Contribution of gestational exposure to ambient traffic air pollutants to fetal cord blood manganese

Environmental Research 112 (2012) 1–7

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Contribution of gestational exposure to ambient traffic air pollutants to fetal cord blood manganese Ying-Ying Lin a, Yaw-Huei Hwang a,b, Pau-Chung Chen a,c, Bing-Yu Chen a, Hui-Ju Wen a, Jyung-Hung Liu a, Yue Leon Guo a,c,n a

Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei, Taiwan Department of Public Health, College of Public Health, National Taiwan University, Taipei, Taiwan c Department of Environmental and Occupational Medicine, National Taiwan University (NTU) College of Medicine and NTU Hospital, Rm. 339, No. 17 Xuzhou Rd., Taipei 10002, Taiwan b

a r t i c l e i n f o

abstract

Article history: Received 18 May 2011 Received in revised form 26 October 2011 Accepted 11 November 2011 Available online 15 December 2011

Motor vehicle emissions have become a major source of air pollution. Contributions of motor vehicle emissions to exposure to toxic metals such as manganese remain inconclusive. This study investigates the relationship between the concentration of manganese in cord blood and exposure to criteria air pollutants during pregnancy. A total of 1526 mother–newborn pairs were recruited by stratified sampling between April, 2004 and July, 2005. The newborns’ mothers completed questionnaires that collected information on their demographic characteristics, medical histories, and living environments. Cord blood samples were collected at birth and analyzed by inductively coupled plasma mass spectrometry for manganese. Information about criteria air pollutants which included CO, NO2, ozone, SO2, and PM10 was obtained from monitoring stations run by the Taiwan Environmental Agency. Using the Arc9 Geographic Information System’s kriging method, the concentration of each criteria pollutant was estimated at each newborn’s residence. The geometric mean for cord blood manganese concentrations was 47.0 mg/L (GSD ¼1.4). After adjusting for confounding factors such as family income, maternal education, maternal smoking, alcohol drinking during pregnancy, maternal age, child gender, parity, gestational age, and birth season, the results of a multiple linear regression model indicated that cord blood manganese concentration was significantly associated with NO2 concentration in each trimester, as well as the whole duration of gestation. Between the pregnant women exposed to the highest and those to the lowest quartile of NO2, a 6 mg/L difference in cord blood manganese concentration was found. This finding suggests that despite other sources of manganese exposure, maternal exposure to ambient NO2, a surrogate for traffic emission, significantly contributed to fetal cord blood manganese level. Further study is warranted to determine whether the contribution of manganese due to traffic emission causes adverse health effects in fetuses. & 2011 Elsevier Inc. All rights reserved.

Keywords: Cord blood Manganese Traffic emission Air pollution Geographic information system Prenatal exposure

1. Introduction Motor vehicle emissions have become a major source of air pollution. More than 19.8 million vehicles were on the road by the end of 2005 in Taiwan (Taiwan Ministry of Transportation and Communications, 2009). Gasoline is a petroleum-derived liquid mixture used primarily as fuel in internal combustion engines. Taiwan completely ceased the use of leaded gasoline in February,

n Corresponding author at: Department of Environmental and Occupational Medicine, National Taiwan University (NTU) College of Medicine and NTU Hospital, Rm. 339, No. 17 Xuzhou Rd., Taipei 10002, Taiwan. Fax: þ 886 2 2327 8515. E-mail address: [email protected] (Y.L. Guo).

0013-9351/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2011.11.006

2000 and used that which might have contained manganese additives. Although MMT (methylcyclopentadienyl manganese tricarbonyl) is not added into gasoline as an antiknock agent, previous reports found detectable Mn in gasoline (1.97 mg/kg) and diesel (1.67 mg/kg). Automobile emissions from motor vehicles using gasoline contained Mn at 0.0370.0076 mg/g, whereas those from diesel users contained 2.6371.56 mg/g (Taiwan EPA). Since diesel accounted for approximately 40% of all fuel used by motor vehicles, exposure to Mn from traffic emissions is of concern. Studies have shown that the concentration of manganese in gasoline is 1.95 mg/L (Chen, 2002). Humans can be exposed to manganese when it is emitted from gasoline combustion in vehicle engines. It is possible that ambient air pollution from traffic emission contributed to human exposure to manganese.

