# ENVIRONMENTAL HEALTH PERSPECTIVES ENVIRONMENTAL HEALTH PERSPECTIVES

National Institutes of Health U.S. Department of Health and Human Services 

# Serum Fluoride Level and Children’s Intelligence Quotient in Two Villages in China

Quanyong Xiang, Youxin Liang, Bingheng Chen, Liansheng Chen, Minghao Zhou, Ming Wu, and Mingsheng Zhou doi: 10.1289/ehp.1003171 (available at http://dx.doi.org/) Online 17 December 2010 ehponline.org 

# ehp1

Serum Fluoride Level and Children’s Intelligence Quotien t in Two Villages in

China

Quanyong Xiang, 1 Youxin Liang, 2 Bingheng Chen, 2 Liansheng Chen, 1 Minghao Zhou, 1 Ming Wu 1 , Mingsheng Zhou 3

1 Department of Environmental Health, Jiangsu Province Center for D iseases Control and Prevention, Nanjing, China;

2 Department of Occupational and Environmental Health, School of Public He alth, Fudan University (formerly Shanghai Medical University), Shanghai, Chi na;

3 Sihong County Center for Diseases Control and Prevention, Sihong County, J iangsu Province, China

For correspondence: Quanyong Xiang, Department of Environmental Health, Jiangsu Province Center for Diseases Control and Prevention. 172 Jiangsu Road, Nanjing, Jiangsu Province , 210009, P.R. China.

Tel: 86-25-83759414 Fax: 86-25-83759411 E-mail: quanyongxiang@yahoo.com.cn Page 1 of 222

Article running title : Fluoride and Intelligence Quotient

Keywords: Children; Fluoride; Intelligence quotient

Acknowledgements: This work was supported by Jiangsu Province Association for Endemic Disease Control and Prevention (X200327). We thank Xipen Jin (School of Public Health, Fudan Univesity), Zumin Shi (Jiangsu Province Center for Diseases Control and Prevention) and Mingfang Zhang (Sihong County Center for Dis ease Control and Prevention) for their valuable suggestions. The authors decla re that they have no competing financial interests.

Abbreviation:

CI Confidence Interval

CRT-RC Combined Raven’s Test for Rural China IQ Intelligence Quotient NCTB Neurobehavioral Core Test Battery

OR Odds Ratios

SD Standard Deviation WPPSI Wechsler Preschool and Primary Scale of Intelligen ce Page 2 of 223 Abstract

Background: Animal studies show that brain fluoride levels increase with incr easing exposure to fluoride. Human studies have indicated an association between high levels of drinking-water fluoride and lower intelligence. Data on the assoc iation between serum fluoride and children’s intelligence quotient (IQ) are limited. Objective: This study was conducted to assess the relationship between serum fluoride and children’s IQ.

Methods: We collected blood samples from 512 children aged 8-13 years from two villages (Wamiao and Xinhuai) in China. We also used minitype fluori de ion selective electrode and Combined Raven’s Test for Rural China (CRT-RC) to measure serum fluoride and children’s IQ.

Results: In Wamiao, the mean (± SD) serum fluoride concentration was 0.081 ± 0.019 mg/L, and average children’s IQ was 92.02 ± 13.00; in Xinhuai, the mean serum

fluoride concentration was 0.041 ± 0.009 mg/L and average IQ was 100.41 ± 13.21. There was a significant inverse association between serum fluori de concentration and IQ scores among children in Wamiao (regression coefficient for a 1-unit change in serum fluoride = –112.61, p = 0.015), the association was not significant in Xinhuai (regression coefficient = 78.85, p = 0.362). Serum fluoride levels were negatively associated with IQ after adjustment for age and sex [odds ratios and 95% confidence intervals for IQ < 80 of 2.22 (1.42-3.47) and 2.48 (1.85-3.32) for serum fluoride concentrations of 0.05–0.08 and > 0.08 mg/L compared with < 0.05 mg/L, respectivel y,

p for trend < 0.001]. IQ was not related to family income or parent’s education level. There was a significant positive relation between serum fluoride and drinking-water

fluoride.

