DETERMINATION OF FLUORIDE IN WATER, MILK, AND DAIRY PRODUCTS

Abstract

The objective of this study was to determine the fluoride ion (F) levels of water, cow's milk, yoghurt, and Herby cheese in some villages, in the Turkish provinces of Van and Ağrı, in areas where high levels of fluorosis in human and animals were present. The range of the F levels in samples of drinking water for human consumption was 2.36-3.56 ppm and in water for animal use it was 1.76-3.73 ppm. These levels were above the WHO guideline value of 1.5 mg/L. The ranges for the F content of the milk, yoghurt, and Herby cheese samples were 0.02-0.18, 0.06-0.19, and 0.02-0.19 ppm, respectively. No significant differences (p>0.05) were present between the F contents of the samples taken from Çaldıran and Doğubeyazıt but the F levels were significantly higher (p<0.05) in spring, compared to autumn, in the Doğubeyazıt district for milk, yoghurt, and Herby cheese. The results indicate that there is no danger of fluorosis developing from the consumption of milk and dairy products produced in the Çaldıran and Doğubeyazıt districts. However, the consumption of drinking water from these districts, with its excess of F may cause fluorosis, including dental fluorosis, skeletal fluorosis, and non-skeletal fluorosis. It is necessary that measures are taken as soon as possible to reduce this risk and provide safe drinking water.

Headnote

ABSTRACT: The objective of this study was to determine the fluoride ion (F) levels of water, cow's milk, yoghurt, and Herby cheese in some villages, in the Turkish provinces of Van and Ağrı, in areas where high levels of fluorosis in human and animals were present. The range of the F levels in samples of drinking water for human consumption was 2.36-3.56 ppm and in water for animal use it was 1.76-3.73 ppm. These levels were above the WHO guideline value of 1.5 mg/L. The ranges for the F content of the milk, yoghurt, and Herby cheese samples were 0.02-0.18, 0.06-0.19, and 0.02-0.19 ppm, respectively. No significant differences (p>0.05) were present between the F contents of the samples taken from Çaldıran and Doğubeyazıt but the F levels were significantly higher (p<0.05) in spring, compared to autumn, in the Doğubeyazıt district for milk, yoghurt, and Herby cheese. The results indicate that there is no danger of fluorosis developing from the consumption of milk and dairy products produced in the Çaldıran and Doğubeyazıt districts. However, the consumption of drinking water from these districts, with its excess of F may cause fluorosis, including dental fluorosis, skeletal fluorosis, and non-skeletal fluorosis. It is necessary that measures are taken as soon as possible to reduce this risk and provide safe drinking water.

Key words: Ağrı province; Cheese; Fluoride; Milk; Turkey; Van province; Water; Yoghurt.

INTRODUCTION

The fluoride ion (F) is the ionic form of fluorine, one of several elements receiving much attention owing its possible harmful effects on health and the environment.1,2 At higher levels F is a potentially toxic element.3

Although it has been considered that the optimal range of F intake is 0.05-0.07 mg F/kg body weight/day2-5 and the World Health Organization (WHO) has stated that low concentrations of F, usually 0.5-1.0 mg/L, provide protection against dental caries in both children and adults, F is neither an essential trace element for human health nor necessary for the development of healthy teeth and bones.6 There is strong scientific evidence for the protective cariostatic effect of topical F application but the respective data for the systemic application of F via drinking water are less convincing.6 Usually, drinking water is the largest contributor to the daily F intake but in coal-burning fluorosis the toxicity results from the contamination of food by the smoke from the domestic burning, for heating and cooking, of briquettes made by mixing powdered coal with high-F clay in stoves lacking a flue for taking the smoke out of the dwelling.7 The World Health Organization (WHO) recommends that the concentration of the F ion in drinking water should be less than 1.5 mg/L.5 In the United Kingdom (UK), F occurs naturally in tap drinking water supplies at the concentrations up to approximately 1.5 mg/L. In Canada, the government recommends a maximum concentration of F in drinking water of 1.5 mg/L. In Australia and China, up to 1.5 and 1.2 mg/L, respectively, are acceptable for naturally occurring F in drinking water.8