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Due to the recognized importance of ambient air pollutants, monitoring of common pollutants is done regularly by Taiwan Environmental Protection Administration (EPA). Most of the monitoring stations recorded carbon monoxide (CO), nitrogen dioxide (NO2), ozone, sulfur dioxide (SO2), and particulates with aerodiameters of 10 um or lower (PM10) (Guo et al., 1999). However, the coverage of monitoring data was considered more reliable within 2 km of the monitoring stations. Therefore, in a population study where not every participant resides within 2 km of a monitoring station, exposure data may not be available. The growth of the Geographic Information System (GIS) technique has facilitated the establishment of an individualized traffic-related air pollution exposure index (Briggs, 2007; Gulliver and Briggs, 2005). Spatial interpolation models such as the kriging and inverse distance weighted (IDW) methods have been widely used to map air pollution (Beelen et al., 2009; Chan et al., 2009; Hwang and Jaakkola, 2008). Individual exposures to air pollutants can therefore be estimated using this approach. Fetuses in the gestational period are generally considered to be highly vulnerable to environmental toxins. Despite being an essential element for humans, high manganese exposure during pregnancy may have toxic effects on fetal development. Manganese crosses the placenta to the fetus via active transport mechanisms (Rossipal et al., 2000). Excessive exposure to manganese was associated with intrauterine growth restriction (IUGR) in fetuses (Vigeh et al., 2008), hyperactive behavior, diminished intellectual capacity, and impaired psychomotor development in developing infants and children (Bouchard et al., 2007; Kim et al., 2009; Takser et al., 2003). Because pregnancy is a well-defined and relatively narrow period of exposure, it is important to identify windows of greater susceptibility to air pollution (Woodruff et al., 2009). To understand many birth outcomes, an appropriate scale of study may be months or trimesters (Ritz et al., 2008). Despite inconsistencies in the methods employed and the results reported, there is growing evidence suggesting that ambient air pollution during pregnancy is associated with adverse birth outcomes (Maisonet et al., 2004;

Slama et al., 2008; Stillerman et al., 2008). For example, previous studies have found that NO2 exposure in the early stages of pregnancy (the first or second trimester) has an adverse effect on birth weight (Aguilera et al., 2009; Bell et al., 2007; Ha et al., 2001; Lee et al., 2003). Other studies have found that exposure to ambient NO2 in the first trimester is associated with preterm birth (Leem et al., 2006) and that each 10 ppb increase in NO2 exposure throughout pregnancy significantly increases the risk of very small gestational age (VSGA) (Rich et al., 2009). Exposure to NO2 is also associated with intrauterine growth retardation (IUGR) (Liu et al., 2003; Liu et al., 2007). 1.1. Study aim In a previous study, we found that the level of manganese in cord blood was related to the density of petrol stations in the area of maternal residence. This study was further conducted to investigate the relationship between cord blood manganese and the ambient air pollutants throughout pregnancy. The contribution of air pollutants to manganese in cord blood was estimated.

2. Methods 2.1. Study populations The study population was described previously (Lin et al., 2011). Briefly, 1526 newborns were enrolled between May, 2004 and July, 2005 from a variety of medical facilities, including three medical centers, one local hospital, and eight clinics from different locations in Taiwan (Fig. 1A). The newborns’ mothers were informed of the content of this research and signed consent forms before their participation in the study. The protocols used in this study were approved by the Institutional Review Board of National Taiwan University Hospital. 2.2. Data collection Demographics, family history, and information related to the gestation period were collected by a structured questionnaire after childbirth. Nurses of the participating hospitals were responsible for collecting information on the

Fig. 1. (A) Geographical locations of study subjects’ residences. (B) Ambient air NO2 concentration and air monitoring sites. Note: Fig. 1B showed an example of spatial distribution of NO2 predicted with the ordinary kriging; derived from the data measured by the 69 monitoring stations that were located in Taiwan.