Conclusions: The results indicated that fluoride in drinking water was highly correlated with serum fluoride, and that higher fluoride exposure m ay affect intelligence among children. Page 3 of 224 Introduction

In 1937, Kaj Roholm published his classic study on 68 cryolite workers c hronically exposed to fluoride, 84% of whom had skeletal fluorosis. Twenty-two percent of these cases had neurologic symptoms such as excessive tiredness, sleepi ness, headache, and giddiness (Roholm 1937). In 1961, Singh and Jolly reported that 10% of patients w ho had skeletal fluorosis also had nervous system damage (Singh and Joll y 1961). Autopsies of patients with skeletal fluorosis indicated nuclear vac uoles; reduction of cells in the anterior horn of the spinal cord, the medical posterior funiculus, and the lateral horns; as well as reduction and partial clotting of the Nissl bodies which are located near the cell membrane. These results suggested that t he damage was directly caused by the effects of fluoride on the nervous system (Franke 1976; Franke et al. 1975). There have been a number of studies in humans on the relationship betw een cognitive function and fluoride in drinking water (Fan et al. 1991; Guo et al . 1991; He et al. 1989; Li et al. 1994; Li et al. 1995; Lu et al. 2000; Qin and Cui 1990; Ren et al. 1989; Zhao et al. 1996, Xiang et al. 2003 ). Studies by Zhao (Zhao et al. 1996), Lu (Lu et al. 2000), and Xiang (Xiang et al. 2003) have indicated an association between hi gher levels of drinking-water fluoride and a lower intelligence quotient (IQ) in childr en, but other studies (WHO 2002) did not find such an association. Serum fluoride concentration has been recognized as a reliable i ndicator of fluoride exposure and is also used as one of the biomarkers to assess the ef fect of endemic fluorosis control and prevention (Lund et al. 1997; WHO 1996; National Standard of P.R. China 2001; Wan et al. 2000). The literature, however, contains a wide range (0.4–2.4 μ mol/L or 0.018–0.101 mg/L) of measures defining “normal” plasma fluori de concentration. The differences in these results may have been att ributable to the testing of fasting individuals in some studies and nonfasting in others (WH O 2002). Currently, there is no cut-off point of serum fluoride which is considered accepta ble by WHO or other academic organizations. Page 4 of 225

The relationship between serum fluoride and children’s IQ has not been r eported. The objective of the present study was to describe the associati on between levels of fluoride in serum and in drinking water and children’s IQ.

Materials and Methods

Selection of study areas Two villages, Wamiao (a severe endemic fluorosis area) and Xinhuai (a non endemic fluorosis area) in Jiangsu Province, People’s Republic of China, were selected for this study Study subjects The study was conducted between September 2002 and June 2003 and included children 8–13 years of age who attended village primary schools in Wami ao and Xinhuai (from grade 2 to grade 6, at an age when dental fluorosis in permanent teeth can be diagnosed clearly) (Xiang et al. 2004). A questionnaire, comple ted with the assistance of parents, provided information on personal characteristic s, medical history including illnesses affecting the nervous system and head trauma, educational level of the children and parents, family socioeconomic status, and lifestyle. There w ere a total of 238 and 305 students in grade 2 through 6 in Wamiao and Xinhuai, respecti vely. After excluding children who had been absent from either village fo r ≥ 2 years or who had a history of brain disease or head injury, 222 children (93%) were enrolled from Wamiao and 290 (95%) were enrolled from Xinhuai (Table 1). Before the investigation, written informed consent was obtained from t he children’s parents. The study was approved by Jiangsu Provincial Center for Disease Contr ol and Prevention, and School of Public Health, Fudan University. Higher levels of lead and iodine deficiency are significant c auses of mental impairment (Burgstahler 2003; National standard of P.R. China 1995; Carpe nter 2005; Chen et al. 2005). To identify whether the blood lead and urinary iodine had significant relationship with children’s IQ or not in these two villages, childre n were randomly selected used random function in Excel 2000 (Microsoft Corporation, Redm ond, USA) Page 5 of 226

