Research Related to the Effects of Elevated Nitrates/Nitrites in Drinking Water

Ward, L.B. Yakima Herald Republic. “Hidden Wells, Dirty Water” describes the nitrate situation in Outlook in 2009. Outlook Elementary School had to drill a new well at a cost of $48,000. http://www.yakima-herald.com/stories/2008/10/11/hidden-wells-dirty-water

U.S. Agency for Toxic Substances and Diseases Registry. 2001. "Case Studies in Environmental Medicine: Nitrate/Nitrite Toxicity." http://www.atsdr.cdc.gov/HEC/CSEM/nitrate/docs/nitrate_nitrite.pdf

World Health Organization. 2006. "International Program on Chemical Safety, Environmental Health Criteria 5: Nitrates, Nitrites, and N-Nitroso Compounds." http://www.inchem.org/documents/pims/chemical/pimg016.htm

Laitinen, S., et al. 1993. "Calculated dietary intakes of nitrate and nitrite by young Finns." Food Addit.Contam 10(4):469-477. http://www.ncbi.nlm.nih.gov/pubmed/8405586

Abstract Dietary intakes of nitrate and nitrite of 1212 Finns aged 9, 12, 15, 18, 21, and 24 years were calculated using food consumption data obtained by the 48-hour recall method in 1986, in connection with the Study on Cardiovascular Risk in Young Finns. Files on nitrate and nitrite content of foods and water were compiled for this study. The mean daily intakes of nitrate (NO3-) and nitrite (NO2-) from food were 54.0 mg and 1.4 mg, respectively. Vegetables including potatoes contributed 86% of nitrate intake and meat products 69% of the nitrite intake. If two litres of water within the mode concentration class of nitrate and nitrite were used, the intake of nitrate from water would have been between 1.0 and 2.0 mg and the intake of nitrite between 0.0 and 0.2 mg.

Reinik, M., et al. 2005. "Nitrites, nitrates and N-nitrosoamines in Estonian cured meat products: intake by Estonian children and adolescents." Food Addit.Contam 22(11):1098-1105. http://www.ncbi.nlm.nih.gov/pubmed/16332632

Abstract The contents of nitrate, nitrite and N-nitrosoamines in commercial cured meat products on the Estonian market were determined for 2000-01 and 2003-04 as part of the Estonian food safety monitoring programme and the Estonian Science Foundation grant research activities. The maximum permitted levels of residual nitrites and nitrates were not exceeded in the samples analysed. However, a great variation in the content of nitrate, nitrite and N-nitrosoamines was 2 found for all the products. The concentrations of these compounds in domestic cured meat products showed a decrease from year to year. The mean intake of nitrate, nitrite and N- nitrosoamines by Estonian children (n=346) from cured meat products was calculated on the basis of individual intake data. The mean daily intake of nitrates was 1.7 mg, that of nitrites was 0.83 mg and that of N-nitrosoamines was 0.073 microg. In the 2000-01 study, the calculated nitrite intake exceeded the acceptable daily intake by up to 140% for 1-6-year-old children and up to 105% in 2003-04.

Sanchez-Echaniz, J., et al. 2001. "Methemoglobinemia and consumption of vegetables in infants." Pediatrics 107(5):1024-1028. http://pediatrics.aappublications.org/content/107/5/1024.abstract

Abstract Objective. To assess clinical and epidemiologic data of 7 infants diagnosed with acquired methemoglobinemia at the pediatric emergency department between 1993 and 1998. All cases were attributed to the consumption of mixed vegetables. Methods. Medical records were reviewed to collect anamnestic data; history of food ingestion; and results of physical examination, pulse oximetry, gasometry, cooximetry, urinalysis, and outcome. Local health authorities provided information on nitrate concentration in running water and in vegetables of common consumption in the area. Results. The mean age of the patients was 8.14 months (range: 7–13). None of the infants was undernourished, had diarrhea, or was given any drug. Drinking water showed a nitrate concentration of 3 to 6 ppm. All were fed homemade purée of mixed vegetables, prepared in advance and kept in the refrigerator for 12 to 27 hours. Silver beets were a common ingredient. No case showed metabolic acidosis. Methemoglobin level ranged between 10% and 58%. Three cases had nitrituria. Silver beets in our area were the vegetables with the highest nitrate concentration (mean: 3200 mg/kg). Conclusions. Consumption of silver beets and incorrect storage of homemade purées of mixed vegetables were potential causes of methemoglobinemia in this series. The disease may occur in children older than 6 months of age. Nitrituria in a cyanotic infant may suggest the diagnosis of methemoglobinemia. methemoglobinemia, nitrates, nitrites, food analysis, infant food, food preservation.

Dusdieker, L.B., et al. 1994. "Nitrate in baby foods. Adding to the nitrate mosaic." Arch.Pediatr.Adolesc.Med. 148(5):490-494. http://archpedi.jamanetwork.com/article.aspx? articleid=517134

Abstract Objective: To identify commercial baby food varieties high in nitrate content using ion chromatography and compare the health risk associated with the consumption of high-nitrate water and high-nitrate commercial baby food. 3

Design: Ion chromatographic determination of nitrate concentration in a variety of commercial baby foods. Setting: University Hygienic Laboratory, University of Iowa College of Medicine, Iowa City. Patients: None. Results: Commercial baby foods with nitrate levels higher than 45 ppm include mixed vegetables, bananas, carrots, garden vegetables, spinach, green beans, and beets. The amount of nitrate in one 113-g (4 oz) jar of beets, for example, is equivalent to the amount of nitrate in nearly 5.5 L of water at 45 ppm nitrate. Conclusions: A controlled clinical trial is needed to clarify how consuming high-nitrate foods correlates with methemoglobin levels in infants younger than 6 months.(Arch Pediatr Adolesc Med. 1994;148:490-494)

Tamme, T., et al. 2006. "Nitrates and nitrites in vegetables and vegetable-based products and their intakes by the Estonian population." Food Addit.Contam 23(4):355-361. http://peer.ccsd.cnrs.fr/docs/00/57/75/75/PDF/PEER_stage2_10.1080%252F0265203050048236 3.pdf

Abstract The content of nitrates were determined in 1,349 samples of vegetables and ready-made food in 2003-2004 as a part of the Estonian food safety monitoring programme and the Estonian Science Foundation grant research activities. The results of manufacturers' analyses carried out for internal monitoring were included in the study. The highest mean values of nitrates were detected in dill, spinach, lettuce and beet root. The mean concentrations were 2,936, 2,508, 2,167 and 1,446 mg kg(-1), respectively. The content of nitrites in samples was lower than 5 mg kg(-1). In total, the mean intake of nitrates by the Estonian population was 58 mg day(-1). The mean content of nitrates in vegetable-based infant foods of Estonian origin was 88 mg kg(-1). The average daily intake of nitrates by children in the age group of 4-6 years was 30 mg. The infants' average daily intake of nitrates from consumption of vegetable-based foods was 7.8 mg.

U.S. Environmental Protection Agency. 1991. "Integrated Risk Information System (IRIS): Nitrate." http://www.epa.gov/iris/subst/0076.htm

Knobeloch, L., et al. 2000. "Blue babies and nitrate-contaminated well water." Environ.Health Perspect. 108(7):675-678. http://ehp03.niehs.nih.gov/article/fetchArticle.action;jsessionid=8735B81065C3E95AD5571033 337E8549?articleURI=info%3Adoi%2F10.1289%2Fehp.00108675

Abstract The use of nitrate-contaminated drinking water to prepare infant formula is a well-known risk factor for infant methemoglobinemia. Affected infants develop a peculiar blue-gray skin color and may become irritable or lethargic, depending on the severity of their condition. The 4 condition can progress rapidly to cause coma and death if it is not recognized and treated appropriately. Two cases of blue baby syndrome were recently investigated. Both cases involved infants who became ill after being fed formula that was reconstituted with water from private wells. Water samples collected from these wells during the infants' illnesses contained nitrate- nitrogen concentrations of 22.9 and 27.4 mg/L.

Preston-Martin, S., et al. 1996. "Maternal consumption of cured meats and vitamins in relation to pediatric brain tumors." Cancer Epidemiol.Biomarkers Prev. 5(8):599-605. http://journals.cambridge.org/download.php?file=%2FPHN %2FPHN4_06%2FS1368980001001215a.pdf&code=e9c02c9577b4c8e21e3a1a9fe14560df

Ward, M.H., et al. 2000. "Dietary exposure to nitrite and nitrosamines and risk of nasopharyngeal carcinoma in Taiwan." Int.J.Cancer 86(5):603-609. http://carcin.oxfordjournals.org/content/30/12/2031.full

Abstract Previous studies of nasopharyngeal carcinoma (NPC) have found elevated risks with higher consumption of salted fish and preserved foods, particularly during childhood. These foods can contain high levels of nitrosamines; however, most studies have not estimated exposure to nitrosamines directly. We conducted a case-control study in Taiwan to evaluate dietary intakes and NPC risk. A total of 375 cases (99% response rate) and 327 controls (88% response rate) were interviewed about their diet as an adult and at age 10 using a food-frequency questionnaire. We interviewed mothers of participants about their child's diet at age 10, age 3 and during weaning and the mother's diet while she was breast-feeding. Mothers of 96 cases and 120 controls were interviewed. Nitrosamine and nitrite levels were assigned to 66 foods based on published values. Intake of nitrosamines and nitrite as an adult was not associated with risk of NPC. High intakes of nitrosamines and nitrite during childhood and weaning were associated with increased risks of NPC for foods other than soy products. Adjusted odds ratios for the highest quartile were 2.2 [95% confidence interval (CI) 0.8-5.6] for age 10, 2.6 (95% CI 1.0-7.0) for age 3 and 3.9 (95% CI 1.4-10.4) for weaning diet. Intakes of nitrite and nitrosamines from soybean products during childhood and weaning were inversely associated with risk. Soybeans contain known inhibitors of nitrosation, and thus may explain the inverse association we observed. Our results suggest that nitrosamine and nitrite intake during childhood may play a role in the development of NPC.

Pogoda, J.M., and S. Preston-Martin. 2001. "Maternal cured meat consumption during pregnancy and risk of paediatric brain tumour in offspring: potentially harmful levels of intake." Public Health Nutr. 4(2):183-189. http://www.ncbi.nlm.nih.gov/pubmed/11299090 Abstract OBJECTIVE: To describe the relationship between specific levels of nitrite intake from cured meat consumption during pregnancy and the relative risk of paediatric brain tumours in the offspring. 5

DESIGN: Exposure data were previously collected for a population-based case-control study of paediatric brain tumours; data on nitrite content were obtained by a comprehensive literature review of surveys of residual nitrite content in cured meats published in the USA and Canada. The level of nitrite intake for each mother was predicted by year of pregnancy based on survey results. Dose- response was evaluated both categorically and continuously using polynomial and quadratic spline regression. SETTING: The US west coast: Los Angeles County, the San Francisco-Oakland Bay Area and the Seattle- Puget Sound area. SUBJECTS: There were 540 cases diagnosed between 1984 and 1990 at ages varying from 0 to 19 years, and 801 controls frequency-matched by geographic area, age and birth year. RESULTS: In general, survey results suggest a trend of decreasing nitrite levels in cured meats over time. We observed a moderate increase in brain tumour risk in the offspring of mothers with relatively low levels of nitrite consumption from cured meats during pregnancy, and a two- to three-fold risk increase in offspring of mothers who consumed 3 mg day-1 nitrite from cured meats (about 125 g day-1 of cured meat consumption throughout the pregnancy). CONCLUSIONS: A substantial risk of paediatric brain tumour appears to be associated with relatively high levels of maternal cured meat consumption during pregnancy. A more scientifically valid approach than a literature review to estimate nitrite intake from cured meats and data from a large group of highly exposed subjects would be useful in determining potentially harmful levels.

Sarasua, S., and D.A. Savitz. 1994. "Cured and broiled meat consumption in relation to childhood cancer: Denver, Colorado (United States)." Cancer Causes Control 5(2):141-148. http://www.ncbi.nlm.nih.gov/pubmed/8167261 Abstract The association between cured and broiled meat consumption by the mother during pregnancy and by the child was examined in relation to childhood cancer. Five meat groups (ham, bacon, or sausage; hot dogs; hamburgers; bologna, pastrami, corned beef, salami, or lunch meat; charcoal broiled foods) were assessed. Exposures among 234 cancer cases (including 56 acute lymphocytic leukemia [ALL], 45 brain tumor) and 206 controls selected by random-digit dialing in the Denver, Colorado (United States) standard metropolitan statistical area were compared, with adjustment for confounders. Maternal hot-dog consumption of one or more times per week was associated with childhood brain tumors (odds ratio [OR] = 2.3, 95 percent confidence interval [CI] = 1.0-5.4). Among children, eating hamburgers one or more times per week was associated with risk of ALL (OR = 2.0, CI = 0.9-4.6) and eating hot dogs one or more times per week was associated with brain tumors (OR = 2.1, CI = 0.7-6.1). Among children, the combination of no vitamins and eating meats was associated more strongly with both ALL and brain cancer than either no vitamins or meat consumption alone, producing ORs of two to seven. The results 6 linking hot dogs and brain tumors (replicating an earlier study) and the apparent synergism between no vitamins and meat consumption suggest a possible adverse effect of dietary nitrites and nitrosamines.

McCredie, M., et al. 1994. "Antenatal risk factors for malignant brain tumours in New South Wales children." Int.J.Cancer 56(1):6-10. http://www.ncbi.nlm.nih.gov/pubmed/8262678

Abstract A population-based case-control study of incident primary malignant brain tumours diagnosed during 1985 to 1989 in children aged 0 to 14 years was carried out in the coastal conurbation of New South Wales comprising Sydney, Wollongong and Newcastle in the period 1988 to 1990. Personal interviews were conducted using a structured questionnaire with mothers of 82 cases and 164 control children individually matched to the cases by sex and age. Among the hypotheses being examined were those related to exposure to parental tobacco smoke, N-nitroso compounds and possible protection from sources of vitamin C. No link was found with tobacco smoking by the mother before or during pregnancy. While exposure during pregnancy of the mother to tobacco smoke of the father appeared to double the risk of childhood brain tumours and a similar risk was found for father (but not mother) smoking before the index pregnancy, there was no "dose-response" and the increased risk was confined to data supplied by the mother (rather than the father himself). The risk of childhood brain tumours rose with reported increasing consumption, during pregnancy, of cured meats, which have high levels of N-nitroso compounds (or their precursors), and fell with rising consumption of vegetables. No association was found between the risk of childhood brain tumours and family history of epilepsy, cancer, or tumours of the nervous system, parental irradiation, previous miscarriage or procedures carried out during pregnancy, maternal consumption of antihistamines, barbiturates or diuretics, or maternal contact with cats or farm- life during pregnancy.