Chronic excessive F intake can result in the chronic systemic disease of fluorosis, which can be called endemic fluorosis when it occurs regularly in a certain region. Fluorosis can be characterized by dental fluorosis with the mottling of teeth, with hypomineralized and porous enamel, when exposure occurs early up to the age of approximately 6 yr; the more severe skeletal fluorosis with lower extremity pain, microfracture, periosteal exostosis, osteosclerosis, osteoporosis, and osteophytosis; and non-skeletal fluorosis with symptoms such as gastrointestinal discomforts, neurological disorders, impaired endocrine and reproductive functions, teratogenic effects, renal effects, genotoxic effects, apoptosis, and excitotoxicity.1,8 Continuous exposure to F may cause damaging effects on body tissues, particularly in the nervous system. Locomotor impairment may occur in children and adults of both sexes. The other clinical effects of fluorosis include cardiac dysfunction, thyroid hormone derangements in children, gastrointestinal discomfort, visual disturbance, muscle weakness, respiratory symptoms, renal impairment, and abnormalities in glucose tolerance. F has also been found to destroy blood cells, blood vessels, and the lining of the stomach and intestine, and to accelerate the calcification of blood vessels. Furthermore, F has been linked to a reduction in the level of reproductive hormones in the serum of men, miscarriage, birth abnormalities, mental retardation, and cancer in bone, lungs, bladder and uterus. F can penetrate into the brain and is a normal component of the cerebrospinal fluid. If its concentration reaches a very high level, as in patients with fluorosis, F produces harmful effects in the brain.9-11

Large animals have been exposed to excess F through the ingestion of high F rock phosphates used as nutritional supplements, by the ingestion of forage contaminated with excess F from industrial pollutants or volcanic emissions, or from the consumption of water containing excess F from industrial pollution or natural sources, such as ground water passing through F rich rocks.102,13

Traditionally, fluorosis has been connected primarily with intake of F through drinking water. Ground water, especially in areas with volcanic base-rock, often contains high concentrations of F.14

Several previous studies have determined the F content of water, baby foods, vegetables, fruits, beverages, cereals, tea, milk, and powdered milk.3,15-19 Since the F in milk is absorbable, measuring the F concentration in the diet of children, including milk, is essential during the years in which children are at risk for fluorosis.17 The mean concentration of F in mature human milk is approximately 18 qg/L. The F content of bovine milk is variable (mean approximately 50 qg/L). About 46-64% of the F in bovine milk occurs as free F ions with the remainder bound to proteins.1

Endemic fluorosis has been found to occur in Turkey (in Ağrı and Van provinces of East Anatolia and in Eskişehir and Kırşehir provinces of Central Anatolia) and in other many countries (India, East Africa Rift Valley, Ethiopia, Lake Elementaita, Pakistan, Thailand, China, Iran, Iraq, and Sudan).11,14,20-28

The objective of this study was to determine the F levels of water, cow's milk, yoghurt, and Herby cheese in some villages, in the Turkish provinces of Van and Ağrı, in areas where high levels of fluorosis in human and animals were present.

MATERIALS AND METHODS

Samples: Water, milk, yoghurt and Herby cheese samples were obtained in the autumn (period I) and spring (period II) periods from the Çaldıran district of Van province and the Doğubeyazıt district of Ağrı province. The samples were collected from specified villages of the districts into polyethylene sample containers, and brought, under cold chain, to the Dairy Technology and Analysis Laboratory of the Department of Food Engineering, for analysis. They were processed, coded, and frozen before any analyses were done, and, at the time of analysis, the processed samples were analyzed in numerical order.

Apparatus: An Orion F ion selective electrode was used for the F analyses together with an Orion pH meter (Thermo Scientific Orion Dual Star 166 Cummings Center Beverly, MA 01915 USA, serial:E02171).

Reagents: All solutions were prepared with analytical-reagent grade chemicals and distilled water. The following reagents were used: 0.01 M HCl solution, prepared by adding 0.83 mL HCl to 1 L distilled water, and concentrated hydrochloric acid (37%; specific gravity 1.19), Merck. A 0.1 M NaF stock standard solution stabilised in NaF, TISAB III, and buffer solutions for pH calibration, purchased from Sigma were also used.