for urine iodine (46 in Wamiao, 40 in Xinhuai) and blood lead (71 in Wamiao, 67 in Xinhuai) measurements. Sampling and testing The drinking-water samples collected from the shallow wells of ea ch child’s household were kept in clean plastic bottles and analyzed within 1 week . A fasting venous blood sample (2–2.5 mL) was obtained from each child and preserved i n a clean plastic centrifuge tube, which was immediately centrifuged by 3,000 r/min for 10 min. The serum was removed to another clean plastic tube and kept in a ref rigerator (–40 ê ), and was subsequently analyzed within 1 week. Fluoride in drinking water and serum was measured with a fluoride ion selective electrode (National Standard of P.R. China 1999; National Standard of P.R. China 2001). Urine samples were collecte d in the early morning before breakfast during 6:00-8:00 AM and kept in clean plastic bo ttles. Iodine in urine was measured with a commercially available Rapid Test Kit, manufa ctured by Hubei Province Center for Diseases Control and Prevention (Wuhan, China ). The blood samples (80 μl) were collected via index finger prick and preserv ed in clean plastic centrifuge tubes which included 0.64 mL Triton X-100, manufactured by Amre sco Inc. Solon, Ohio, USA. The blood lead was measured by atomic absorption spectrophotometer (National Standard of China 1996). IQ measures We used the Combined Raven’s Test for Rural China (CRT-RC) which was published by East China Normal University (1989) to measure the IQ of each child (Li and Che n 1989). CRT-RC was based on the Combined Raven’s Test (CRT), and revised by East China Normal University from 1986 to 1987. In the CRT-RC validation stu dy by East China Normal University, 4,212 children 5-14 years of age from 12 provinces i n China were selected from villages using stratified random sampling ( Li and Chen 1989; Wang et al. 1989). The test included items with different difficulty index to evaluate sample standardization, reliability, and validity. The results of relia bility indicated that the correlation coefficient was 0.92 (Li and Chen 1989; Wang et al. 1989). In the present study, we tested the children’s IQ in the school cla ssroom. The children finished the test independently within 40 min under the supervision of an exa miner and Page 6 of 227

two assistants according to the guidelines of the CRT-RC manual for the test administration conditions, instructions, and the test environment (Li and C hen 1989). The examiner (trained by East China Normal University) and as sistants were blinded to

the levels of fluoride in each child’s serum and the household shallow we ll when they conducted the IQ test. A normal model in the instruction manual of C RT-RC divides the IQ into seven categories according to the IQ scores: mental re tardation (IQ < 70), borderline (IQ 70–79), dull normal (IQ 80–89), normal (IQ 90–109), bright norm al (IQ 110–119), superior (IQ 120–129), and very superior (IQ > 129). The IQ was a scaled score that was adjusted by age in the normal model. Statistical analysis The 512 subjects were classified into quartiles according to level of serum fluoride. We used the chi-square test to compare differences in categorica l variables, and analysis of variance to compare differences in continuous variables among groups . We analyzed the association between serum fluoride levels and the ri sk of low IQ using logistic regression models, adjusting for age and sex. In the logis tic regression, first and second quartiles of serum fluoride were combined and used as reference group. Tests of linear trend across levels of serum fluoride were computed using o rdinal scoring. Values were considered significant when p < 0.05 (two sided). The analyses were performed using STATA software (version 9; StataCorp, College S tation, TX, USA) and SAS software (version 8.2; SAS Institute Inc., Cary, NC, USA).

Results

Wamiao and Xinhuai are located 64 km apart in Sihong County. Wamiao villa ge, in northeast Sihong County, about 32 km northeast of Sihong, is a severe endemic fluorosis area. The mean fluoride level in Wamaio drinking water wa s 2.47 ± 0.79 mg/L (range 0.57–4.50 mg/L), and the prevalence of dental fluorosis and defect ed dental fluorosis was 88.56% and 38.98%, respectively. Xinhuai village, in the southwe st part of Sihong county, about 32 km southwest of Sihong, is a non endemic fluorosis a rea. The mean level of fluoride in Xinhuai drinking water was 0.36±0.15 mg/L (range Page 7 of 228