Preston-Martin, S., et al. 1982. "N-Nitroso compounds and childhood brain tumors: a case- control study." Cancer Res. 42(12):5240-5245. http://cancerres.aacrjournals.org/content/42/12/5240.abstract

Abstract We questioned mothers of 209 young brain tumor patients and mothers of 209 controls about experiences of possible etiological relevance which they had during pregnancy or which their children had while growing up. Long-suspected brain tumor risk factors such as head trauma and X-rays appeared to be factors for relatively few cases. Increased risk was associated with maternal contact with nitrosamine- containing substances such as burning incense (odds ratio, 3.3; p = 0.005), sidestream cigarette smoke (odds ratio, 1.5; p = 0.03), and face makeup (odds ratio, 1.6; p = 0.02); with maternal use of diuretics (odds ratio, 2.0; p = 0.03) and antihistamines (odds ratio, 3.4; p = 0.002); and with the level of maternal consumption of cured meats (p = 0.008). These drugs contain nitrosatable amines and amides, and the cured meats contain nitrites, chemicals which are precursors of N-nitroso compounds. We propose a hypothesis that brain tumors in these young people are related to in utero exposure to N-nitroso compounds and their precursors, the most potent nervous system carcinogens known in experimental animals. 7

Volkmer, B.G., et al. 2005. "Influence of nitrate levels in drinking water on urological malignancies: a community-based cohort study." BJU.Int 95(7):972-976. http://www.ncbi.nlm.nih.gov/pubmed/15839916

Abstract OBJECTIVE: To evaluate the effect of nitrate levels in the drinking water on the incidence of urological malignancies in a German community. PATIENTS AND METHODS: For 28 years (1957-86) the community of Bocholt, Germany (70,000 inhabitants) had a drinking water supply with different nitrate levels, i.e. 60 mg/L in group A (57,253 inhabitants) and 10 mg/L in group B (10,037 inhabitants). All newly diagnosed cases of urological malignancies were registered from 1986 to 1997. The incidence was calculated using an age standardization based on the German population. Results: In all, there were 527 urological malignancies recorded (urothelial cancer 39.8%, renal cell carcinoma 10.8%, testicular tumours 8.0%, penile carcinoma 1.7%, prostate cancer 39.7%). The incidence per 100,000 inhabitants/year of urinary tract tumours was 33.8 in group A and only 17.1 in group B (relative risk, RR 1.98, 95% confidence interval, CI, 1.10-3.54). The RR was 0.87 (0.34-2.22) for renal tumours, 0.66 (0.14-2.88) for penile cancer and 1.06 (0.76-1.48) for prostate cancer. For testicular tumours there was an inverse association with nitrate level, with a RR of 0.43 (0.21-0.90). CONCLUSION: This study showed an association of nitrate load in drinking water and the incidence of urothelial cancer in both genders, with an inverse correlation to testicular tumours and no correlation with renal, penile and prostatic tumours.

Bunin, G.R., et al. 1993. "Relation between maternal diet and subsequent primitive neuroectodermal brain tumors in young children." N.Engl.J.Med. 19;329(8):536-541. http://www.ncbi.nlm.nih.gov/pubmed/8167265

Abstract N-nitroso compounds and their precursors, nitrites and nitrates, have been hypothesized as risk factors, and vitamins C and E, which inhibit N-nitroso formation, as protective factors for brain tumors. A case- control study of maternal diet during pregnancy and risk of astrocytoma, the most common childhood brain tumor, was conducted by the Childrens Cancer Group. The study included 155 cases under age six at diagnosis and the same number of matched controls selected by random-digit dialing. A trend was observed for consumption of cured meats, which contain preformed nitrosamines (a class of N-nitroso compounds) and their precursors (adjusted odds ratio [OR] for highest quartile of intake relative to lowest = 1.7, P trend = 0.10). However, no strong trends were observed for nitrosamine (OR = 0.8, P = 0.60); nitrite (OR = 1.3, P = 0.54); nitrate (OR = 0.7, P = 0.43); vitamin C (OR = 0.7, P = 0.37); or vitamin E (OR = 0.7, P = 0.48). Iron supplements were associated with a significant decrease in risk (OR = 0.5, 95 percent confidence interval = 0.3-0.8). The effect of several dietary factors differed by income level, making interpretation of the results difficult. Future research should investigate the effect of dietary 8 components not assessed in this study, as these may explain the disparate effects by income level. The results of this study provide limited support for the nitrosamine hypothesis.

Mueller, B.A., et al. 2001. "Residential water source and the risk of childhood brain tumors." Environ.Health Perspect. 109(6):551-556. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1240334/pdf/ehp0109-000551.pdf

Abstract Gestation may represent a window of susceptibility to transplacental effects of environmental exposures, including chemicals in water. The N-nitroso compounds (NNC), a class of chemicals with demonstrated neurocarcinogenic potential, include substances detected in drinking water. We used data from a study of possible risk factors for childhood brain tumors (CBT) to investigate the association of source of residential drinking water during pregnancy and CBT occurrence among offspring. In addition, dipstick measurements were made of nitrates and nitrites in tap water for the subset of women living in the same home they had lived in during their pregnancies. Population-based CBT cases (n = 540) and controls (n = 801) were identified in three regions including Los Angeles County, and the San Francisco Bay Area of California, and the Seattle-Puget Sound area of western Washington state. Overall, we observed no increased risk of CBT in offspring associated with wells as the source of residential water. However, an increased risk of CBT [odds ratio (OR) = 2.6; 95% confidence interval (CI), = 1.3-5.2] was observed in western Washington among offspring of women who relied exclusively on well water, and a decreased risk of CBT (OR = 0.2; 95% CI, 0.1-0.8) was observed in Los Angeles County. Among the small subset of subjects for whom dipstick measurements of tap water were available, the risk of CBT associated with the presence of either measurable nitrite and/or nitrate was 1.1 (95% CI, 0.7-2.0). Given the crude measurement method employed and because measurements often were obtained years after these pregnancies occurred, the relevance of the dipstick findings is unclear. The lack of consistency in our findings related to residential water source does not support the hypothesis of increased risk related to consumption of well water; however, regional differences in well water content may exist, and the increased risk observed in western Washington deserves further evaluation.

Howe, G.R., et al. 1989. "An exploratory case-control study of brain tumors in children." Cancer Res. 49(15):4349-4352. http://cancerres.aacrjournals.org/content/49/15/4349.long

Abstract An exploratory case-control study of childhood brain tumors was conducted in southern Ontario between 1977 and 1983, on 74 cases and 138 age- and sex-matched population controls. A significantly elevated risk (perhaps due to early case symptoms) was seen for skull X-rays at least 5 years prior to diagnosis, and for head or neck injuries which required medical attention. However, no evidence of an increased risk appeared for exposure to sick pets or to pesticides, maternal or paternal history of smoking, and various birth characteristics or antenatal exposure of the child, though these have previously been reported to be associated with childhood brain tumors. With respect to the hypothesis that N-nitroso compounds may be involved in the etiology of childhood brain tumors, most exposures of this type were not associated with risk, though a significant positive association was seen for consumption of beer by the mother during pregnancy, and a significant negative association was seen with consumption of fruit juice by the child. Other findings in the present study include an association with developmental problems relating to height 9 and weight and with certain socioeconomic characteristics of the mother. Further investigation of these results in future studies is warranted.

Kuijten, R.R., et al. 1990. "Gestational and familial risk factors for childhood astrocytoma: results of a case-control study." Cancer Res. 50(9):2608-2612. http://www.ncbi.nlm.nih.gov/pubmed/2328486

Abstract Gestational and familial risk factors were investigated for their association with astrocytoma, the most frequently occurring brain tumor in children. A case-control study of 163 matched pairs was performed. Cases under 15 years of age at diagnosis in 1980-1986 were identified through the tumor registries of 8 hospitals in Pennsylvania, New Jersey, and Delaware. Controls were selected by random digit dialing and were matched to cases for age, race, and telephone area code and exchange. Maternal antinausea medications increased the risk of childhood astrocytoma [OR (odds ratio) = 2.0, P = 0.04]. Cured meat consumption during pregnancy was more common among cases (OR = 1.9, P = 0.07), and a significant trend with increasing frequency of consumption was observed (P = 0.04). Results for gestational exposure to marijuana (OR = 2.8, P = 0.07) were of borderline significance. Gestational exposure to neurally active medications, alcohol, and tobacco were not risk factors. There was a significant trend for cases to be of higher birth weight (P = 0.03). Mental retardation (OR = 3.0, P = 0.04) and cancer (OR = 1.7, P = 0.02) in a relative of the child significantly increased the risk of astrocytoma. Significantly increased risks were observed for brain tumors in relatives of children 0-4 years of age at diagnosis (OR = 6/0, P = 0.04). A significant protective effect was observed for maternal history of miscarriage or stillbirth (OR = 0.5, P = 0.01). The results of this study suggest that some gestational and familial factors may increase the risk of childhood astrocytoma.

Kean-Cowdin, R., et al. 2003. "Maternal prenatal exposure to nitrosatable drugs and childhood brain tumours." Int.J.Epidemiol. 32(2):211-217. http://www.ncbi.nlm.nih.gov/pubmed/12714539

Abstract BACKGROUND: A compelling hypothesis was proposed that childhood brain tumours are associated with maternal exposure to N-nitroso compounds during the prenatal period. Many common drugs, such as antihistamines, aspirin, and antibiotics, are nitrosatable and depending upon the product, potentially carcinogenic. We hypothesized that maternal ingestion of certain subgroups of nitrosatable drug products during pregnancy increases the risk of brain tumour development in offspring. METHODS: Data were collected as part of a population-based case-control study of childhood brain tumours and mothers' self-reported exposure to therapeutic drugs and dietary nitrites. Cases were enrolled from three US West Coast SEER tumour registries: Seattle-Puget sound, Los Angeles County, and the San Francisco-Oakland Bay Area. Tumours were grouped into three major histological tumour subtypes: astroglial, primitive neural ectodermal tumours, and all remaining glial tumours ('other glial'). Therapeutic drugs reported by mothers were translated into active chemical compounds and classified as 10 secondary amines, tertiary amines, amides, or none of the three. Risk estimates were computed according to classes of nitrosatability, potential carcinogenicity, teratogenicity, and predicted end product. RESULTS: We found no significant association between maternal use of nitrosatable drugs, either overall or within any of the nitrosatable drug classifications, and subsequent development of brain tumours in children. Nitrite consumption from cured meats was not an effect modifier. However, exposure to nitrosoephedrine during pregnancy was associated with significantly increased risk of 'other glial' tumours (OR = 3.1; 95% CI: 1.1-9.2). CONCLUSIONS: These findings do not support an association between maternal use of nitrosatable drugs during pregnancy and brain tumour risk in offspring. While exposure to the nitrosation end product nitrosoephedrine was associated with increased risk for other glial tumours, the finding was not specific to any one type of tumour.

Mueller, B.A., et al. 2004. "Household water source and the risk of childhood brain tumours: results of the SEARCH International Brain Tumor Study." Int.J.Epidemiol. 33(6):1209-1216. http://www.ncbi.nlm.nih.gov/pubmed/15567873

Abstract BACKGROUND: The period in utero is a time of increased vulnerability. Offspring of pregnant women exposed to carcinogenic substances in drinking water may be more likely to develop cancer. We examined whether household water source and the presence of nitrates or nitrites in residential water were associated with increased risks of childhood brain tumours (CBT). METHODS: We used data from a multicentre, case-control study with maternal information on residential water source, and nitrate/nitrite levels of tap water measured by dipstick. Subjects included 836 CBT cases and 1485 controls from five countries. RESULTS: The risks of CBT associated with reliance on well water (versus public water) during pregnancy varied widely, with significantly increased risks noted in two (of seven) regions and a decreased risk observed in one region. CBT risk did not increase with increasing nitrate levels. However, our results based on tap water tested in the pregnancy residences suggest the risk of astrocytoma may be associated with increasing levels of nitrite (odds ratio [OR] = 4.3, 95% CI: 1.4, 12.6 for nitrite levels of 1-<5 mg/l nitrite ion; OR = 5.7, 95% CI: 1.2, 27.2 of nitrite > or =5 mg/l). CONCLUSIONS: These results should be interpreted with caution because women's recollection of water sources may have contained inaccuracies, and nitrate and nitrite measurements, available for only a portion of subjects, were often obtained years after the pregnancies occurred. However, our results suggest a need for closer evaluation of well water content in some regions and the possibility that a nitrite-related water exposure may be associated with CBT. 11

Infante-Rivard, C., et al. 2001. "Drinking water contaminants and childhood leukemia." Epidemiology 12(1):13-19. http://www.ncbi.nlm.nih.gov/pubmed/11138808

Abstract We conducted a population-based case-control study to evaluate the relation between exposure to drinking water contaminants (total and specific trihalomethanes and certain metals and nitrates) and childhood acute lymphoblastic leukemia. We compared 491 cases 0-9 years of age with 491 controls. We developed a municipality-exposure matrix based on municipal and provincial historical data, a tapwater survey in 227 homes, and information about residential history. We used average level of exposure and cumulative average over the period as exposure indices, and we measured risk for the pregnancy period as well as for the postnatal period. We show that risks were generally not increased for the prenatal period nor with average levels of exposure. Postnatal cumulative exposure for total trihalomethanes at above the 95th percentile of the distribution for cases and controls was associated with an odds ratio of 1.54 (95% confidence interval = 0.78-3.03); for that same period, risk associated with exposure to chloroform was increased (odds ratio = 1.63; 95% confidence interval = 0.84-3.19) as well as that for exposure to zinc (odds ratio = 2.48; 95% confidence interval = 0.99-6.24). Risks were also increased for exposure to cadmium and arsenic, but not for other metals nor for nitrates.

Law, G., et al. 1999. "Non-Hodgkin's lymphoma and nitrate in drinking water: a study in Yorkshire, United Kingdom." J.Epidemiol.Community Health 53(6):383-384. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1756892/pdf/v053p00383.pdf

Knobeloch, L., and M. Proctor. 2001. "Eight blue babies." WMJ. 100(8):43-47. http://www.wisconsinmedicalsociety.org/_WMS/publications/wmj/issues/wmj_v100n8/100-8- SA-Knobeloch.pdf

Abstract Methemoglobinemia is a serious medical condition that affects hundreds of infants in the United States each year. The condition involves the oxidation of red cell hemoglobin to a state that is unable to transport oxygen. Affected infants appear cyanotic and may have altered mental status. The condition is readily reversible if recognized and treated appropriately. The Wisconsin Division of Public Health investigates all cases of infant methemoglobinemia in an attempt to determine their cause. Between January 1990 and September 1999, 8 infants were diagnosed with this condition. Review of their hospitalization records found that 3 of these cases involved infants whose formula was prepared with water from nitrate-contaminated wels. Risk factors identified in the remaining cases included use of folk remedies, misuse of over-the-counter analgesics, and an inherited enzyme deficiency. Causes were not identified for 2 of the cases. All of the affected infants recovered.