Analysis of milk, yoghurt, and Herby cheese: The F determinations were carried out by the method of diffusion, as described by Taves.29 Firstly, a set of standards (ranging between 0.19 M and 19 M) was prepared, using serial dilution from a 0.1 M NaF stock solution. Then, to 3 mL or 3 g of milk, yoghurt, and Herby cheese, an equal volume of 0.01 M HCl was added and left at 37°C for 1 hr. Next, the samples were centrifuged at 3000 x g for 5 min to obtain a supernatant and sediment. Before analysing the supernatant, 0.1 mL 0.1 M NaOH and 0.11 mL TISAB III were added to 1 mL of supernatant. The standard solutions and sample solutions were analysed using a fluoride ion-selective electrode. The electrode was soaked in distilled water and blotted dry before each measurement. All samples were analysed in duplicate.

Analysis of water: Before the F analysis of the water samples, 5 mL of TISAB III was added to a 5 mL water sample to obtain a pH of 5.2-5.4, which is the optimal pH range for F determination. The prepared water solution was analysed using a fluoride ion-selective electrode.

Statistical analysis: Data were analyzed using the SAS statistical program, with variance analysis conducted for all the data. The differences between samples were determined using the Kruskal-Wallis Range Test and the differences between the periods were determined using the Wilcoxon- Mann-Whitney Range Test.30

RESULTS AND DISCUSSION

F concentrations the drinking water samples available for human consumption:

The mean F content of the water samples available for human consumption from the different villages in Çaldıran district of Van province and Doğubeyazıt district of Ağrı province, in areas where high levels of fluorosis in human and animals were present, were 3.43±0.40 ppm (mg/L) and 2.41±0.30 ppm (mg/L), respectively, and did not differ significantly from one another (p=0.0629). The rainfall is lower in autumn (period I) than in spring (period II) and although the mean F levels in the villages in the two districts were higher in autumn than in spring, the seasonal differences were not significantly different, (p=0.9214 for Çaldıran district and p=0.9350 for Doğubeyazıt district) (Table 1).

According to the WHO, the F concentration in drinking water should not exceed 1.5 mg/L.5 The same is true for EU drinking water guidelines.28 In this study, all the water sources, except for one village, contained F at a level above the WHO guideline value.

Şendil and Bayşu21 reported endemic fluorosis in Eastern Anatolia, in villages in the Doğubeyazıt district of Ağrı province where the drinking water F concentration range was 10.3-12.5 ppm and in the Çaldıran district of Van province where the drinking water F concentration range was 5.7-15.2 ppm. Oruç31 reported that in the Doğubeyazıt district of Ağrı province the drinking water F concentration range was 6.5-12.5 ppm and in the Çaldıran district of Van province the range was 2.0-7.5 ppm. Ergun et al.32 were found the F concentration of water in the same area to be 7.6 ppm. Similar results were reported by Fidancı et al.22 who studied waters in natural and industrial fluorosis areas of Central Anatolia.

Although the mean water F level was higher in autumn than in spring in both districts, the seasonal differences were not significantly different, (p=0.9214 for Çaldıran district and p=0.9350 for Doğubeyazıt district) (Table 1). Oto26 also found higher water F concentrations in autumn which may be the result of the reduced rainfall in autumn compared to spring.

F concentrations the drinking water samples available for animal consumption:

The mean F content of the water samples available for animal consumption, in areas where high levels of fluorosis in human and animals were present, were significantly higher (p=0.0306) in the villages in Çaldıran district of Van province than in the villages in Doğubeyazıt district of Ağrı province, 3.34±0.42 ppm (mg/L) and 2.02±0.39 ppm (mg/L), respectively. No seasonal differences in F levels were present, (p=0.2720 for Çaldıran district and p=0.6874 for Doğubeyazıt district) (Table 2).