0.18-0.76 mg/L), and the prevalence of dental fluorosis was 4.48%, with no ca ses of defected dental fluorosis identified (Xiang et al. 2004, National Sta ndard of P.R. China 1997). Neither village has fluoride pollution from burning coal or other industrial sources, and the residents do not drink brick tea, which has been known to cause fluorosis (Sun and Liu 2005; Wang et al. 1997; Zhao et al. 2000). Wamiao and Xinhuai village are of comparable size (total populations of 3,097 and 3,208, respectively in 2002) and are both characterized by low economic d evelopment (the average family income was 1500–2000 Yuan/year/person), limited educa tion (average education level of elementary school) and lack of communicat ion with the

outside world. The mean age of the 222 children in Wamiao was significantly higher than the mean age of the 290 children in Xinhuai (11.56 ± 1.68 and 10.75 ± 1.76 years, respectively,

p <0.01) (Table 1). IQ scores, urine iodine concentrations, and blood lead leve ls were normally distributed, but drinking-water fluoride (Kolmogorov-Smirnov Z=6.095, p <0.01) and serum fluoride (Kolmogorov-Smirnov Z = 3.965, p <0.01) concentrations

were not

. Drinking-water fluoride (2.47 ± 0.79 versus 0.36 ± 0.11 mg/L) and serum fluoride (0.081 ± 0.019 versus 0.041 ± 0.009 mg/L) were significantly higher in Wamiao than Xinhuai, but urine iodine and blood lead levels were comparable between ch ildren from the two villages (Table 2). There were significant differences between the two villages in IQ scores among males ( p <0.002), and females ( p <0.001) (Figure 1). There was also significant

difference within Wamiao between male and female children’s I Q scores ( p <0.001) (Figure 1), but not in Xinhuai (p=0.664). Most children’s IQs were distri buted within the normal range of 90-109 (47.30% in Wamiao, 51.72% in Xinhuai). In Xinhuai, 27.59% of the children had a CRT-RC IQ score indicating bright normal or higher intelligence ( ≥ 110), whereas only 8.11% of Wamiao children had IQ within this ran ge. In fact, in Wamiao, 15.31% of children had a CRT-RC IQ score indicati ng mental retardation (<70) and borderline (70-79), whereas only 6.20% in Xinhuai vill age had IQ in this range. The k-density distribution of IQ scores shows a shif t in the distribution Page 8 of 229

indicating higher IQ scores among children in Xinhuai compared with Wamiao (Figure 2). Linear regression models indicated a significant inverse associat ion between serum fluoride concentration and IQ scores among children in Wamiao (regr ession coefficient for a 1-unit change in serum fluoride = –112.61[95% confidence interval (C I) from -203.15 to -22.06, r 2 =0.0266, p =0.015] (Figure 3). In contrast, the association was not significant among children in Xinhuai (regression coefficient = 79.85, 95% CI from -92.15 to 251.84, r 2 =0.0029, p =0.362). Further adjustment for age and sex increased the

r 2 to 0.088 and 0.034 in Wamiao and Xinhuai, respectively. The regression coeffi cient were: -104.70 (95% CI from -192.41 to -16.98) in Wamiao; 59.82 (95% CI from -110.68 to 230.32) in Xinhuai. When the data from 512 children from the two villages were combined, serum fluoride levels were negatively associated with IQ. The proportion of children with IQ scores <80 increased with increasing quartiles of serum fluoride. Adjusted odds ratios (OR) for IQ score <80 versus ≥ 80 were 2.22 (95% CI, 1.42–3.47) and 2.48 (95% CI, 1.85–3.32) for serum fluoride levels of 0.05-0.08 and >0.08 mg/L compared with <0.05 mg/L, respectively (

p for trend < 0.001) (Table 3). Using likelihood ratio test, we found no significant interaction between village and serum fluoride level ( p = 0.092). There was a significant positive relation between fluoride in drinki ng water and fluoride in serum of children 8–13 years of age (Spearman correlation coefficient = 0.811, p < 0.001, r 2 = 0.741) (Figure 4). Linear regression showed no significant associations between urinary iodine and IQ ( β =0.011, p =0.942 in Wamiao; β =-0.006, p =0.971 in Xinhuai,) or between blood lead levels and IQ ( β =-0.160, p =0.181 and β =-0.092, p =0.460, respectively). Similar results were obtained when data for both villages were combined ( β =0.036, p =0.785 for urinary iodine and β =-0.142, p =0.096 for blood lead).