Y.-Y. Lin et al. / Environmental Research 112 (2012) 1–7 newborns. This includes gender, gestational age, delivery time, birth weight, length, and head circumference. Measurement of cord blood Mn was described previously (Lin et al., 2011). Briefly, cord blood samples were pretreated with a modifying solution of 5 g/L of 25% ammonia, 0.5 g/L Triton X-100, and 0.5 g/L EDTA in double deionized water with a sample to solution volume ratio of 1:9 (Barany et al., 1997). After passing through a filter with a pore size of 45mm, the diluted blood samples were analyzed for manganese concentrations using inductively coupled plasma mass spectrometry (7500C, Agilent Technologies, Inc., Japan). 2.3. Air pollution data and spatial mapping Measurements of air pollutants were based on data routinely collected at 69 Environmental Protection Administration (EPA) air monitoring stations in Taiwan (Taiwan EPA, 2004). Each monitoring station provided hourly readings of the concentrations of NO2, CO, SO2, O3, and PM10. With the data from each monitoring station, the ordinary kriging method was used to estimate levels of pollutants in the ambient air at each mother’s residence during pregnancy by month from August, 2003 to July, 2005. In general, the kriging method was used as a statistical mapping technique that used data collected at each point location to predict concentration in each grid cell over a spatial domain. We used ArcView GIS (version 9.3) and its Geostatistical Analyst Extension (ESRI Inc., Redlands, CA) for semivariogram determination and cross-validation. This software was also used to estimate monthly location-specific concentrations and geocode all newborns’ addresses. We used 0.25 km by 0.25 km grids to partition each mother’s area and determine average ambient concentration per month during pregnancy. Three frequently-referenced spatial models (spherical, exponential, and Gaussian) were used to obtain the ‘‘optimal’’ daily semivariogram parameters (range, partial sill, and nugget) (Liao et al., 2006). A good-fitting semivariogram and kriging model should result in an average prediction error (PE) and standardized prediction error (SPE) near 0 and a root mean square standardized (RMSS) value near 1.0. 2.4. Exposure assessment We used residential address at the time of childbirth to assess exposure during gestational period. Ambient air pollutant concentrations were estimated for each calendar month. Trimesters were calculated by subtracting gestational periods from the day of childbirth. The average of the air pollutant levels in the calendar months responsible for the trimester was taken as the air pollutant level in that trimester. 2.5. Statistic analysis The fundamental hypothesis of this study was that the temporal and spatial variations in traffic-related air pollution levels would be associated with the concentration of manganese in fetal cord blood. This association was calculated after adjusting for family income, maternal education, alcohol consumption during pregnancy, maternal smoking, maternal age, child gender, gestational age, and birth season (Lin et al., 2011). We used a multiple linear regression model to determine the association between cord blood manganese level and ambient air pollutant concentrations after adjusting for the above-mentioned variables by one-pollutant model. While the blood level was undetectable, onehalf of the method detection limit value, i.e., 1.5 mg/L, was assigned for the statistical analysis. For any of those air pollutants significantly associated with manganese in fetal cord blood by one-pollutant model, a two-pollutant model was employed to clarify the interaction between each pollutant and cord blood manganese after adjusting for the above-mentioned variables. To determine the gestational period most responsible for cord blood level of manganese, the air pollutant level of each trimester was entered into the model. The strength of association was compared among the trimesters using multiple linear regression after adjusting for the potential confounding factors mentioned above. All statistical analyses were performed using SAS software version 9.1 (SAS, Institute Inc., Cary, NC). Statistical significance was set at p o0.05 based on a twotailed calculation.

3. Results In total, 1 526 mother–newborn pairs were recruited between May, 2004 and July, 2005. Among them, 1 407 mothers (92.2%) completed questionnaires and had cord blood samples collected. All cord manganese levels were above detection level. Home address matching with currently available addresses on GIS failed for 64 pairs. Thus, the final birth cohort consisted of 1 343 mother-newborn pairs. Fig. 1A shows the geographical locations

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of these study subjects’ residences. Fig. 1B shows an example of the spatial distribution of NO2 predicted with the ordinary kriging method based on data measured by the 69 monitoring stations of Taiwan. In this specific case, the average NO2 concentration of years 2004 and 2005 is shown. The demographics and information related to gestation are shown in Table 1. The average maternal age was 28.4 years. Most of the mothers (95.7%) had at least junior or senior high school education. Approximately 82.4% of the mothers were Taiwanese. The median annual household income was US$12,000–18,000 per year. Most of the mothers did not smoke (97.8%) or drink alcohol (96.9%) during pregnancy. Almost half (41.7%) of the mothers were exposed to environmental tobacco smoke during pregnancy. The percentage of boys among study newborns was 51.5%. Almost half of the newborns were their mothers’ firstborn children. The average gestational age, birth weight, birth length, and head circumference were 38.7 weeks, 3097 g, 49.7 cm, and 33.8 cm,

Table 1 Demographic data and information related to gestation in study mothers and children. Variable

Mean 7SD or na (%)

Maternal age (yrs) Maternal education Elementary school and below Junior and senior high school College and above Maternal occupation Agro-forestry and livestock Manufacturing and business Service industry Housewife Others Maternal nationality Taiwanese Aboriginal Mainland China Foreigners Family income (US $/year) o 12,000 12,000–18,000 18,000–30,000 4 30,000 Maternal smoking No Yes ETS exposure during pregnancy No Yes Alcohol consumption during pregnancy No Yes Gender Boy Girl Parity 1 2 42 Birth Season Spring Summer Fall Winter Gestational age (weeks) Birth weight (g) Birth length (cm) Head circumference (cm) Cord blood manganese (mg/L)b