Savino, F., et al. 2006. "Methemoglobinemia caused by the ingestion of courgette soup given in order to resolve constipation in two formula-fed infants." Ann Nutr.Metab 50(4):368-371. http://www.ncbi.nlm.nih.gov/pubmed/16809905 12

Abstract Methemoglobinemia is not a rare condition arising from the exposure to hemoglobin-oxidizing agents such as nitrates-nitrites present in well water or vegetables. Infants < 3 months of age are more susceptible than adults because of lower amounts of a key enzyme, NADH-cytochrome b5 reductase, which converts methemoglobin back to hemoglobin. We report 2 infants, aged respectively 2 and 1 months, suffering from methemoglobinemia, fed with a formula that was reconstituted with a high concentration of courgette soup to resolve constipation. They developed a severe cyanosis with methemoglobinemia (respectively 30.4 and 27%) and were hospitalized and treated with methylene blue at 1%. After 12 h the syndrome was completely resolved. Home-prepared infant foods containing vegetables are potential causes of methemoglobinemia. It is important not to feed infants with vegetables having a high nitrate content (e.g., courgette, spinach, beets and green beans) to resolve constipation since, particularly in the first months of life, they may cause severe methemoglobinemia.

Jones, J.H., et al. 1973. "Grandmother's poisoned well: report of a case of methemoglobinemia in an infant in Oklahoma." J.Okla.State Med.Assoc. 66(2):60-66. http://www.ncbi.nlm.nih.gov/pubmed/4688467

MMWR. 1997. "Methemoglobinemia Attributable to Nitrite Contamination of Potable Water Through Boiler Fluid Additives - New Jersey, 1992-1996." Morbidity and Mortality Weekly Report March 7, 1997. http://www.cdc.gov/mmwr/preview/mmwrhtml/00046656.htm

Abstract Nitrite and nitrate ions are naturally occurring forms of nitrogen that can be present in ground and surface water and can be used as a food preservative because they inhibit the growth of Clostridium botulinum. Exposure to excessive levels of nitrite or nitrate may result in the acute syndrome of methemoglobinemia (MetHb), in which nitrite binds to hemoglobin. This report summarizes the findings of investigations of two incidents in which unintentional exposure to high doses of nitrite occurred through drinking potable water contaminated with additives to boiler conditioning fluids.

Bukowski, J., et al. 2001. "Agricultural contamination of groundwater as a possible risk factor for growth restriction or prematurity." J.Occup.Environ.Med. 43(4):377-383. http://www.ncbi.nlm.nih.gov/pubmed/11322099

Abstract Agricultural activity on Prince Edward Island poses a potential hazard to groundwater, which is the sole source of drinking water on the island. This study investigates the potential impact of groundwater nitrate exposure on prematurity and intrauterine growth restriction on Prince Edward Island. A total of 210 intrauterine growth restriction cases, 336 premature births, and 4098 controls were abstracted from a database of all Island births. An ecological measure of groundwater nitrate level was used to gauge potential exposure to agriculturally contaminated drinking water. The higher nitrate exposure categories were positively associated with intrauterine growth restriction and prematurity, and significant dose- response trends were seen, even after adjustment for several important covariates. Nevertheless, these 13 risks must be interpreted cautiously because of the ecological nature of this exposure metric. An investigation using nitrate levels for individual study subjects is needed to confirm this association.

George, M., et al. 2001. "Incidence and geographical distribution of sudden infant death syndrome in relation to content of nitrate in drinking water and groundwater levels." Eur.J.Clin.Invest 31(12):1083-1094. http://www.ncbi.nlm.nih.gov/pubmed/11903496

Abstract BACKGROUND: Previous studies indicate that the enteral bacterial urease is inhibited in victims of sudden infant death syndrome (SIDS). One possible inhibitor of this bacterial activity is nitrate. If ambient pollution by nitrate is involved in the etiology of SIDS only a fraction of the nitrate concentration not infrequently found in drinking water would be enough for this inhibition. METHODS: Occurrence of SIDS (n = 636) in Sweden during the period 1990 through 1996 were analysed regarding geographical and seasonal distribution in relation to the nitrate concentration in drinking water and changes in the groundwater level. RESULTS: Both the birth rate and the incidence of SIDS decreased during the study period. One quarter of the municipalities constituting 11% of the population had no cases, the maximum incidence being 6.5 per 1000 live births. Seasonality: The northernmost parts of the country had its highest incidence when the rest of the country had its lowest incidence, and the occurrence of individual deaths was associated with the recharge of groundwater which increases its nitrate content. The local incidence of SIDS was correlated (rs = 0.34-0.87) to maximally recorded concentrations of nitrate in drinking water. CONCLUSIONS: The seasonal distribution of SIDS was widely different from the south to the north of the country and seems to be associated with differences in the groundwater level changes subsequent to precipitation, frost penetration, and melting of snow. Use of drinking water with high peak concentrations or great variations in nitrate concentration was correlated to the incidence of SIDS.

Cedergren, M.I., et al. 2002. "Chlorination byproducts and nitrate in drinking water and risk for congenital cardiac defects." Environ.Res. 89(2):124-130. http://www.ncbi.nlm.nih.gov/pubmed/12123645

Abstract Drinking water disinfection byproducts have been associated with an increased risk for congenital defects including cardiac defects. Using Swedish health registers linked to information on municipal drinking water composition, individual data on drinking water characteristics were obtained for 58,669 women. Among the infants born, 753 had a cardiac defect. The risk for a cardiac defect was determined for ground water versus surface water, for different chlorination procedures, and for trihalomethane and nitrate concentrations. Ground water was associated with an increased risk for cardiac defect when crude 14 rates were analyzed but after suitable adjustments this excess rate was found to be determined by chlorination procedures including chlorine dioxide. Chlorine dioxide appears itself as an independent risk factor for cardiac defects (adjusted odds ratio 1.61 (95%CI 1.00-2.59)). The risk for cardiac defects increased with increasing trihalomethane concentrations (P=0.0005). There was an indicated but statistically nonsignificant excess risk associated with nitrate concentration. The individual risk for congenital cardiac defect caused by chlorine dioxide and trihalomethanes is small but as a large population is exposed to public drinking water, the attributable risk for cardiac defects may not be negligible. Manassaram, D.M., Backer, L.C. & Moll, D.M. (2006) A Review of Nitrates in Drinking water: Maternal Exposure and Adverse Reproductive and Developmental Outcomes. Environ Health Perspect. 2006 March; 114(3): 320–327. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1392223/

Abstract In this review we present an update on maternal exposure to nitrates in drinking water in relation to possible adverse reproductive and developmental effects, and also discuss nitrates in drinking water in the United States. The current standard for nitrates in drinking water is based on retrospective studies and approximates a level that protects infants from methemoglobinemia, but no safety factor is built into the standard. The current standard applies only to public water systems. Drinking water source was related to nitrate exposure (i.e., private systems water was more likely than community system water to have nitrate levels above the maximum contaminant limit). Animal studies have found adverse reproductive effects resulting from higher doses of nitrate or nitrite. The epidemiologic evidence of a direct exposure– response relationship between drinking water nitrate level and adverse reproductive effect is still not clear. However, some reports have suggested an association between exposure to nitrates in drinking water and spontaneous abortions, intrauterine growth restriction, and various birth defects. Uncertainties in epidemiologic studies include the lack of individual exposure assessment that would rule out confounding of the exposure with some other cause. Nitrates may be just one of the contaminants in drinking water contributing to adverse outcomes. We conclude that the current literature does not provide sufficient evidence of a causal relationship between exposure to nitrates in drinking water and adverse reproductive effects. Future studies incorporating individual exposure assessment about users of private wells—the population most at risk—should be considered.

Arbuckle, T.E., et al. 1988. "Water nitrates and CNS birth defects: a population-based case- control study." Arch.Environ.Health 43(2):162-167. http://www.ncbi.nlm.nih.gov/pubmed/3377550

Abstract The relation between maternal exposure to nitrates in drinking water and risk of delivering an infant with a central nervous system (CNS) malformation was examined by means of a case-control study in New Brunswick, Canada. All cases of CNS defects for a "high" and a "low" prevalence area of New Brunswick, for the years 1973-1983, were included in the study. Controls were selected randomly from the livebirth files for the province, matched on county of maternal residence and date of birth. One hundred and thirty (130) cases were identified and individually matched with two controls each. Individual water samples were collected from the case and control mother's address given on the birth or stillbirth records. The study revealed that the effect of nitrate exposure in water was modified by whether the source of the drinking water was a private well or a public municipal distribution system. Compared 15 to a baseline nitrate level of 0.1 ppm, exposure to nitrate levels of 26 ppm from private well water sources was associated with a moderate, but not statistically significant, increase in risk (risk odds ratio = 2.30; 95% confidence interval = 0.73-7.29). If the source of drinking water was a municipal distribution system or a private spring, an increase in nitrate exposure was associated with a decrease in risk of delivering a CNS-malformed infant; however, these effect estimates were not statistically significant. The positive increase in risk with nitrate exposure from well water sources requires further study using a larger case series and a larger proportion of exposures to nitrate levels exceeding 5 ppm.

Dorsch, M.M., et al. 1984. "Congenital malformations and maternal drinking water supply in rural South Australia: a case-control study." Am.J.Epidemiol. 119(4):473-486. http://www.ncbi.nlm.nih.gov/pubmed/6711537

Abstract A case-control study, carried out in the Mount Gambier region of South Australia, investigated the relationship between mothers' antenatal drinking water source and malformations in offspring. It was prompted by earlier descriptive findings of a statistically significant, and localized, increase in the perinatal mortality rate in Mount Gambier, due principally to congenital malformations affecting the central nervous system and multiple organ systems. Available for statistical analysis were 218 case- control pairs, from the period 1951-1979, individually matched by hospital, maternal age (+/- 2 years), parity and date of birth (+/- 1 month). Compared with women who drank only rainwater during their pregnancy (relative risk (RR) = 1.0), women who consumed principally groundwater had a statistically significant increase in risk of bearing a malformed child (RR = 2.8). Statistically significant risk increases occurred specifically for malformations of the central nervous system and musculoskeletal system. Reanalysis of the data by estimated water nitrate concentration demonstrated a nearly threefold increase in risk for women who drank water containing 5-15 ppm of nitrate, and a fourfold increase in risk for those consuming greater than 15 ppm of nitrate. A seasonal gradient in risk was evident among groundwater consumers, ranging from 0.9 for babies conceived in winter, 3.0 in autumn, to 7.0 and 6.3 for spring and summer conceptions, respectively. Linear logistic regression analysis, controlling for risk factors not accounted for in the study design, showed that maternal water supply, infant's sex, and mother's area of residence all contributed significantly to the risk of malformation. These results are discussed in relation to previous experimental and human descriptive studies, suggesting a plausible mechanism for nitrate-induced teratogenesis.

Croen, L.A., et al. 2001. "Maternal exposure to nitrate from drinking water and diet and risk for neural tube defects." Am.J.Epidemiol. 153(4):325-331. http://aje.oxfordjournals.org/content/153/4/325.full.pdf

Brender, J.D., et al. 2004. "Dietary nitrites and nitrates, nitrosatable drugs, and neural tube defects." Epidemiology 15(3):330-336. http://www.ncbi.nlm.nih.gov/pubmed/15097014

Abstract 16

BACKGROUND: Amine-containing (nitrosatable) drugs can react with nitrite to form N-nitroso compounds, some of which are teratogenic. Data are lacking on whether dietary intake of nitrates and nitrites modifies the association between maternal nitrosatable drug exposure and neural tube defects (NTDs) in offspring. METHODS: We examined nitrosatable drug exposure and NTD-affected pregnancies in relation to dietary nitrite and total nitrite intake in a case-control study of Mexican American women. We interviewed 184 women with NTD-affected pregnancies and 225 women with normal live births, including questions on periconceptional drug exposures and dietary intake. For 110 study participants, nitrate was also measured in the usual source of drinking water. RESULTS: Women who reported taking drugs classified as nitrosatable were 2.7 times more likely to have an NTD- affected pregnancy than women without this exposure (95% confidence interval [CI] = 1.4-5.3). The effect of nitrosatable drugs was observed only in women with higher intakes of dietary nitrite and total nitrite (dietary nitrite + 5% dietary nitrate). Women within the highest tertile (greater than 10.5 mg/day) of total nitrite were 7.5 times more likely to have an NTD-affected pregnancy if they took nitrosatable drugs (95% CI = 1.8-45.4). The association between nitrosatable drug exposure and NTDs was also stronger in women whose water nitrate levels were higher. CONCLUSIONS: Findings suggest that effects of nitrosatable drug exposure on risk for neural tube defects in offspring could depend on the amounts of dietary nitrite and total nitrite intake.

Virtanen, S.M., et al. 1994. "Nitrate and nitrite intake and the risk for type 1 diabetes in Finnish children. Childhood Diabetes in Finland Study Group." Diabet.Med. 11(7):656-662. http://www.ncbi.nlm.nih.gov/pubmed/7955990

Abstract The intakes of nitrate and nitrite of children and their parents from food and drinking water were estimated in a Finnish nation-wide case-control study on the epidemiology of Type 1 diabetes. The study population consisted of 684 case and 595 control children; 548 case-control pairs of fathers; and 620 case-control pairs of mothers. The consumption frequencies of foods which are important sources of nitrate and nitrite were assessed by structured questionnaire. Nitrate and nitrite concentration data were collected from Finnish water works. Diabetic children's and their mothers' daily dietary intake of nitrite was greater compared with that of control children and mothers (for case and control children 0.9 mg vs 0.8 mg, for case and control mothers 0.9 mg vs 0.8 mg, p < 0.001). Case mothers compared with control mothers received less (p < 0.05) nitrate from their diet. No differences were observed in the intake of nitrate or nitrite from drinking water. Dietary nitrite intake of children (odds ratios and 95% confidence intervals for the second, third, and fourth quartile 1.16, 0.82-1.65; 1.49, 1.06-2.10; 2.32, 1.67-3.24, respectively) and mothers (odds ratios and 95% confidence intervals for the second, third, and fourth quartile 1.15, 0.76-1.74; 1.29, 0.87-1.91; 1.98, 1.35-2.90, respectively) was positively associated with the risk for Type 1 diabetes independently from length of mother's education, child's or mother's age, place of residence or mother's smoking status.(ABSTRACT TRUNCATED AT 250 WORDS) 17

Gupta, S.K., et al. 2001. "Recurrent diarrhea in children lining in areas with high levels of nitrate in drinking water." Arch of Environ Health 56(4): 369 – 373. http://www.ncbi.nlm.nih.gov/pubmed/11572282

Abstract Given that there was documented evidence of an association between diarrhea and high nitrate ingestion, the authors examined drinking water nitrate concentration and its possible correlation(s) with methemoglobin levels, cytochrome b5 reductase activity, and recurrent diarrhea. In addition, the authors studied histopathological changes in the intestines of rabbits in an animal model. Five village areas were studied, and nitrate concentrations (expressed in mg of nitrate per liter of water) of 26, 45, 95, 220, and 459 existed in the respective villages. The study included 88 randomly selected children who were 8 yr of age or younger; they represented 10% of the total population of each of the areas. Detailed histories of recurrent diarrhea were noted, and medical examinations were conducted. Cytochrome b5 reductase activity and methemoglobin levels were estimated biochemically. Collected data were analyzed statistically with Microsoft Excel software. In addition, the authors exposed rabbits to various levels of nitrate, and histopathological changes of the stomach and intestine (small and large) were evaluated. There was a strong relationship between nitrate concentration and recurrent diarrhea; 80% of the recurrent diarrhea cases were explained by nitrate concentration alone. In the rabbit intestines, lymphocytic infiltration and hyperplasia characterized the submucosa as nitrate concentrations increased.