Discussion As previously noted (Xiang et al. 2003), the average IQ of the children i n Xinhuai was higher than that in Wamiao. The absence of significant ass ociations between IQ Page 9 of 2210

and family income or father’s education level in either village (Xiang et al . 2003) May be explained by the parents’ low educational level and family income in both villages. However, these results are not consistent with those of another st udy conducted in China (Zhao et al. 1996), which reported significant associations betw een children’s IQ scores and their parents’ educational level (the total children we re 320 from 7 to 14 years old in the study of Zhao et al.). The results of this cross-sectional study when the data of two vi llages were combined indicated that serum fluoride level was associated with IQ scores. As the fluoride level in children’s serum increased, the children’s IQs fell and the rat es of borderline or lower intelligence increased. In this study we found a significant dose–response relation between fluori de level in serum and children’s IQ. Fluoride is able to cross the blood–brain barr ier, producing biochemical and functional impairment of the nervous system during dev elopmental periods of infancy and childhood (Guan et al. 1998; He et al. 1989; Mullenix et al. 1995; Zhang et al. 2000). The findings of Guan et al. and those of other studie s suggest that higher fluoride intake during the prenatal period can cause nervous s ystem damage in the fetus (Cui and Wang 2001; Guan et al. 1986; He et al. 1989; WHO 1996). The results of a study of 4- to 7-year old children by Wang et al. ( 1996) indicated that the higher fluoride intake was mainly associated with Performan ce IQ rather than Verbal IQ scores from the WPPSI (Wechsler Preschool and Primary Sca le of Intelligence). Guo et al. (2001) suggested that occupational exposure to fluoride affect ed not only cognitive function but also motor function; NCTB (Neurobehavioral Core T est Battery) scores were significantly lower in the exposed group compared with the control group, except for scores on the Benton Visual Retention Test. The CRT-RC is a test of observational ability and logical thinking rather than verbal IQ (L i and Chen 1989; Zhang et al. 2007). Therefore our findings suggest an effect of serum fluoride on performance IQ, consistent with Wang et al. and Guo et al. (Wang et al. 1996; Guo et al. 2001), but do not provide information regarding effects on verbal IQ. We found no significant difference between the two villages in uri ne iodine levels, and did not find associations between urine iodine and IQ. In addition, neither village Page 10 of 2211

has been identified as being in an area of endemic iodine deficiency (National standard of P.R. China 1995). Thus, differences in urinary iodine levels do not appear to explain the differences in IQ between the two villages in this study. Prenatal and postnatal exposure to lead is now well recognized as an important cause of mental impairment (Burgstahler 2003; Carpenter 2005; Chen et al. 2005). We measured 138 blood samples for blood lead and found no significant differe nce between the two villages in blood lead levels, and no association betwee n blood lead and IQ in study participants. This result could be explained by the l ow level of blood lead in our study population compared with other studies (Ai et al. 2006; Jusko et al. 2008). In our previous study (Xiang et al. 2003), we found a significant dose–re sponse relation between fluoride in drinking water and children’s IQ in both Wamiao and Xinhuai. In the present study we found a positive dose–response relation be tween the level of fluoride in drinking water and the level of fluoride in se rum of children ( r 2 =0.741), which suggests that fluoride in drinking water is the main sour ce of fluoride intake in Wamiao and Xinhuai village. In conclusion, we found an inverse association between serum fluoride leve l and IQ among children in an area containing a high level of fluoride in the water. Further research should be done in more varied (e.g., urban and village) population t o determine if the relationship between IQ and fluoride is independent o f family income and parent’s level of education. In addition, the results of our cross-se ctional study should be confirmed in a larger study population with a longitudinal study design.