The analyses showed that the F concentrations in the water samples, available to humans and animals, were at levels considered to be harmful to both people and animals in each district of the two provinces during both the periods studied. Similar results have been found previously in studies done in the area.21,26.32

Prolonged exposure to a toxic concentration of F in drinking water has been found to produce skeletal lesions and adverse changes in the bone structure of both human beings and animals.11,33,34 The prolonged ingestion of drinking water containing 1.5-4.0 ppm of F produced deleterious effects on the bones of cattle, buffaloes, sheep, and goats in villages in Rajasthan, India.11 The consumption of drinking water with 2 mg F/L may produce some tooth mottling, but Carlson considered that this level was not excessive with respect to animal health or the deposition of F in meat, milk, or eggs.35

F concentrations the milk samples:

The ranges of F concentrations in the milk samples from the different villages in Çaldıran district of Van province and Doğubeyazıt district of Ağrı province, in areas where high levels of fluorosis in human and animals were present, were 0.01-1.58 ppm and 0.01-0.26 ppm, respectively. The mean F concentrations in the milk samples from the different villages in Çaldıran district of Van province and Doğubeyazıt district of Ağrı province were 0.13±0.05 ppm (mg/L) and 0.07±0.09 ppm (mg/L), respectively, and did not differ significantly from one another (p=0.3239). The mean level was significantly higher (p=0.0003) in spring (period II) than in autumn (period I) for the Doğubeyazıt district but no significant difference was present in the Çaldıran district (p=0.3844) (Table 3).

Similar variations in the F levels of milk (0.05-0.22 pg/mL, 0.05-0.22 ppm) in the Lake Elementaita region of Kenya were reported by Kahama et al.36 Buzalaf et al.37 analysed the F concentrations of whole milk, skim milk (defatted milk), and chocolate milk sold in markets in Brazil and found the range for all the milks was 0.02-1.6 pg/mL (0.02-1.6 ppm), for the whole and skim milks the range was lower at 0.02-0.07 pg/mL (0.02-0.07 ppm), and for the chocolate milks it was higher at 0.041.6 pg/mL (0.04-1.6 ppm).

The concentrations of F in cow's milk are higher than the concentrations in human breast milk.38 The concentration of F in human milk generally ranges from 2-5 pg/L (0.002-0.005 ppm). Hossny et al. found the mean F concentration in the milk of cows fed with a normal diet was 0.103 mg/L (0.103 ppm) and 0.283 mg/L (0.283 ppm) in those fed on F-contaminated pastures.39 Jackson et al. considered that bovine milk contains very low levels of F, generally less than 0.05 ppm.15

F concentrations the yoghurt samples:

The ranges of F concentrations in the yoghurt samples from the different villages in Çaldıran district of Van province and Doğubeyazıt district of Ağrı province, in areas where high levels of fluorosis in human and animals were present, were 0.02-0.50 ppm and 0.01-0.27 ppm, respectively. The mean F concentrations in the yoghurt samples from the different villages in Çaldıran district of Van province and Doğubeyazıt district of Ağrı province were 0.12±0.02 ppm (mg/L) and 0.11±0.10 ppm (mg/L), respectively, and did not differ significantly from one another (p=1.000). The mean level was significantly higher (p=0.0008) in spring (period II) than in autumn (period I) for the Doğubeyazıt district but no significant difference was present in the Çaldıran district (p=0.6144) (Table 4).

In a study carried out by Gutmanis and Chen in India, a total of 441 brand-name food and beverage items were purchased and individually analyzed for F. The F content of the milk and milk products (milk and milk drinks, yogurt, cheese, cream, sour cream, milk sauces, milk deserts, and ice cream) had a range of 0.23-1.36 pg/g (ppm) with a mean value of 0.31±0.29 pg/g (ppm).40 The levels of F we found in milk (mean F concentrations in the Çaldıran and Doğubeyazıt districts of 0.13±0.05 ppm [pg/g, mg/L] and 0.11±0.10 ppm [pg/g, mg/L], respectively) and yoghurt (mean F concentrations in the Çaldıran and Doğubeyazıt districts of 0.12±0.02 ppm [pg/g, mg/L] and 0.07±0.09 ppm [pg/g, mg/L], respectively) were lower than the those found by Gutmanis and Chen.