Gupta, S.K., et al. 2000. "Recurrent acute respiratory tract infections in areas with high nitrate concentrations in drinking water." Environ.Health Perspect. 108(4):363-366. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1638033/

Abstract A review of the literature indicated an association among high nitrate ingestion, methemoglobinemia, and pathologic changes in bronchi and lung parenchyma. The present study examined a possible correlation among drinking water nitrate concentration, methemoglobin levels, cytochrome b(5) reductase activity, and acute respiratory tract infection with a history of recurrence (RRTI). Our study was conducted in five village units in the state of Rajasthan, India, with nitrate concentrations of 26, 45, 95, 222, and 459 mg NO(3) ion/L. We randomly selected 88 children. The children were up to 8 years of age, age matched, and represented 10% of the total population of these areas. We obtained detailed RRTI histories and conducted medical examinations. Methemoglobin levels and cytochrome b(5) reductase activity were estimated biochemically. The data collected were statistically analyzed using spreadsheet software on a personal computer. We observed strong interdependence between methemoglobin levels and RRTI in children up to 8 years of age. Methemoglobin levels alone explained 80% of the variation in the RRTI cases. This study indicates that methemoglobinemia, secondary to high nitrate ingestion in drinking water, causes RRTI. Increased production of methemoglobin and free radicals of nitric oxide and oxygen due to nitrate metabolism in the body lead to alveolar damage and mismatching of ventilation and perfusion, which may be the reason for high mortality in children due to RRTI. 18

Craun, G.F., et al. 1981. "Methaemoglobin levels in young children consuming high nitrate well water in the United States." Int.J.Epidemiol. 10(4):309-317. http://ije.oxfordjournals.org/content/10/4/309.abstract

Abstract

The EPA limit for nitrate (10 mg/l NO, -N) in drinking water was esteblished to prevent infantile methaemoglo-binemia, an acute condition confined almost exclusively to infants less than three months. This condition is clinically detectable at methaemoglobin levels of approximately 10%. Several studies in the Soviet Union have suggested that elevated methaemoglobin levels from ingested nitrate may not be confined to the young infant and have reported an association between increased methaemoglobin levels of up to 7% in schoolchildren and drinking water with a nitrate concentration of 23–204 mg/l NO,-N. An epidemiologic study of 102 children aged 1–8 conducted in Washington County, Illinois, did not show that ingestion of water with a nitrate concentration of 22–111 mg/l NO, –N was related to increasing methaemoglobin levels nor that the children had high or above normal methaemoglobin levels. The potential for transmission of infectious waterborne disease in this area was demonstrated, however, as a large percentage of the wells used for drinking water contained high numbers of total and faecal conforms.

Zeman, C.L., et al. 2002. "Exposure methodology and findings for dietary nitrate exposures in children of Transylvania, Romania." J.Expo.Anal.Environ.Epidemiol. 12(1):54-63. http://www.ncbi.nlm.nih.gov/pubmed/11859433

Abstract AIMS: The primary focus of this exposure assessment work involved developing an exposure model and determining a numerical point estimate of the amount of biologically relevant nitrate/nitrite exposure that occurred for each child in the study. This assessment was done in support of two epidemiological studies. The first study was an epidemiological cohort study (three cohorts based on nitrate/nitrite exposure) that explored the relationship between high nitrate/nitrite exposure and neuropsychological development. The second study was a nested case/control study (cases of methemoglobinemia versus disease-free controls) that sought to explore the relationship between MHG and various risk factors for the disease. METHODS: This work uses both dietary survey and environmental sampling and modeling in order to develop two point estimates of nitrate exposure in milligrams per kilogram per day of nitrite (the biologically active form of the hemoglobin-oxidizing agent) for the first 6 months of the child's life (2-months-of-age and 6- months-of-age point estimates). Methodologies included proxy interviews of primary caregivers, review of existing medical and environmental sampling and analysis. RESULTS: Exposure to nitrate--nitrogen (with final calculations converted to the biologically active form of the toxin, nitrite) was categorized as high, medium, and low as determined from the distribution of the data derived from final exposure calculations at both the 2-months-of-age point estimate and at the 6-months- of-age point estimate. These tertiles correspond to greater-than-or-equal1.5 mg/kg/day nitrite-nitrogen for high-exposure individuals, <1.5-> or = 0.1 mg/kg/day for medium-exposure individuals, and <0.1 19 mg/kg/day for low-exposure individuals. Analyses illustrate that over 90% of the nitrate exposure occurred through the consumption of liquids (water) at the 2-months-of-age point estimate while at the 6- months-of-age point estimate, a 10-fold change in the amount of solid consumables occurred. CONCLUSIONS: Final exposure calculations were well differentiated into three tertiles based on a point estimate of average daily intake of nitrite in milligrams per kilogram body weight per day at roughly 2 and 6 months of age. These categories of exposure, based on the exposure model point estimate, correspond well with the exposure estimates as estimated only on the basis of cohort status and their corresponding nitrate/nitrite well water levels. Comparisons of these two sets of data illustrate that following the MHG incident, Cohort II shifted places with Cohort I to become the high-exposure cohort. Further, the predictive ability of the exposure assessment in regard to the outcome of MHG was estimated using a Likelihood Ratio and Pearson's Crosstab analysis. This was performed on the 2-month-of-age point estimate. Likelihood Ratio and Pearson's chi-square were 39.40 and 33.74, respectively, with a probability of achieving these fits by chance alone of <0.0001. This indicates clearly that the children who experienced MHG were also the children at the 2-month-of-age point estimate who had received the highest exposure to nitrate/nitrite through their diet.

Gupta, S.K., et al. 1999. "Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water." Bull.World Health Organ 77(9):749-753. http://www.ncbi.nlm.nih.gov/pubmed/10534899

Abstract An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents.

Shearer, L.A., et al. 1972. "Methemoglobin levels in infants in an area with high nitrate water supply." Am.J.Public Health 62(9):1174-1180. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1530503/pdf/amjph00731-0006.pdf

A study of methemoglobin levels in infants from birth through six months showed that even healthy babies not exposed to excessive nitrate levels in diets have higher levels when young. Babies with diarrhea or respiratory illness had the highest levels in this population. Ingestion of 20 water or formula high in nitrates appears to increase the frequency of elevated methemoglobin. More than 60% of formulae showed bacterial contamination. Long-term consequences should be investigated.

MMWR. 1996. "Spontaneous Abortions Possibly Related to Ingestion of Nitrate-Contaminated Well Water - LaGrange County, Indiana, 1991-94." Morbidity and Mortality Weekly Report 45(26):July 5, 1996. http://www.cdc.gov/mmwr/preview/mmwrhtml/00042839.htm

Tsezou, A., et al. 1996. "High nitrate content in drinking water: cytogenetic effects in exposed children." Arch.Environ.Health 51(6):458-461. http://www.ncbi.nlm.nih.gov/pubmed/9012325

Abstract The potential genotoxicity of nitrates and nitrites-contaminants of drinking water that have been implicated in carcinogenesis-was investigated in this study. Sister chromatid exchanges and frequency of chromatid/chromosome aberrations were studied in peripheral blood lymphocytes of 70 children who were 12-15 y of age. These children were permanent residents in geographical areas of Greece, where elevated concentrations of nitrates (i.e., 55.70-87.98 mg/l) existed in drinking water. The control group comprised 20 healthy children who resided in areas with very low nitrate concentrations (i.e., 0.7 mg/l). A significant increase in the mean number of chromatid/chromosome breaks was observed in children exposed to nitrate concentrations that exceeded 70.5 mg/l (p < .01), but there was no significant increase in the mean number of sister chromatid exchanges per cell. The results indicate that chronic administration of elevated concentrations of nitrate in drinking water has the capability of inducing cytogenetic effects.

Thorpe, N., and A. Shirmohammadi. 2005. "Herbicides and nitrates in groundwater of Maryland and childhood cancers: a geographic information systems approach." J Environ Sci Health C.Environ Carcinog.Ecotoxicol.Rev. 23(2):261-278. http://www.ncbi.nlm.nih.gov/pubmed/16291529

Abstract This hypothesis-generating study explores spatial patterns of childhood cancers in Maryland and investigates their potential associations with herbicides and nitrates in groundwater. The Maryland Cancer Registry (MCR) provided data for bone and brain cancers, leukemia, and lymphoma, for ages 0-17, during the years 1992-1998. Cancer clusters and relative risks generated in the study indicate higher relative risk areas and potential clusters in several counties. Contingency table analysis indicates a potential association with several herbicides and nitrates. Cancer rates for the four types have a crude odds ratio (OR) = 1.10 (0.78-1.56) in relationship to atrazine, and an OR = 1.54 (1.14-2.07) for metolachlor. Potential association to mixtures of three compounds give an OR = 7.56 (4.16-13.73). A potential association is indicated between leukemia and nitrates, OR = 1.81 (1.35-2.42), and bone cancer with metolachlor, OR = 2.26 (0.97-5.24). These results give insight to generate a hypothesis of the potential association between exposure to these herbicides and nitrates and specific types of childhood cancer. 21

Moller, H. 1997. "Work in agriculture, childhood residence, nitrate exposure, and testicular cancer risk: a case-control study in Denmark." Cancer Epidemiol.Biomarkers Prev. 6(2):141- 144. http://www.ncbi.nlm.nih.gov/pubmed/9037566

Abstract A population-based case-control study in Denmark investigated the hypothesis that parental occupation in agriculture increases the risk of testicular cancer in the offspring. Other factors investigated were: childhood residence on a farm, in the country, or in an area with high nitrate concentration in ground water, and the subjects' own occupation in agriculture. The only association that emerged was with childhood residence in the high-nitrate area. The excess risk was, however, confined entirely to men who did not grow up on a farm or in the country. This makes it very unlikely that nitrate exposure per se should be responsible. Further analyses revealed that the excess risk was confined largely to men who grew up in nonrural areas within Arhus County, where Denmark's second largest city is located, and an excess risk was also seen among men who grew up in Copenhagen Municipality, which is the center of the most urban area in Denmark. The geographic pattern of incidence is stronger for the area of childhood residence than for the area of residence at the time of diagnosis. This supports indirectly the idea that testicular cancer is caused by unidentified factors early in life. van Maanen, J.M., et al. 2000. "Does the risk of childhood diabetes mellitus require revision of the guideline values for nitrate in drinking water?" Environ.Health Perspect. 108(5):457-461. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1638059/

Abstract

In recent years, several studies have addressed a possible relationship between nitrate exposure and childhood type 1 insulin-dependent diabetes mellitus. The present ecologic study describes a possible relation between the incidence of type 1 diabetes and nitrate levels in drinking water in The Netherlands, and evaluates whether the World Health Organization and the European Commission standard for nitrate in drinking water (50 mg/L) is adequate to prevent risk of this disease. During 1993-1995 in The Netherlands, 1,104 cases of type 1 diabetes were diagnosed in children 0-14 years of age. We were able to use 1,064 of these cases in a total of 2,829,020 children in this analysis. We classified mean nitrate levels in drinking water in 3,932 postal code areas in The Netherlands in 1991-1995 into two exposure categories. One category was based on equal numbers of children exposed to different nitrate levels (0.25-2.08, 2.10-6.42, and 6.44- 41.19 mg/L nitrate); the other was based on cut-off values of 10 and 25 mg/L nitrate. We determined standardized incidence ratios (SIRs) for type 1 diabetes in subgroups of the 2,829,020 children with respect to both nitrate exposure categories, sex, and age and as compared in univariate analysis using the chi-square test for trend. We compared the incidence rate ratios (IRRs) by multivariate analysis in a Poisson regression model. We found an effect of increasing age of the children on incidence of type 1 diabetes, but we did not find an effect of sex or of nitrate concentration in drinking water using the two exposure categories. For nitrate levels > 25 mg/L, an increased SIR and an increased IRR of 1.46 were observed; however, this increase was not statistically significant, probably because of the small number of cases (15 of 1,064). We concluded that there is no convincing evidence that nitrate in drinking water at current exposure levels is a risk factor for childhood type 1 diabetes mellitus in The Netherlands, although a threshold value > 25 mg/L for the occurrence of this disease can not be excluded. 22

Moltchanova, E., et al. 2004. "Zinc and nitrate in the ground water and the incidence of Type 1 diabetes in Finland." Diabet.Med. 21(3):256-261. http://www.ncbi.nlm.nih.gov/pubmed/15008836

Abstract AIMS: In Finland, the risk of childhood Type 1 diabetes varies geographically. Therefore we investigated the association between spatial variation of Type 1 diabetes and its putative environmental risk factors, zinc and nitrates. METHODS: The association was evaluated using Bayesian modelling and the geo-referenced data on diabetes cases and population. RESULTS: Neither zinc nor nitrate nor the urban/rural status of the area had a significant effect on the variation in incidence of childhood Type 1 diabetes. CONCLUSIONS: The results showed that although there was no significant difference in incidence between rural and urban areas, there was a tendency to increasing risk of Type 1 diabetes with the increasing concentration of NO3 in drinking water. The fact that no significant effect was found may stem from the aggregated data being too crude to detect it.

