nitric oxide therapy for acute respiratory stress syndrome and persistent hypertension in newborns. Rapidity of methemoglobin formation may lead to severe symptoms. Acquired methemoglobinemia can also result from exposure to other chemicals and pharmaceuticals. People with glucose-6phoshate dehydrogenase deficiency and infants aged 6 months and less are at increased risk. Methemoglobinemia should be suspected in patients with central cyanosis and low oxygen saturations, which are unresponsive to oxygen therapy. Treatment should be guided by the severity of the symptoms initially, aimed at decreasing the level of methemoglobin found in the blood, and accompanied by removal from exposure. Methylene blue is used when significant symptoms are present (dizziness, confusion, seizure, somnolence, and headache), with clinical observation of lowering methemoglobin levels. However, methylene blue will not be responsive in patients with G6PD deficiency, and it has not been FDA approved for the pediatric population.
Other Health Effects Associations between nitrate in drinking water and hypertension, immunologic effects, recurrent respiratory tract infection, diarrhea, and childhood onset of diabetes mellitus have been reported. The data are very limited and not convincing, and results were conflicting in studies of childhood diabetes mellitus.
Exposure Standards and Guidelines The WHO guideline for nitrate in drinking water is 50 ppm. The US MCL for nitrate in drinking water is 45 ppm as nitrate, or 10 ppm as nitrate-N, and 1 ppm for nitrite as nitrite-N. The combined MCL is 10 ppm nitrate/nitrite-N.
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See also: Ammonium Nitrate; Butyl Nitrite; Nitrites; Amyl Nitrite; Nitrite Inhalants.
Further Reading Aly, H.A., Mansour, A.M., Abo-Salem, O.M., Abd-Ellah, H.F., Abdel-Naim, A.B., 2010. Potential testicular toxicity of sodium nitrate in adult rats. Food Chem. Toxicol. 48 (2), 572–578. Aschebrook-Kilfoy, B., Cross, A., Stolzenberg-Solomon, R.Z., Schatzkin, A., Hollenbeck, A.R., Sinha, R., Ward, M.H., August 1, 2011. Pancreatic cancer and exposure to dietary nitrate and nitrite in the NIH-AARP diet and health study. Am. J. Epidemiol. 174 (3), 305–315. Aschebrook-Kilfoy, B., Ward, M.H., Gierach, G.L., Schatzkin, A., Hollenbeck, A.R., Sinha, R., Cross, A.J., 2012. Epithelial ovarian cancer and exposure to dietary nitrate and nitrite in the NIH-AARP Diet and Health Study. Eur. J. Cancer Prev. 21 (1), 65–72. Eskiocak, S., Dundar, C., Basoglu, T., Altaner, S., 2005. The effects of taking chronic nitrate by drinking water on thyroid functions and morphology. Clin. Exp. Med. 5, 66–71. Fan, A.M., 2011. Nitrate and nitrite in drinking water: a toxicological review. In: Nriagu, J.O. (Ed.), Encyclopedia of Environmental Health, vol. 4. Elsevier, Burlington, pp. 137–145.
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Fan, A.M., Steinberg, V.E., 1996. Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. Reg. Toxicol. Pharmacol. 23, 35–43. Kilfoy, B.A., Zhang, Y., Park, Y., Holford, T.R., Schatzkin, A., Hollenbeck, A., Ward, M.H., 2011. Dietary nitrate and nitrite and the risk of thyroid cancer in the NIH-AARP Diet and Health Study. Int. J. Cancer 129 (1), 160–172. L’hirondel, J., L’hirondel, J.L., 2001. Nitrate and Man, Toxic, Harmless or Beneficial? CABI Publishing, Wallingford, UK. Ward, M.H., Kilfoy, B.A., Weyer, P.J., Anderson, K.E., Folsom, A.R., Cerhan, J.R., 2010. Nitrate intake and the risk of thyroid cancer and thyroid disease. Epidemiology 21 (3), 389–395. Zaki, A., Ait Chaoui, A., Talibi, A., Derouiche, A.F., Aboussaouira, T., Zarrouck, K., Chait, A., Himmia, T., 2004. Impact of nitrate intake in drinking water on the thyroid gland activity in male rat. Toxicol. Lett. 147, 27–33.
Relevant Website http://www.who.int/water_sanitation_health/dwq/chemicals/nitratenitrite2ndadd.pdf – Background Document for Development of WHO Guidelines for Drinking-water Quality. World Health Organization, Geneva, Switzerland.