References

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Table 1 Sample characteristics in Wamiao and Xinhuai Village * Page 14 of 2215 Characteristics Wamiao Xinhuai p Age (years) (mean, SD) 11.56± 1.68(222) 10.75 ± 1.76(290) <0.010 ‡

Boys (%) 55.41(123/222) 54.83(159/290) 0.896 †

Age (years) (mean, SD) 11.50±1.51 10.81±1.61 <0.010 ‡

Girls (%) 44.59(99/222) 45.17(131/290) 0.896 †

Age (years) (mean, SD) 11.63±1.47 10.68±1.78 <0.010 ‡

Family income (RMB/year/ person) (%)

Very Low (<1000) 18.92(42/222) 13.79(40/290) 0.117 †

Low (1001-2000) 57.65(128/222) 56.66(173/290) 0.649 †

Medium (2001-3000) 17.57(39/222) 20.34(59/290) 0.429 †

High (>3000) 5.86(13/222) 6.21(18/290) 0.869 †

Education of father (%)

Primary and below 33.33(74/222) 41.73(121/290) 0.053 †

Junior 53.16(118/222) 45.17(131/290) 0.073 †

High school and above 13.51(30/222) 13.10(38/290) 0.892 †

Having medical history (%) ** 1.26(3/238) 0.98(3/305) 0.759 †

Absent from village for ≥ 2 years(%) 5.46(13/238) 4.14(12/305) 0.399 †

* Data collected in Oct. 2002. ** History of head trauma or illnesses affecting the nervous system

† Chi square Test

‡ Independent–samples T Test Page 15 of 2216

Table 2 Fluoride in drinking water and serum, iodine in urine, and lead in serum in two villages’ children (Mean ± SD)* Drinking water Fluoride Serum Fluorid e Urine iodine Blood lead Village —————————— ——————————————————— ————————— Samples mg/L Samples mg/L Samples μ g/L Samples μ g/L

Wamiao 222 2.47 ± 0.79 222 0.081 ± 0.019 46 280.70 ± 87.16 71 21.95±13.65

Xinhuai 290 0.36 ± 0.11 290 0.041 ± 0.009 40 300.96 ± 92.88 67 23.61±14.17

p <0.001 † <0.001 † 0.447 ‡ 0.487 ‡

* Compared with Wamiao village.

† Mann-Whitney Test

‡ Independent–samples T Test Page 16 of 2217

Table 3 Association between serum fluoride and children’s IQ a

N Mean IQ SD IQ p b IQ<80 (%) p c OR(95%CI) for IQ<80 Serum fluoride levels (quartiles) Q1/Q2(<0.05 mg/L) 259 100.1 13.4 7.0 1 Q3(0.05- 0.08 mg/L) 126 95.9 13.7 <0.001 15.1 0.004 2.22(1.42-3.47) Q4(>0.08 mg/L) 127 92.1 13.4 17.3 2.48(1.85-3.32) P trend<0.001 d

a Adjusted for age and gender using Logistic regression analysis. The dat a from two villages were combined.

b ANOVA

c Chi square Test

d Tests of linear trend were computed using ordinal scoring. Page 17 of 2218

Figure Legends

Figure 1 Boxplot of children’s IQ in Wamiao and Xinhuai village by genders. p values

were for c omparing IQs between villages within the same gender using T-t est. The dots

in the female parts of the graph indicated that there are two f emales’ IQ lower than

mean-3SD in Xinhuai, and one in Wamiao.

Figure 2 Kdensity distribution of children’s IQ in Wamiao and Xinhuai village

.

Figure 3 Association between serum fluoride and children’s IQ in Wamiao and

Xinhuai

.

Figure 4 Association between the drinking water fluoride and serum fluoride

. Page 18 of 22p=0.002 p<0.0016080100120140IQ

Male Female Xinhuai Wamiao Xinhuai Wamiao

Figure 1 Boxplot of children’s IQ in Wamiao and Xinhuai village by genders. p values were for comparing IQs between villages within the same gender using T-test . The dots in the female parts of the graph indicated that there are two females’ IQ lowe r than mean-3SD in Xin huai, and one in Wamiao. Page 19 of 220.01.02.03Kdensity

60 80 100 120 140 IQ

Xinhuai W amiao

Figure 2 Kdensity distribution of children’s IQ in Wamiao and Xinhuai village Page 20 of 22r=0.054 p=0.362 r=-0.163 p=0.0156080100120140

0 .05 .1 .15 0 .05 .1 .15 Xinhuai Wamiao

IQ Fitted values Serum Fluoride

Figure 3 Association between serum fluoride and children’s IQ in Wamiao and Xinhuai Page 21 of 22Figure 4 Association between the drinking water fluoride and serum fluoride Page 22 of 22