Tajtakova, M., et al. 2006. "Increased thyroid volume and frequency of thyroid disorders signs in schoolchildren from nitrate polluted area." Chemosphere 62(4):559-564. http://www.ncbi.nlm.nih.gov/pubmed/16095667

Abstract Thyroid volume (ThV) and echogenicity by ultrasound were estimated in 324 schoolchildren (aged between 10 and 13-years) from high nitrate area (HNA) located in agricultural lowland with high nitrate drinking water supply (51-274 mg/l) from shallow wells. The data were compared to children of the same age from low nitrate area (LNA) consisting of 168 children from the neighboring area with very low nitrate (< 2 mg/l) drinking water and of 596 children from the city of Kosice located in a vicinity of LNA and also supplied by low nitrate water. Blood samples were obtained from 315 willing children from HNA and 109 children from LNA and the levels of thyrotropin (TSH), total thyroxine (TT4), free triiodothyronine (FT3) and thyroperoxidase antibodies (anti-TPO) in serum were determined. ThV (mean +/- SE) in 10-year (5.10 +/- 0.14 ml) and 13-year (5.97 +/- 0.11 ml) old children from HNA was significantly higher than that in two groups of respective age from LNA, 4.58 +/- 0.17 (p < 0.02) and 5.23 +/- 0.15 ml (p < 0.05), and from the city of Kosice, 4.77 +/- 0.10 ml (p < 0.05) and 4.87 +/- 0.1 0ml (p < 0.0001). The frequency of hypoechogenicity in HNA was also significantly higher than that in pooled LNA plus Kosice, 13.7% vs. 4.7% (p < 0.01) in 10-year and 10.6% vs. 5.7% (p < 0.03) in 13-year, respectively. The frequency of TSH level in the range of subclinical hypothyroidism (> 4.0 mU/l) in pooled age groups from HNA was 13/324 (4.0%) and that of positive anti-TPO was 8/324 (2.5%), while 23 no case of either increased TSH or positive anti-TPO was found in 109 children from LNA. Finally, no differences in the levels of TT4 and FT3 were found between HNA and LNA. Roth, A.C., et al. 1987. "Evaluation of the developmental toxicity of sodium nitrite in Long- Evans rats." Fundam.Appl.Toxicol. 9(4):668-677. http://www.ncbi.nlm.nih.gov/pubmed/3692023

Abstract Sodium nitrite administered in the drinking water to Long-Evans rats during pregnancy and lactation severely affected erythropoietic development, growth, and mortality in their offspring. Pregnant rats were maintained throughout gestation on 0.5, 1, 2, or 3 g NaNO2/liter. There were no significant differences between treated and control litters at birth. Thereafter, pups of treated dams on 2 and 3 g NaNO2/liter gained less weight, progressively became severely anemic, and began to die by the third week postpartum. By the second week postpartum, hemoglobin levels, RBC counts, and mean corpuscular volumes of these pups were all drastically reduced compared to controls. Blood smears showed marked anisocytosis and hypochromasia. Gross chylous serum lipemia and fatty liver degeneration were noted. Histopathology demonstrated cytoplasmic vacuolization of centrilobular hepatocytes and decreased hematopoiesis in bone marrow and spleen. Administration of 1 g NaNO2/liter resulted in hematological effects but did not affect growth or mortality. NaNO2 (0.5 g/liter) was at or near the no observed effect level. Cross-fostering indicated that treatment during the lactational period was more instrumental in producing lesions than treatment during the gestational period. The data presented are consistent with the lactational induction of severe iron deficiency in the neonate.

Inoue, T., et al. 2004. "Increases in serum nitrite and nitrate of a few-fold adversely affect the outcome of pregnancy in rats." J.Pharmacol.Sci. 95(2):228-233. http://www.ncbi.nlm.nih.gov/pubmed/15215647

Abstract The objective of this study was to evaluate serum nitrite and nitrate (nitrite/nitrate) concentrations that affect adversely pregnancy outcome. Pregnant rats, from day 2 to day 8 of pregnancy, were daily given subcutaneously several doses (5, 10, and 30 mg/rat) of diethylenetriamine-nitric oxide (DETA/NO). Serum nitrite/nitrate concentrations were measured using an HPLC system. Serum nitrite/nitrate concentrations increased dose-dependently with DETA/NO. Effects of DETA/NO on pregnancy outcome were assessed on day 14 of pregnancy. In rats given 5 mg DETA/NO, there was a significant increase in serum nitrite/nitrate concentrations (49.2 vs 24.6 micromol/l, P<0.001), and both placental weight and fetal weight decreased compared to control rats. Macroscopic bleeding in placenta was frequently observed in rats given DETA/NO. We further studied effects of DETA/NO on cultured trophoblastic BeWo cells. DETA/NO added to the culture medium increased nitrite/nitrate concentrations in the medium in a dose-dependent manner. Nitrite/nitrate concentrations in the medium over four times the concentration of control decreased progesterone in the medium at 24 h after the application of DETA/NO. The hormonal secretion was not affected by DETA only. This study shows for the first time nitrite/nitrate concentrations affecting adversely pregnancy outcome and function of the trophoblastic cells.

Vorhees, C.V., et al. 1984. "Developmental toxicity and psychotoxicity of sodium nitrite in rats." Food Chem.Toxicol. 22(1):1-6. http://www.ncbi.nlm.nih.gov/pubmed/6537931 24

Abstract Sodium nitrite (NaNO2) was fed to male and female rats before and during breeding, to females only during gestation and lactation, and to their offspring after weaning (day 21 after birth) through day 90, at levels of 0, 0.0125, 0.025 or 0.05% (w/w) of the diet. Dams in a fifth group (positive controls) were given 4 mg/kg ip of the anti-mitotic/embryotoxic drug 5-azacytidine on day 16 of gestation. All offspring were reared by their natural dams and were evaluated blind with respect to treatment in a battery of standardized behavioural tests between 3 and 90 days of age. NaNO2 produced no significant reductions in parental body weight or food consumption, though it significantly increased offspring mortality and decreased weight gain at the two highest doses during the preweaning period. Functionally, NaNO2 delayed swimming development and decreased open-field activity. The open-field effect was not linearly dose dependent. In rats killed on day 90 after birth, NaNO2 produced no effects on brain or body weights. 5-Azacytidine produced evidence of substantially greater developmental toxicity than did NaNO2. NaNO2 produced a moderate degree of developmental toxicity, but no evidence was found to suggest that the central nervous system was the target organ for the toxic effects. The inclusion of tests of functional development added useful confirmatory evidence to the overall picture of NaNO2 toxicity.

Fan, A.M., and V.E. Steinberg. 1996. "Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity." Regul.Toxicol.Pharmacol. 23(1 Pt 1):35-43. http://www.ncbi.nlm.nih.gov/pubmed/8628918

Abstract In 1987, an evaluation of the nitrate drinking water standard was performed with a primary focus on the effects of nitrate on methemoglobinemia and reproductive/developmental effects (Fan et al. (1987). Regul. Toxicol. Pharmacol. 7, 135-148). The present review presents an updated overview and evaluation of the available information on the same health effects of nitrate and nitrite with an emphasis on data not included in the previous review, which should be used as a compendium to this report. Recent epidemiologic data have suggested an association between developmental effects in offspring and the maternal ingestion of nitrate from drinking water, but a definite conclusion on the cause and effect relationship cannot be drawn. Animal experimental data have shown reproductive toxicity associated with high exposure levels to nitrate or nitrite, which are not likely to be encountered in drinking water. No teratogenic effects were observed in rats, mice, rabbits, and hamsters tested. Several cases of methemoglobinemia have been reported in infants in the United States using water containing nitrate at levels higher than the current maximum contaminant level (MCL) of 45 ppm (mg/liter) nitrate (NO3) or 10 ppm nitrate-nitrogen (nitrate-N), but none at or lower than the MCL. The uncertainties in the data base are discussed, noting that no uncertainty factor was applied in deriving the MCL in order to account for the uncertainties that exist in the data base.

Heindel, J.J., et al. 1994. "Assessment of the reproductive and developmental toxicity of pesticide/fertilizer mixtures based on confirmed pesticide contamination in California and Iowa groundwater." Fundam.Appl.Toxicol. 22(4):605-621. http://www.ncbi.nlm.nih.gov/pubmed/8056207

Abstract 25

Pesticides and fertilizers, as used in modern agriculture, contribute to the overall low-level contamination of groundwater sources. In order to determine the potential of pesticide and fertilizer mixtures to produce reproductive or developmental toxicity at concentrations up to 100 x the median level found in groundwater, we prepared and studied two mixtures of pesticides and a fertilizer (ammonium nitrate). One mixture containing aldicarb, atrazine, dibromochloropropane, 1,2-dichloropropane, ethylene dibromide, and simazine plus ammonium nitrate was considered to be a representative of groundwater contamination in California (CAL). The other, containing alachlor, atrazine, cyanazine, metolachlor, metribuzin, and ammonium nitrate, simulated groundwater contamination in Iowa (IOWA). Each mixture was administered in the drinking water of either Swiss CD-1 mice during a Reproductive Assessment by Continuous Breeding study or pregnant Sprague-Dawley rats (gd 6-20) at three dose levels (1x, 10x, and 100x) where 1x was the median concentration of each pesticide component as determined in the groundwater surveys in California or Iowa. Unlike conventional toxicology studies, the purpose of this study was to evaluate the health effects of realistic human concentrations. Thus, the testing concentrations are probably well below the maximally tolerated dose. Propylene glycol was used as the solubilizer for the pesticides in drinking water formulations in both studies. In the reproductive study, neither mixture caused any clinical signs of toxicity, changes in food or water consumption, or body weight in either F0 or F1 mice at doses up to 100x the median groundwater concentrations. There were no treatment-related effects on fertility or any measures of reproductive performance of either the F0 or the F1 generation mice exposed to either CAL or IOWA at up to 100x. Similarly, measures of spermatogenesis, epididymal sperm concentration, percentage motile sperm, percentage abnormal sperm, and testicular and epididymal histology were normal. In the developmental study, CAL- or IOWA-exposed females did not exhibit any significant treatment-related clinical signs of toxicity. No adverse effects of CAL or IOWA were observed for measures of embryo/fetal toxicity, including resorptions per litter, live litter size, or fetal body weight. CAL or IOWA did not cause an increased incidence of fetal malformations or variations. In summary, administration of these pesticide/fertilizer mixtures at levels up to 100-fold greater than the median concentrations in groundwater supplies in California or Iowa did not cause any detectable reproductive (mice), general, or developmental toxicity (rats).

Shimada, T. 1989. "Lack of teratogenic and mutagenic effects of nitrite on mouse fetuses." Arch.Environ.Health 44(1):59-63. http://www.ncbi.nlm.nih.gov/pubmed/2916857

Abstract To assess embryotoxic effects of sodium nitrite, pregnant ICR mice were given drinking water containing sodium nitrite at a concentration of either 100 or 1000 mg/L on days 7-18 of gestation. There were no significant differences between treated and control groups in measures of developmental toxicity, e.g., litter size, fetal weight, and number of resorbed or dead fetuses. The incidences of external and skeletal malformations in fetuses of treated groups were not significantly different from those in the controls. No significant increase was observed in the frequency of gaps and breaks of liver cell chromosomes in fetuses exposed in utero to sodium nitrite. Teratogenic and mutagenic effects of sodium nitrite were absent in mice at the doses used

Globus, M., and D. Samuel. 1978. "Effect of maternally administered sodium nitrite on hepatic erythropoiesis in fetal CD-1 mice." Teratology 18(3):367-378. http://www.ncbi.nlm.nih.gov/pubmed/741389

Abstract 26

A commonly used food preservative, sodium nitrite, was administered to pregnant CD-1 mice at a concentration of 0.5 mg/mouse/day. Embryotoxic and teratogenic effects on the hemopoietic tissues and skeletons of their offspring, were evaluated. Fetal mortality, resorptions, the mean number of offspring per litter, the mean weight per embryo and the incidence of skeletal malformations, were not significantly different from controls. Hemopoietic cell suspensions, prepared from the livers of treated and control 14-, 16- and 18-day embryos, were cytocentrifuged onto microscope slides and differential counts were performed after staining with benzidine and Wright-Giemsa stain. The results indicate that maternally administered Na nitrite, stimulates fetal hepatic erythropoiesis. This was manifested in a statistically significant increase in the percentage of polychromatophilic erythroblasts and mature erythrocytes at 14 and 16 days of gestation, respectively. The possibility that Na nitrite may induce fetal methemoglobinemia is discussed and mechanisms responsible for the observed erythroid stimulation, are considered.

Gruener, N., et al. 1973. "Methemoglobinemia induced by transplacental passage of nitrites in rats." Bull.Environ.Contam Toxicol. 9(1):44-48. http://www.ncbi.nlm.nih.gov/pubmed/4780432

In the framework of the paper the incidence of infant methemoglobinemia in Cluj county, Romania was evaluated from medical records. The incidence rate ranged from 42 to 239 per 100,000 live births between 2000 and 2004. Bottle-babies less than 4 months are the age group most at risk of acquired methemoglobinemia. Well water was the main source of nitrate, though early incorporation of vegetable juices and table food into infant diet were also incriminated. Some recommendations for the management of methemoglobinemia in this area are described: introduce a low nitrate water supply scheme specially for infant feeding, education program for mothers to increase duration of breast feeding, further studies on long-term development effects on infants.

Dreosti, I.E., et al. 1983. "Teratogenic effect of extended administration of N-nitrosoethylurea and ethylurea/nitrite in rats." Res.Commun.Chem.Pathol.Pharmacol. 41(2):265-281. http://www.ncbi.nlm.nih.gov/pubmed/6635320

Abstract N-Nitrosoethylurea (ENU) and ethylurea (EU) with sodium nitrite (NaNO2) were administered daily by mouth to Hooded Wistar rats. Doses ranged from 1-50 mg ENU/kg body weight (day-1) and from 20-70 mg EU/kg body weight day-1 and extended over days 7 to 16 inclusive of pregnancy. Severe teratogenesis occurred in animals which received from 10 mg ENU/kg day-1. Fetal death predominated at intakes above 12.5 mg ENU/kg body wt. Many organ systems were affected by ENU, but developmental anomalies of the nervous system were most common, especially anophthalmia and hydrocephalus. With EU and nitrite severe teratogenesis was noted at levels above 50 mg EU/kg body weight. Maximum teratogenic potency was obtained when nitrite was administered at approximately 50% of the level of the EU dose. Terata arising from treatment with EU/nitrite were similar to those caused by ENU. However, unlike ENU-treated animals, litters from the EU/nitrite study were either severely malformed or barely affected at all. The erratic nature of the teratogenesis following treatment with EU/nitrite was not influenced by the presence of food in the stomach at the time of dosing. For both substances administered over a 10-day period, the threshold dose needed for obvious teratogenicity was considerably less than in all previously reported single-dose studies. 27

Rustia, M. 1975. "Inhibitory effect of sodium ascorbate on ethylurea and sodium nitrite carcinogensis and negative findings in progeny after intestinal inoculation of precursors into pregnant hamsters." J.Natl.Cancer Inst. 55(6):1389-1394. http://www.ncbi.nlm.nih.gov/pubmed/1206758

Abstract To assess their carcinogenic effects, the ethylnitrosourea (ENU) precursors, ethylurea and sodium nitrite, [were administered to pregnant hamsters as a single intragastic] dose on day 15 of gestation, or introduced into the cecum on day 14. Since sodium ascorbate (NaASC) inhibits the biosynthesis of nitrosamides, identical doses of the precursors were given concomitantly with NaASC. Progeny of mothers treater intragastrically developed significant incidences of neurogenic tumors of the peripheral nervous system, with a predominance in females. The concurrent administration of NaASC with ENU precursors prevented carcinogenic effects in the progency, whereas the simultaneous inoculation of the precursors into the cecum produced no carcinogenic effects in the offspring.

Rustia, M., and J. Schenken. 1976. "Transplacental effects of ethylnitrosourea precursors ethylurea and sodium nitrite in hamsters." Cancer Res.Clin.Oncol. 85(3):201-207. http://www.ncbi.nlm.nih.gov/pubmed/178112

Abstract Four simultaneous dosages of the ethylnitrosourea precursors, ethylurea and sodium nitrite, were administered intragastrically to pregnant hamsters at 100 mg/kg and 50 mg/kg respectively, from the 12- 15th days of pregnancy. The treatment induced multiple neurogenic tumors of the peripheral nervous system in the offspring. Female progeny developed a greater incidence and multiplicity of peripheral nervous system tumors with significantly shorter latencies than males, thus establishing evidence that the tumors were age and sex dependent. The tumors presented varied morphological patterns and upon transplantation, grew regularly, exhibiting their malignant nature. The possible influence of estrogenic hormones on the development and growth of peripheral nervous system tumors and comparative aspects of the relationship between prenatal and postnatal carcinogenesis with regard to the ensuing tumor spectra as a consequence of exposure to the same chemical agent, are discussed.