The bioavailability of F in milk and milk products is another point to be considered due to the high calcium concentrations in milk. There is the possibility of diminished F absorption from milk and milk products in the gastro-intestinal tract, due to the high calcium content, leading to the calculations of the F available from these products being overestimated. On the other hand, milk is also rich in fats, which are known to increase the lag time of the food or beverages in the stomach. Although the high calcium content in milk may interfere with the rate of F absorption, it has been demonstrated that 67 to 82% of total F in milk is absorbed.37

F concentrations the Herby cheese samples:

The ranges of F concentrations in the Herby cheese samples from the different villages in Çaldıran district of Van province and Doğubeyazıt district of Ağrı province, in areas where high levels of fluorosis in human and animals were present, were 0.01-1.04 ppm and 0.01-0.27 ppm, respectively. The mean F concentrations in the Herby cheese samples from the different villages in Çaldıran district of Van province and Doğubeyazıt district of Ağrı province were 0.13±0.03 ppm (mg/L) and 0.06±0.01 ppm (mg/L), respectively, and did not differ significantly from one another (p=0.1060). The mean level was significantly higher (p=0.0003) in spring (period II) than in autumn (period I) for the Doğubeyazıt district but no significant difference was present in the Çaldıran district (p=0.1394) (Table 5).

The range of the mean F content in several Italian cheeses was found by Gutmanis and Chen40 to be 4-7 pg F/g of dry cheese solids (ppm, mg/kg). Gonzales et al41 reported that the F concentration in Spanish cheese was lower than 0.7 mg/kg (pg/g, ppm). Maguire et al.42 reported that the range of F levels in dairy products in grocery stores and health food shops in three cities in the north-east of England was 0.0330.245 pg/g (ppm, mg/kg).

The F in milk is associated with calcium and theoretically CaF2 may be less well absorbed from the gut.36 The F in milk is in a bound state, with either the organic components of the milk or with calcium ions, and this can lead to decreased F bioavailability over time.43,44 Furthermore, the percentage of F bound with calcium may have increased during storage. At pH 6.6 and 5.5, none of the F was bound to proteins. At pH 3.9, a-lactalbumin combined with the F.

CONCLUSION

We conclude that the waters consumed by humans and animals in the villages studied in the Çaldıran district of Van province and the Doğubeyazıt district of Ağrı province, in areas where high levels of fluorosis in human and animals were present, contained excessively high mean levels of F, 3.43±0.40 ppm (mg/L) and 2.41±0.30 ppm (mg/L), respectively. However, the mean F levels in the milk and the milk products of yoghurt and Herby cheese produced in these villages in the Çaldıran and Doğubeyazıt districts, were not elevated with the mean F levels being: milk: 0.13±0.05 ppm (mg/L) and 0.07±0.09 ppm (mg/L); yoghurt: 0.12±0.02 ppm (mg/L) and 0.11±0.10 ppm (mg/L); and Herby cheese: 0.13±0.03 ppm (mg/L) and 0.06±0.01 ppm (mg/L), respectively. This indicates that there is no danger of fluorosis developing from the consumption of the milk and dairy products in this region. But this is not the case for the drinking water in the Çaldıran and Doğubeyazıt districts, where the mean F values were 3.43±0.40 ppm (mg/L) and 2.41±0.30 ppm (mg/L), respectively, higher than the upper permissible limit recommended by the WHO of 1.5 mg/L.5 The consumption of drinking water with an excess of F may cause fluorosis, including dental fluorosis, skeletal fluorosis, and non-skeletal fluorosis. It is necessary that measures are taken as soon as possible to reduce this risk and provide safe drinking water. In several nations around the world health problems occur due to the interaction of excessive F exposure and dietary calcium deficiency. Interestingly, the use of selenium has been suggested as an antidote against fluorosis.11'45

ACKNOWLEDGEMENTS

This project was supported by funds from the Yuzuncu Yil University Scientific Researches Project Presidency (Project code 2010-FBE-YL040). We thank the local people who support us for their assistance with the trials.

References

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