U.S. Food and Drug Administration. 1998. "A Fresh Look at Food Preservatives." http://www.cfsan.fda.gov/~dms/fdpreser.html

U.S. Department of Agriculture: Food Safety and Inspection Service. 2001. "Fact Sheets: Food Labeling - Additives in Meat and Poultry Products." http://www.fsis.usda.gov/Fact_Sheets/Additives_in_Meat_&_Poultry_Products/index.asp

U.S. Environmental Protection Agency. 2006. "2006 Edition of the Drinking Water Standards and Health Advisories." http://www.epa.gov/waterscience/criteria/drinking/dwstandards.pdf

U.S. Environmental Protection Agency. 2006. "Drinking Water Contaminants." http://www.epa.gov/safewater/contaminants/index.html 28

U.S. Environmental Protection Agency. 2005. "Priority List of Hazardous Substances for the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) Section 104(i)." http://www.atsdr.cdc.gov/cercla/

U.S. Environmental Protection Agency. 2001. "Lists of Lists: Consolidated List of Chemicals Subject to the Emergency Planning and Right-to-Know Act (EPCRA) and Section 112(r) of the Clean Air Act." http://www.epa.gov/ceppo/pubs/title3.pdf

Arbuckle TE, Hewitt D, Sherman GJ. Re: “Congenital malformations and maternal drinking water supply in rural South Australia: a case-control study” [Letter] Am J Epidemiol. 1986;124:344. http://www.ncbi.nlm.nih.gov/pubmed/3728454 Arbuckle TE, Sherman GJ, Corey PN, Walters D, Lo B. Water nitrates and CNS birth defects: a population-based case-control study. Arch Environ Health. 1988;43:162–167. http://www.ncbi.nlm.nih.gov/pubmed/3377550 Abstract The relation between maternal exposure to nitrates in drinking water and risk of delivering an infant with a central nervous system (CNS) malformation was examined by means of a case- control study in New Brunswick, Canada. All cases of CNS defects for a "high" and a "low" prevalence area of New Brunswick, for the years 1973-1983, were included in the study. Controls were selected randomly from the livebirth files for the province, matched on county of maternal residence and date of birth. One hundred and thirty (130) cases were identified and individually matched with two controls each. Individual water samples were collected from the case and control mother's address given on the birth or stillbirth records. The study revealed that the effect of nitrate exposure in water was modified by whether the source of the drinking water was a private well or a public municipal distribution system. Compared to a baseline nitrate level of 0.1 ppm, exposure to nitrate levels of 26 ppm from private well water sources was associated with a moderate, but not statistically significant, increase in risk (risk odds ratio = 2.30; 95% confidence interval = 0.73-7.29). If the source of drinking water was a municipal distribution system or a private spring, an increase in nitrate exposure was associated with a decrease in risk of delivering a CNS-malformed infant; however, these effect estimates were not statistically significant. The positive increase in risk with nitrate exposure from well water sources requires further study using a larger case series and a larger proportion of exposures to nitrate levels exceeding 5 ppm.

ATSDR 2001. Case Studies in Environmental Medicine: Nitrate/Nitrite Toxicity. Atlanta, GA:Agency for Toxic Substances and Disease Registry. http://www.atsdr.cdc.gov/csem/nitrate/docs/nitrate_nitrite.pdf

Avery AA. Infantile methemoglobinemia: reexamining the role of drinking water nitrates. Environ Health Perspect. 1999;107:583–586. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1566680/ 29

Abstract Ingestion of nitrates in drinking water has long been thought to be a primary cause of acquired infantile methemoglobinemia, often called blue baby syndrome. However, recent research and a review of historical cases offer a more complex picture of the causes of infantile methemoglobinemia. Gastrointestinal infection and inflammation and the ensuing overproduction of nitric oxide may be the cause of many cases of infantile methemoglobinemia previously attributed to drinking water nitrates. If so, current limits on allowable levels of nitrates in drinking water, which are based solely on the health threat of infantile methemoglobinemia, may be unnecessarily strict Bruning-Fann CS, Kaneene JB. The effects of nitrate, nitrite and N-nitroso compounds on human health: a review. Vet Hum Toxicol. 1993;35:521–538. http://www.ncbi.nlm.nih.gov/pubmed/8303822 Abstract The effects of nitrate, nitrite, and N-nitroso compounds on human health are reviewed. Special emphasis has been placed on the role of these compounds on infant methemoglobinemia and gastric cancer. The discussion on methemoglobinemia includes the source of nitrate or nitrite, diagnosis, treatment, prevention and the contributions of age, gastric pH, gastrointestinal illness, and ingestion of vitamin C to this illness. The maternal transfer of these compounds and the potential effect on fetal death and malformation are also described. The etiology and development of gastric cancer is reviewed as well as the roles of nitrate, nitrite, and N-nitroso compounds in this disease. Endogenous nitrosation and the experimental and epidemiologic evidence linking these compounds to gastric cancer is examined. Other sections include adult methemoglobinemia and acute toxicity, hypo- and hypertension, Balkan nephropathy, slowing of motor reflexes in children, nitrate esters dependence. Sources of nitrate, nitrite, and N-nitroso compounds are detailed. Future areas of research are given.

Comly HH. Landmark article Sept 8, 1945: cyanosis in infants caused by nitrates in well-water. JAMA. 1987;257:2788–2792. http://www.ncbi.nlm.nih.gov/pubmed/3553637 or http://jama.jamanetwork.com/article.aspx?articleid=366259 Abstract Two examples of a previously unrecognized condition which may be confused with congenital heart disease are cited in this report. The condition may occur anywhere in rural areas where well water is used in infant feeding.

Croen LA, Todoroff K, Shaw GM. Maternal exposure to nitrate from drinking water and diet and risk for neural tube defects. Am J Epidemiol. 2001;153:325–331. http://www.ncbi.nlm.nih.gov/pubmed/11207149 Abstract In this population-based case-control study conducted in California between June 1989 and May 1991, the authors investigated the association between maternal periconceptional exposure to nitrate from drinking water and diet and risk for neural tube defects. The mothers of 538 cases and 539 nonmalformed controls were interviewed regarding residential history, consumption of 30 tap water at home, and dietary intake during the periconceptional period. Dietary nitrate exposure was not associated with increased risk for neural tube defects. Exposure to nitrate in drinking water at concentrations above the 45 mg/liter maximum contaminant level was associated with increased risk for anencephaly (odds ratio (OR) = 4.0, 95% confidence interval (CI): 1.0, 15.4), but not for spina bifida. Increased risks for anencephaly were observed at nitrate levels below the maximum contaminant level among groundwater drinkers only (OR = 2.1, 95% CI: 1.1,4.1 for 5-15 mg/liter; OR = 2.3, 95% CI: 1.1, 4.5 for 16-35 mg/liter; and OR = 6.9, 95% CI: 1.9, 24.9 for 36-67 mg/liter compared with <5 mg/liter). Adjustment for identified risk factors for anencephaly did not substantially alter these associations, nor did control for maternal dietary nitrate, total vitamin C intake, and quantity of tap water consumed. The lack of an observed elevation in risk for anencephaly in association with exposure to mixed water containing nitrate at levels comparable with the concentration in groundwater may indicate that something other than nitrate accounts for these findings.

Felsot AS. Re-examining the link between nitrates and “blue baby” syndrome: a necessary first step for managing ground water quality to protect public health. Agric Environ News. 1998;150:1–14. http://aenews.wsu.edu/Oct98AENews/aenewsoctober98.htm#anchor545063

Gelperin A, Moses VK, Bridger C. 1975. Relationship of high nitrate community water supply to infant and fetal mortality Ill Med J 147155–157.157, 186. http://www.ncbi.nlm.nih.gov/pubmed/236221 Environmental Working Group. Pouring it on: the health effects of Nitrate Exposure. http://www.ewg.org/node/7712

Hanukoglu A, Danon PN. Endogenous methemoglobinemia associated with diarrheal disease in infancy. J Pediatr Gastroenterol Nutr. 1996;23:1–7. http://journals.lww.com/jpgn/Abstract/1996/07000/Endogenous_Methemoglobinemia_Associate d_with.1.aspx Summary: Infantile diarrhea is sometimes associated with methemoglobinemia. To determine the significance of intestinal bacterial infection or overgrowth and other predisposing factors in this entity, we evaluated prospectively 45 consecutive patients who were admitted for gastroenteritis and methemoglobinemia between March 1980 and September 1992. All the patients were younger than 3 months of age. In 95% of them, methemoglobinemia occurred between the ages of 15 days to 2 months. The peak mean methemoglobin concentration was 9.4% (range, 2.4- 57%). Although stool cultures were positive in only 22% of the infants, the epidemiologic data strongly suggested a bacterial or viral etiology in our study population: for 12 years, there was a significant decrease in the annual incidence of methemoglobinemia associated with diarrhea in parallel to the decrease in infantile diarrhea due to known pathogens throughout the country in the same period. There was also a marked seasonal variation in the incidence of the disease, with two peaks in January and the summer months when viral and bacterial infections, respectively, are prevalent. Failure to thrive and low admission-weight percentiles were associated with methemoglobinemia in most of the patients and diarrhea lasting ≥7 days in 22 (49%) patients. The blood pH and the degree of acidosis did not correlate with the severity of methemoglobinemia. All the patients were formula fed. In the etiology of methemoglobinemia in 31 infants with enteritis, viral and bacterial pathogens appear to play an important role by altering intestinal flora. Breast feeding appears to protect against this entity. Hegesh E, Shiloah J. Blood nitrates and infantile methemoglobinemia. Clin Chim Acta. 1982;125:107–115. http://www.ncbi.nlm.nih.gov/pubmed/7139953 Abstract Infantile methemoglobinemia is of much more frequent occurrence among hospitalized newborns and young infants than is generally appreciated. When present, acute diarrhoea of different etiologies is almost exclusively the cause of hospitalization. No correlation between this condition and ingestion of food or water containing high concentrations of nitrates or nitrites was found. This study demonstrates for the first time that high blood nitrates are a regular part of the syndrome. The evidence presented is interpreted as indicating that diarrhoea results in endogenous, de novo synthesis of nitrites, and this is the principal cause of infantile methemoglobinemia

Johnson CJ, Kross BC. Continuing importance of nitrate contamination of groundwater and wells in rural areas. Am J Ind Med. 1990;18:449–456. http://www.ncbi.nlm.nih.gov/pubmed/2248249 Abstract The contamination of groundwater and rural drinking water supplies by nitrates from livestock and human excrement, other organic waste, or chemical fertilizers is a potential hazard throughout the world. Infant illness and death from nitrate-induced methemoglobinemia is probably often misdiagnosed, perhaps as sudden infant death syndrome, and certainly contributes to the national infant death rate statistics. A 1950 report listed 144 cases of infant methemoglobinemia with 14 deaths in one 30-month period in Minnesota. Infant deaths resulting from misdiagnosis of this preventable, treatable intoxication were still occurring as recently as 1986 in South Dakota. In this state, about 39% of dug or bored wells were unsafe due to high nitrate content, compared with 22% of drilled wells and 16% of driven wells. Properly constructed wells more than 30 m deep are more likely to be safe. Groundwater concentrations of nitrate may be unsafe for consumption, and standards are needed to regulate such contamination. Such standards could serve as guidelines and could be enforceable in the case of water systems dependent on wells.

Schmitz JT. Methemoglobinemia—a cause of abortions? Preliminary report. Obstet Gynecol. 1961;17:413–415. http://journals.lww.com/greenjournal/Citation/1961/04000/Methemoglobinemia_A_Cause_of_A bortions__.2.aspx

Scragg RK, Dorsch MM, McMichael AJ, Baghurst PA. Birth defects and household water supply. Epidemiological studies in the Mount Gambier region of South Australia. Med J Aust. 1982;2:577–579. http://www.ncbi.nlm.nih.gov/pubmed/7162445 Abstract 32

We report a descriptive study indicating a localised excess of congenital malformations in Mount Gambier, South Australia, and summary results of a subsequent case-control study showing an association between the occurrence of congenital malformations and the consumption of underground water by pregnant women. The internal cohesion of the data analyses, and the plausibility conferred by experimental evidence, suggests that the underground water, and its elevated concentration of nitrates, may warrant further consideration as a source of human teratogens.

Tabacova S, Balabaeva L, Little RE. Maternal exposure to exogenous nitrogen compounds and complications of pregnancy. Arch Environ Health. 1997;52:341–347. http://www.ncbi.nlm.nih.gov/pubmed/9546756 Abstract Increased lipid peroxidation and reduced antioxidant activity have been reported with pregnancy complications. Given that exogenous oxidants stimulate formation of lipid peroxides, the authors investigated the relationship between exposure to nitrogen-oxidizing species and pregnancy complications and took into account markers of antioxidant and oxidant status. The study sample included pregnant women who were from an area polluted by oxidized nitrogen compounds. Methemoglobin, a biomarker of individual exposure, was determined, as were measures of oxidant/antioxidant status, including glutathione balance and lipid peroxide levels. Only 10 women experienced normal pregnancies. The most common complications were anemia (67%), threatened abortion/premature labor (33%), and signs of preeclampsia (23%). Methemoglobin was elevated significantly in all three conditions, compared with normal pregnancies. Reduced:total glutathione decreased, whereas lipid peroxide levels increased. These results suggest that maternal exposure to environmental oxidants can increase the risk of pregnancy complications through stimulation of the formation of cell-damaging lipid peroxides and from a decrease in maternal antioxidant reserves.

Walton G. Survey of literature relating to infant methemoglobinemia due to nitrate-contaminated water. Am J Public Health. 1951;41:986–996. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1525621/pdf/amjphnation00426-0083.pdf

Shuval HI, Gruener N. Epidemiological and toxicological aspects of nitrates and nitrites in the environment. Am J Public Health 1992; 62(8):1045–52. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1530374/pdf/amjph00730-0007.pdf

Abstract Studies reported here are part of a program to reevaluate current nitrate and nitrite standards for water and food. Included are field studies among infants in areas with varying concentrations of nitrates in water supplies as well as experimental studies on laboratory animals. Findings are reported and discussed. The detection of irreversible electrical brain activity changes in rats receiving low doses of nitrites in drinking water is of particular concern 33

National Research Council, Committee on Toxicology. Nitrate and nitrite in drinking water. Washington, DC: National Academies Press; 1995.

Nolan BT, Hitt KJ, Ruddy BC. Probability of nitrate contamination of recently recharged ground waters in the conterminous United States. Environ Sci Technol 36(10):2138–45. http://water.usgs.gov/nawqa/nutrients/pubs/est_v36_no10/

Kross BC, Ayebo AD, Fourtes LJ. Methemoglobinemia: nitrate toxicity in rural America. Am Fam Physician 1992;46:183–88. http://www.ncbi.nlm.nih.gov/pubmed/1621630

Abstract Nitrates are frequently found in vegetables and ground water. Nitrate levels in ground water have increased over the past two decades because of the heightened use of nitrogenous fertilizers. Following ingestion, nitrates are converted to nitrites by fecal organisms. Nitrites are absorbed and form methemoglobin, which interferes with the oxygen-carrying capacity of hemoglobin. Infants are particularly susceptible to nitrate poisoning because fetal hemoglobin is more readily oxidized to methemoglobin. In infants, the most common source of nitrate exposure is well water, which is mixed with infant formula. Affected infants may present with asymptomatic cyanosis, which can progress to dyspnea and lethargy or coma. Blood methemoglobin concentrations are elevated. Treatment consists of the administration of oxygen and intravenous and oral methylene blue.

Keating JP, Lell ME, Strauss AW, Zarkowsky H, Smith GE. Infantile methemoglobinemia caused by carrot juice. N Engl J Med 1973;288(16):824–6. http://www.ncbi.nlm.nih.gov/pubmed/4693932

Sadeq, M., Moe, C.L.,Attarassi, B., Cherkaouil, L., Elauad, R., & Idrissi, L. (2008) Drinking water nitrate and prevalence of methemoglobinemia among infants and children agec 1-7 years I Moroccan areas. International Journal of Environmental Health http://www.ncbi.nlm.nih.gov/pubmed/18155958

Abstract

CONTEXT: Nitrate is ubiquitous in environmental media (air, water and soil) and other sources (some medicines, inorganic fertilizers and household's chemicals). It is a hemoglobin-oxidizing agent that can cause methemoglobinemia. The effect of nitrate on infants is well known but less is known about nitrate-induced methemoglobinemia in young children. 34

METHOD: Two cross-sectional studies were carried out in Salé, Morocco to determine the prevalence of methemoglobinemia among 411 infants and children aged 1-7 years in two adjacent areas that were similar in terms of the air quality, available vegetables and medicines but different in terms of the drinking water quality (nitrate-contaminated well water versus municipal water).

RESULTS: In the exposed area, nitrate concentration was measured in 78 wells and ranged from 15.39 to 246.90mg/l as NO3-. Nitrate levels were higher than 50mg/l in 69.2% of the surveyed wells, and 64.2% of the participants were drinking nitrate contaminated well waters. The prevalence of methemoglobinemia among study children was 36.2% in the exposed area, and 27.4% in the non-exposed area. Study children drinking well water with a nitrate concentration >50mg/l were significantly more likely to have methemoglobinemia than those drinking well water with a nitrate concentration <50mg/l (p=0.001 at 95% CI=[1.22-2.64]) or than those drinking municipal water (p<0.01 at 95% CI=[1.16-2.21]). In the exposed area, the mean methemoglobin (MetHb) level increased with age (R2= 0.79, p=0.04), whereas in the unexposed area, the mean MetHb level remained relatively stable in the first 6 years of life (R2=0.21, p=0.44). Mean MetHb was normal when the nitrate concentration in water was below 50mg/l as NO3-, and reached an abnormal level, when the nitrate concentration in water ranged between 50 and 90mg/l as NO3-. This last level was statistically similar to mean MetHb at nitrate level above 90mg/l as NO3- (up to 246.9mg/l as NO3-). No association was observed between methemoglobinemia prevalence and gender. This is the first study about methemoglobinemia conducted in Morocco.

Fewtrell, L. (2004) Drinking-water nitrate, methemoglobinemia, and global burden of disease: a discussion. Environmental Health Perspectives. http://www.ncbi.nlm.nih.gov/pubmed/15471727

Abstract On behalf of the World Health Organization (WHO), I have undertaken a series of literature- based investigations examining the global burden of disease related to a number of environmental risk factors associated with drinking water. In this article I outline the investigation of drinking-water nitrate concentration and methemoglobinemia. The exposure assessment was based on levels of nitrate in drinking water greater than the WHO guideline value of 50 mg/L. No exposure-response relationship, however, could be identified that related drinking-water nitrate level to methemoglobinemia. Indeed, although it has previously been accepted that consumption of drinking water high in nitrates causes methemoglobinemia in infants, it appears now that nitrate may be one of a number of co-factors that play a sometimes complex role in causing the disease. I conclude that, given the apparently low incidence of possible water-related methemoglobinemia, the complex nature of the role of nitrates, and that of individual behavior, it is currently inappropriate to attempt to link illness rates with drinking- water nitrate levels. 35

Pollack, E.S. & Pollack, C.V. (1994) Incidence of subclinical methemoglobinemia in infants with diarrhea. Annals of Emergency Medicine. http://www.ncbi.nlm.nih.gov/pubmed/8092592

Abstract

STUDY HYPOTHESIS: Infants with diarrhea are at a greater-than-recognized risk of developing methemoglobinemia.

DESIGN: Prospective clinical study.

SETTING: A university hospital pediatric emergency department.

PARTICIPANTS: Consecutive infants under 6 months of age with a history of diarrhea of more than 24 hours' duration not associated with vomiting.

INTERVENTIONS: Blood samples were obtained for methemoglobin (MHgb) assay (normal, 0.4% to 1.5%) and electrolytes. Treatment interventions were performed as clinically indicated. Patients with elevated MHgb levels subsequently underwent hemoglobin electrophoresis to exclude congenital methemoglobinemia.

RESULTS: Forty-three patients were studied; 27 (64%) had elevated MHgb levels and 13 were cyanotic. Five patients received infusions of methylene blue for methemoglobinemia. All patients recovered without sequelae. There was a strong correlation between weight at or below the tenth percentile for age and the development of methemoglobinemia. Contrary to previous studies, there was no correlation between incidence or severity of methemoglobinemia and acidosis, hyperchloremia, or positive microbiologic studies.

CONCLUSION: In ill infants with diarrhea, particularly those who are small for age, consideration should be given to screening for methemoglobinemia.

CDC (1993) Methemoglobinemia in an infant - - Wisconsin, 1992 Morbidity & Mortality Weekly http://www.ncbi.nlm.nih.gov/pubmed/8450825

Abstract Methemoglobinemia among infants is a rare and potentially fatal condition caused by genetic enzyme deficiencies, metabolic acidosis, and exposure to certain drugs and chemicals. The most widely recognized environmental cause of this problem is ingestion of nitrate-containing water. 36

Ingestion of copper causes abdominal discomfort, nausea, diarrhea, and in cases of high-level exposure, vomiting. This report summarizes an investigation by the Division of Health, Wisconsin Department of Health and Social Services of methemoglobinemia associated with ingestion of nitrate- and copper-containing water in an infant during 1992.

Durosev, D. (1979) Toxic methemoglobinemia in newborns and infants. Bilt Hematol Transfuz http://www.ncbi.nlm.nih.gov/pubmed/552247

Abstract It has been analised cases of toxic methemoglobinemia in newborns and sucklings on patients of children department city hospital Sombor in period from 1968 to 1979. In all patients methemoglobinemia was caused with wellwater containing nitrates. Originate of this state depends on: growth of child because in first few month of life they have immature methemoglobin reductase in erythrocyte, hypovitaminosis C and gastrointestinal disfunction. Diagnosis was based on anamnestical data that was used water from unhygenic well, on cyanosis of various intensity and that have disappeared during the treatment with vitamin C and not give any recurrence later. In last years number of such cases is decreasing because of better suply with proper drinking water. Further decrease can be achieved with health education and prophylactic peroral consumption of vitamin C in predisposing regions. Venkateswari R, Ganesh R, Deenadayalan M, Mahender E, Ramachandran B, Janakiraman (2007) Transient Methemoglobinemia in an infant. Indian Journal of pediatrics. http://www.ncbi.nlm.nih.gov/pubmed/18057688

Abstract We report a case of transient methemoglobinemia in an infant due to gastroenteritis. Methemoglobinemia should be suspected in infants with a history of diarrhea and cyanosis that is out of proportion to the history and clinical examination. Methemoglobinemia can be life threatening, but outcome is good when treated with IV methylene blue.

Benini, D., Vino, L., & Fanos, V. (1998) Acquired methemoglobinemia: a case report. Pediatr Med Chir 20(6): 411-413. http://www.ncbi.nlm.nih.gov/pubmed/10335542

Abstract When an infant presents severe cyanosis which is not associated with respiratory distress, methaemoglobinemia should always be suspected. In children its main inducers are contaminated water or vegetable broths with high nitrate levels (especially spinach and carrots) used to prepare powdered formula or soups. Children affected with methaemoglobinemia have a peculiar lavender colour. Blood from the heel sticks is chocolate-brown and does not become pink when exposed to room air. Diagnosis can be confirmed by excluding other causes of cyanosis and by spectrophotometric analysis of blood for methaemoglobin. When methaemoglobin's levels reach 60% or more, the patient will collapse and become comatose and may die. Therapy with methylene blue results in prompt relief. In this article we report a case of methaemoglobinemia due to the administration of powdered formula mixed with vegetable broths to a newborn aged 37

16 days. Furthermore we will present a short review of literature regarding methaemoglobinemia caused by toxic agents over the last 10 years.

Saito T, Takeichi S, Osawa M, Yukawa N, Huang XL. (2000) A case of fatal methemoglobinemia of unknown origin but presumably due to ingestion of nitrate. Int J Legal Med 113(3): http://www.ncbi.nlm.nih.gov/pubmed/10876989 Abstract A case of fatal methemoglobinemia (MetHb-emia) which was presumably due to ingestion of nitrate is presented. An unidentified man was taken to a local emergency hospital suffering from deep cyanosis and 7 h later he was found dead in the hospital bed. The post-mortem examination of the blood revealed a methemoglobin (MetHb) concentration of 78% and the concentrations of nitrate and nitrite were 1.50 and 0.76 microg/mL, respectively. Capillary gas chromatography coupled to mass spectrometry (GC-MS) and capillary gas chromatography with a nitrogen- phosphorus detector (NPD) were used to detect nitrates and nitrites in the blood.

Hanukoglu A, Danon PN. (1996) Endogenous methemoglobinemia associated with diarrheal disease in infancy. J Pediatr Gastroenterol Nutr. 23(1): 1-7 http://www.ncbi.nlm.nih.gov/pubmed/8811515 Abstract Infantile diarrhea is sometimes associated with methemoglobinemia. To determine the significance of intestinal bacterial infection or overgrowth and other predisposing factors in this entity, we evaluated prospectively 45 consecutive patients who were admitted for gastroenteritis and methemoglobinemia between March 1980 and September 1992. All the patients were younger than 3 months of age. In 95% of them, methemoglobinemia occurred between the ages of 15 days to 2 months. The peak mean methemoglobin concentration was 9.4% (range, 2.4- 57%). Although stool cultures were positive in only 22% of the infants, the epidemiologic data strongly suggested a bacterial or viral etiology in our study population: for 12 years, there was a significant decrease in the annual incidence of methemoglobinemia associated with diarrhea in parallel to the decrease in infantile diarrhea due to known pathogens throughout the country in the same period. There was also a marked seasonal variation in the incidence of the disease, with two peaks in January and the summer months when viral and bacterial infections, respectively, are prevalent. Failure to thrive and low admission-weight percentiles were associated with methemoglobinemia in most of the patients and diarrhea lasting > or = 7 days in 22 (49%) patients. The blood pH and the degree of acidosis did not correlate with the severity of methemoglobinemia. All the patients were formula fed. In the etiology of methemoglobinemia in infants with enteritis, viral and bacterial pathogens appear to play an important role by altering intestinal flora. Breast feeding appears to protect against this entity.

Chan, T.Y. (1996) Food-borne nitrates and nitrites as a cause of methemoglobinemia. Southeast Asian J Trop Med Public Health 27(1); 1890192 http://www.ncbi.nlm.nih.gov/pubmed/9031426

Abstract 38

Methemoglobinemia is a potentially fatal condition. Previous reports of toxic methemoglobinemia due to food-borne nitrates and nitrites are reviewed. Contamination of food during manufacture or degradation of nitrates in vegetables appear to be the most important factors. Some food items, such as refrigerated "dim-sum", stuffed pork and Chinese sausages, are very popular among some Asian populations; a stringent control against the excessive use of nitrates and nitrites is required in order to prevent outbreaks of toxic methemoglobinemia. Patients with glucose-6-phosphate dehydrogenase deficiency, a common condition in some Asian populations, may present with methemoglobinemia and intravascular hemolysis following exposure to oxidant drugs or chemicals. Methylene blue is inefficient and may exacerbate hemolysis in these patients; partial exchange transfusion may be required.

Gupta SK, Gupta RC, Seth AK, Gupta AB, Bassin JK, Gupta A. (2000) Methaemoglobinemia in areas with high nitrate concentration in drinking water. Natl Med J India 12(2): 58-61 http://www.ncbi.nlm.nih.gov/pubmed/10835850 Abstract

BACKGROUND: An epidemiological investigation was undertaken in all age groups to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking water.

METHODS: Five areas were selected with an average nitrate concentration (as nitrate) of 26, 45, 95, 222 and 459 mg nitrate ions/litre in drinking water. These areas were visited and the house schedule (containing name, age, sex and weight of the family members) prepared in accordance with the statistically designed protocol. In all, 178 persons, matched for age and weight, were selected and arranged in five age groups. They constituted 10% of the total population of each of these areas. A detailed history of the selected population was taken, medical examination conducted and blood samples taken to ascertain the level of methaemoglobin. The collected data were subjected to statistical analysis to ascertain a relationship between nitrate concentration and methaemoglobinaemia.

RESULTS: High nitrate concentrations cause severe methaemoglobinaemia (7%-27% of Hb) in all age groups, especially in the age group of less than 1 year and above 18 years. The lower levels of methaemoglobin in the age group of 1-18 years is probably due to better reserve of cytochrome b5 reductase activity and its adaptation to increasing nitrate concentration in water to compensate for methaemoglobinaemia in this age group.

CONCLUSION: We conclude that high nitrate ingestion causes methaemoglobinaemia in all age groups. Cytochrome b5 reductase activity and its adaptation with increasing water nitrate ingestion plays a role in compensating for the methaemoglobinaemia. 39

Stoewsand, G.S.., Anderson, & Lee, G.Y. J.L.Nitrite-induced Methemoglobinemia in Guinea Pigs: Influence of Diets Containing Beets with Varying Amounts of Nitrate, and the Effect of Ascorboc Acid and Methionine. J. Nutr. 103: 419-424 http://jn.nutrition.org/content/103/3/419.long

Abstract Experiments were conducted with young, male guinea pigs to investigate the influence of feeding a vegetable with naturally occurring low and high amounts of nitrate, and the influence of dietary supplementation of ascorbic acid and methionine on nitrite-induced methemoglobinemia. Freeze-dried red table beets were incorporated into experimental diets. They contained either a low or high amount of nitrate from manipulation of nitrogen fertilization rates during their growing period. High nitratecontaining beet diets, or nitrate added to diets, did not induce methemoglobinemia. High nitrate diets fed to guinea pigs enhanced nitrite-induced methemoglobinemia. Low nitrate-containing beet diets seemed to "protect" guinea pigs from nitrite toxicosis. Purified diets containing \% added L-ascorbic acid slightly reduced nitrite-induced methemoglobin blood levels of normal guinea pigs compared to those fed 0.02% ascorbic acid, but there was an additional 50% reduction when 1% methionine and 1% or 2% ascorbic acid were included in the diet.

Abu Naser AA, Ghbn N, Khoudary R. (2007) Relation of nitrate contamination of groundwater with methaemoglobin level among infants in Gasa East Mediterr Health J. 12(5): 994-1004 http://www.ncbi.nlm.nih.gov/pubmed/18290391

Dean BS, Lopez G, Krenzelok EP. (1992) Environmentally-induced methemoglobinemia in an infant. Toxicol Clin Tociclo 30(1): 127-133 http://www.ncbi.nlm.nih.gov/pubmed/1542142 Abstract Acquired methemoglobinemia results from the exposure to various chemicals and drugs able to oxidize hemoglobin at a rate exceeding the normal enzymatic capacity for hemoglobin reduction. Levels of methemoglobin exceeding 60-70% may be associated with coma and death. We describe a case of complete, uneventful recovery involving a 10 week-old infant who presented to the Emergency Department with profound sudden onset of cyanosis, irritability, metabolic acidosis, and a lethal methemoglobin level of 71.4%. Intravenous administration of 12 mg methylene blue resulted in immediate resolution of the cyanosis and reduction of measured methemoglobin to 1.3%. The carboxyhemoglobin was negative. Sodium bicarbonate successfully corrected the acidosis. RBC reductase measurement was within normal limits, ruling out congenital methemoglobinemia. Family history revealed a wood-burning stove which emitted pine tar fumes as the potential environmental methemoglobin-producing source. The infant's cradle was situated five feet from the stove. The infant was discharged on day three of hospitalization with a methemoglobin level of 0.2%. Lebby T, Roco JJ, Arcinue EL (1993) Infantile methemoglobinemia associated with acute diarrheal illness. Ann J Emerg Med 11(5): 471-472 40 http://www.ncbi.nlm.nih.gov/pubmed/8363684

Abstract A case of an infant with diarrhea and acidosis associated with methemoglobinemia is presented. Of interest is that the association of diarrhea and acidosis with methemoglobinemia is more common than previously thought and can produce dangerously high methemoglobin levels. A review of the literature of this unusual occurrence along with clues to making the diagnosis are discussed. Ward MH, deKok TM, Levallois P, Brender J, Gulis G, Nolan BT, VanDerslice J (2005) Workgroup report: Drinking-water nitrate and health - - recent findings and research needs. Environ Health Perspect 113(11) 1607-1614 http://www.ncbi.nlm.nih.gov/pubmed/16263519 Abstract Human alteration of the nitrogen cycle has resulted in steadily accumulating nitrate in our water resources. The U.S. maximum contaminant level and World Health Organization guidelines for nitrate in drinking water were promulgated to protect infants from developing methemoglobinemia, an acute condition. Some scientists have recently suggested that the regulatory limit for nitrate is overly conservative; however, they have not thoroughly considered chronic health outcomes. In August 2004, a symposium on drinking-water nitrate and health was held at the International Society for Environmental Epidemiology meeting to evaluate nitrate exposures and associated health effects in relation to the current regulatory limit. The contribution of drinking-water nitrate toward endogenous formation of N-nitroso compounds was evaluated with a focus toward identifying subpopulations with increased rates of nitrosation. Adverse health effects may be the result of a complex interaction of the amount of nitrate ingested, the concomitant ingestion of nitrosation cofactors and precursors, and specific medical conditions that increase nitrosation. Workshop participants concluded that more experimental studies are needed and that a particularly fruitful approach may be to conduct epidemiologic studies among susceptible subgroups with increased endogenous nitrosation. The few epidemiologic studies that have evaluated intake of nitrosation precursors and/or nitrosation inhibitors have observed elevated risks for colon cancer and neural tube defects associated with drinking-water nitrate concentrations below the regulatory limit. The role of drinking-water nitrate exposure as a risk factor for specific cancers, reproductive outcomes, and other chronic health effects must be studied more thoroughly before changes to the regulatory level for nitrate in drinking water can be considered.

Lundberg JO, Weitzberg E, Cole JA, Benjamin N. Nitrate, bacteria and human health. Nat Rev Microbiol 2004;2(7):593–602. http://www.nature.com/nrmicro/journal/v2/n7/abs/nrmicro929.html 41

Abstract Nitrate is generally considered a water pollutant and an undesirable fertilizer residue in the food chain. Research in the 1970s indicated that, by reducing nitrate to nitrite, commensal bacteria might be involved in the pathogenesis of gastric cancers and other malignancies, as nitrite can enhance the generation of carcinogenic N-nitrosamines. More recent studies indicate that the bacterial metabolism of nitrate to nitrite and the subsequent formation of biologically active nitrogen oxides could be beneficial. Here, we will consider the evidence that nitrate- reducing commensals have a true symbiotic role in mammals and facilitate a previously unrecognized but potentially important aspect of the nitrogen cycle.

Hill MJ. Nitrate toxicity: myth or reality? Br J Nutr 1999;81(5):343–4. http://journals.cambridge.org/download.php?file=%2FBJN %2FBJN81_05%2FS0007114599000616a.pdf&code=5a95311f8c48180b67d2aaa56a4c8e16

Wright RO, Lewander WJ, Woolf AD. Methemoglobinemia: etiology, pharmacology, and clinical management. Ann Emerg Med 1999;34(5):646–56. http://www.annemergmed.com/article/S0196-0644%2899%2970167-8/abstract

Abstract Methemoglobin (MHb) may arise from a variety of etiologies including genetic, dietary, idiopathic, and toxicologic sources. Symptoms vary from mild headache to coma/death and may not correlate with measured MHb concentrations. Toxin-induced MHb may be complicated by the drug’s effect on other organ systems such as the liver or lungs. The existence of underlying heart, lung, or blood disease may exacerbate the toxicity of MHb. The diagnosis may be complicated by the effect of MHb on arterial blood gas and pulse oximeter oxygen saturation results. In addition, other dyshemoglobins may be confused with MHb. Treatment with methylene blue can be complicated by the presence of underlying enzyme deficiencies, including glucose-6-phosphate dehydrogenase deficiency. Experimental antidotes for MHb may provide alternative treatments in the future, but require further study. [Wright RO, Lewander WJ, Woolf AD: Methemoglobinemia: Etiology, pharmacology, and clinical management. Ann Emerg Med November 1999;34:646-656.]

Harris JC, Rumack BH, Peterson RG, McGuire BM. Methemoglobinemia resulting from absorption of nitrates. JAMA 1979;242(26):2869–71. http://jama.jamanetwork.com/article.aspx? articleid=368092

Abstract 42

Three cases of toxic methemoglobinemia resulted from absorption of nitrate salts through burned skin areas. In addition to the unusual route of intoxication, this report emphasizes the occupational hazard of methemoglobinemia in workers exposed to nitrogen salts and the importance of rapid diagnosis and treatment. The diagnosis of methemoglobinemia should be suspected in any case of cyanosis refractory to oxygen therapy. Appropriate treatment should include oxygen, methylene blue, and exchange transfusion in clinically severe cases.

American Academy of Pediatrics Committee on Nutrition. Infant methemoglobinemia: the role of dietary nitrate. Pediatrics 1970;46(3):475–8. http://www.pediatricsdigest.mobi/content/116/3/784.short

Abstract Infants for whom formula may be prepared with well water remain a high-risk group for nitrate poisoning. This clinical report reinforces the need for testing of well water for nitrate content. There seems to be little or no risk of nitrate poisoning from commercially prepared infant foods in the United States. However, reports of nitrate poisoning from home-prepared vegetable foods for infants continue to occur. Breastfeeding infants are not at risk of methemoglobinemia even when mothers ingest water with very high concentrations of nitrate nitrogen (100 ppm).

Parslow RC, McKinney PA, Law GR, Staines A, Williams R, Bodansky HJ. Incidence of childhood diabetes mellitus in Yorkshire, northern England, is associated with nitrate in drinking water: an ecological analysis. Diabetologia 1997; 40(5):550–6. http://www.springerlink.com/content/7d21fcmtmgjj54pt/

Abstract

The relationship between the incidence of childhood-onset insulin-dependent diabetes mellitus and levels of nitrate in drinking water in the former Yorkshire Regional Health Authority was investigated by means of an ecological analysis. A population-based register contributed 1797 0– 16-year-olds diagnosed with diabetes between 1978 and 1994. Nitrate data were based on 9330 samples of drinking water tested between 1990 and 1995 in 148 water supply zones, for which 1991 census small area statistics were taken on population density, ethnicity and socio-economic status. Diabetes incidence was positively associated with raised mean nitrate levels with a standardised incidence ratio of 115 in zones with greater than 14.85 mg · l–1 (χ2 = 26.81, 1 df, p < 0.001). Significant negative trends were found between standardised incidence ratios and proportion of non-whites in the population (χ2 = 33.57, 1 df, p < 0.001), childhood population density (χ2 = 30.81, 1 df, p < 0.001) and the Townsend deprivation score (χ2 = 33.89, 1 df, p < 0.001). Poisson regression modelling, adjusting for the other factors, showed a significant increase in relative incidence rate ratio from a baseline of 1 at nitrate levels below 3.22 mg · l–1 to 1.27 (95 % confidence interval 1.09,1.48) for mean nitrate levels above 14.85 mg · l–1. An association between higher nitrate levels in domestic drinking water and incidence of childhood diabetes has been demonstrated. This was not explained by the ethnic composition of the 43 population, population density or socioeconomic status. Nitrate in drinking water may be a precursor of chemicals which are toxic to the pancreas. [Diabetologia (1997) 40: 550–556]

Tricker AR, Preussmann R. Carcinogenic N-nitrosamines in the diet: occurrence, formation, mechanisms and carcinogenic potential. Mutat Res 1991;259:277–89. http://www.sciencedirect.com/science/article/pii/0165121891901234

Abstract Nitrosamines form a large group of genotoxic chemical carcinogens which occur in the human diet and other environmental media, and can be formed endogenously in the human body. N- Nitroso compounds can induce cancer in experimental animals. Some representative compounds of this class induce cancer in at least 40 different animal species including higher primates. Tumours induced in experimental animals resemble their human counterparts with respect to both morphological and biochemical properties. Extensive experimental, and some epidemiological data suggest that humans are susceptible to carcinogenesis by N-nitroso compounds and that the presence of these compounds in some foods may be regarded as an aetiological risk factor for certain human cancers including cancers of the oesophagus, stomach and nasopharynx.

Eichholzer M., Gutzwiller F. Dietary nitrates, nitrites, and N-nitroso compounds and cancer risk: a review of the epidemiologic evidence. Nutr Rev 1998; 56:95–105. http://onlinelibrary.wiley.com/doi/10.1111/j.1753-4887.1998.tb01721.x/abstract

Abstract

Experimental animal studies have shown N-nitroso compounds (NOC) to be potent carcinogens. Epidemiologic evidence of the carcinogenic potential of dietary NOC and precursor nitrates and nitrites in humans remains inconclusive with regard to the risk of stomach, brain, esophageal, and nasopharyngeal cancers. Inadequate available data could obscure a small to moderate effect of NOC

Barrett JH, Parslow RC, McKinney PA, Law GR, Forman D. Nitrate in drinking water and the incidence of gastric, esophageal, and brain cancer in Yorkshire, England. Cancer Causes Control 1998;9:153–9. http://www.springerlink.com/content/l587899574222218/

Objectives: This small-area ecologic study in Yorkshire, northern England, examines the hypothesis that exposure to higher levels of nitrate in drinking water increases the risk of stomach, esophageal, or brain cancer in adults. Methods: Nitrate levels over the period 1990-95 and numbers of incident cancers from 1975-94 were available for 148 water supply zones, geographically defined areas each supplying water of homogeneous chemical composition to an average population of around 20,000. Results: No relationship was found between nitrate 44 concentrations and the incidence of stomach or esophageal cancers. The incidence of cancer of the brain and central nervous system was found to be higher in areas with higher nitrate levels, with a relative risk of 1.18 (95 percent confidence interval = 1.08-1.30) in the quartile of the population with the highest average levels (mean 29.8 mg/l) compared with the lowest quartile (mean 2.4 mg/l).

Van Loon AJ, Botterweck AA, Goldbohm RA, Brants HA, van Klaveren JD, van den Brandt PA. Intake of nitrate and nitrite and the risk of gastric cancer: a prospective cohort study. Br J Cancer 1998; 78:129–35. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2062934/

Abstract

The association between the intake of nitrate or nitrite and gastric cancer risk was investigated in a prospective cohort study started in 1986 in the Netherlands, of 120,852 men and women aged 55-69 years. At baseline, data on dietary intake, smoking habits and other covariates were collected by means of a self-administered questionnaire. For data analysis, a case-cohort approach was used, in which the person-years at risk were estimated from a randomly selected subcohort (1688 men and 1812 women). After 6.3 years of follow-up, 282 microscopically confirmed incident cases of stomach cancer were detected: 219 men and 63 women. We did not find a higher risk of gastric cancer among people with a higher nitrate intake from food [rate ratio (RR) highest/lowest quintile = 0.80, 95% CI 0.47-1.37, trend-P = 0.18], a higher nitrate intake from drinking water (RR highest/lowest quintile = 0.88, 95% CI 0.59-1.32, trend-P = 0.39) or a higher intake of nitrite (RR highest/lowest quintile = 1.44, 95% CI 0.95-2.18, trend-P = 0.24). Rate ratios for gastric cancer were also computed for each tertile of nitrate intake from foods within tertiles of vitamin C intake and intake of beta-carotene, but no consistent pattern was found. Therefore, our study does not support a positive association between the intake of nitrate or nitrite and gastric cancer risk.

Xu G, Song P, Reed PI. The relationship between gastric mucosal changes and nitrate intake via drinking water in a high-risk population for gastric cancer in Moping County, China. Eur J Cancer Prev 1992; 1(6):437–43. http://journals.lww.com/eurjcancerprev/Abstract/1992/10000/The_relationship_between_gastric _mucosal_changes.7.aspx

Abstract

There are conflicting reports on whether nitrate intake is related to gastric carcinogenesis. In this laboratory/field study from a high-risk area for gastric cancer, we analysed 178 samples of drinking water for nitrate and nitrite, and examined the relationship between gastric mucosal lesions (including gastric cancer) and quality of different types of drinking water and nitrate intake via water. The results showed that the nitrate content in the local drinking water was generally very high, with a mean of 109.6 mg/l (range 4.4-497.2 mg/l). There were significant differences in the nitrate content in drinking water from different wells in qualitatively different types of water. The histological changes were closely related to the quality of drinking water and 45 its nitrate content. The results suggest that nitrate in drinking water probably plays an important role in gastric carcinogenesis and that in future aetiological studies of gastric cancer should include more information on well depth, the presence of public or private wells and nitrate content of water.