28.4 7 5.0

a b

57 (4.3) 725 (54.9) 539 (40.8) 69 (5.3) 217 (16.7) 420 (32.2) 450 (34.5) 147 (11.3) 714 (82.4) 24 (2.8) 32 (3.7) 96 (11.1) 317 390 342 242

(24.6) (30.2) (26.5) (18.8)

1275 (97.8) 29 (2.2) 747 (58.3) 534 (41.7) 1270 (96.9) 41 (3.1) 678 (51.5) 638 (48.5) 555 (48.6) 403 (35.3) 183 (16.0) 259 (19.8) 322 (24.6) 296 (22.6) 431 (33.0) 38.7 (1.4) 3097 (475) 49.7 (3.3) 33.8 (4.4) 47.0 (1.4)

Total number of subjects under each item may differ due to missing data. GM (GSD).

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respectively. The geometric mean of cord blood manganese concentrations for all study newborns was 47.0 mg/L. Table 2 shows the correlation matrix of ambient air pollutants. There was a high correlation between NO2 and CO (r ¼0.95, p o0.0001). On the contrary, there was a negative correlation between NO2 and O3 (r ¼ 0.77, p o0.0001). Multiple regression analysis was done using ambient air pollutants (NO2, CO, SO2, O3 and PM10) in each trimester as well as those in total gestational period as independent variables and cord blood manganese as the dependent variable, adjusting for potential confounders, including family income, maternal age, maternal education, maternal smoking, alcohol consumption during pregnancy, newborn gender, parity, gestational age, and birth season. The results of the single pollutant model are shown in Table 3. It was found that fetal cord blood manganese was most associated with the NO2 concentration in ambient air during the first trimester, followed by those in the second and the third trimesters. A strong relationship between NO2 for total gestational period and cord blood

Table 2 Correlation matrix of ambient air pollutants.

CO SO2 O3 PM10 n

NO2

CO

SO2

O3

0.949nnn 0.440nnn  0.767nnn  0.493nnn

0.257nnn  0.796nnn  0.623nnn

 0.258nnn 0.238nnn

0.566nnn

po 0.05. p o 0.01.

Table 4 Relationship between fetal cord blood manganese concentration (log-transformed) and ambient air pollutant concentration during the whole period of gestation, by regression analysis (two pollutants model). Log (Cord Blood Manganese, mg/L)a 95%CI

b Nitrogen dioxide With CO With SO2 With O3 With PM10

0.0124 0.0074 0.0098 0.0094

(0.0025, (0.0039, (0.0052, (0.0058,

p value

0.0222) 0.0108) 0.0144) 0.0130)

0.0137 o .0001 o .0001 o .0001

Carbon monoxide With NO2 With SO2 With O3 With PM10

 0.1632 0.1683 0.2282 0.2684

(  0.4472, 0.1209) (0.0752, 0.2614) (0.0926, 0.3638) (0.1552, 0.3817)

Sulfur dioxide With NO2 With CO With O3 With PM10

 0.0028 0.0037 0.0078 0.0117

(  0.0168, (  0.0094, (  0.0051, (  0.0008,

0.0112) 0.0167) 0.0207) 0.0241)

0.6909 0.5813 0.2346 0.0661

Ozone With With With With

0.0067 0.0042  0.0056  0.0086

(  0.0016, (  0.0042, (  0.0114, (  0.0153,

0.0150) 0.0127) 0.0001)  0.0018)

0.1156 0.3280 0.0540 0.0129

NO2 CO SO2 PM10

0.2600 0.0004 0.0010 o .0001

a Using multiple linear regression, adjusting for family income, maternal education, maternal smoking, alcohol drinking during pregnancy, maternal age, child gender, parity, gestational age, and birth season.

nn

nnn

po 0.001.

Table 3 Relationship between fetal cord blood manganese concentration (log-transformed) and ambient air pollutant concentration, by trimester, using regression analysis (single pollutant model). Trimester

Log (Cord Blood Manganese, mg/L)a 95%CI

b

p value

Nitrogen dioxide First trimester Second trimester Third trimester All trimester

0.0068 0.0036 0.0038 0.0070

(0.0045, (0.0013, (0.0007, (0.0040,

Carbon monoxide First trimester Second trimester Third trimester All trimester

0.2172 0.1525 0.0658 0.1768

(0.1410, 0.2934) (0.0741, 0.2309) (  0.0132, 0.1448) (0.0887, 0.2649)

o .0001 0.0001 0.1024 o .0001

Sulfur dioxide First trimester Second trimester Third trimester All trimester

0.0170 0.0149 0.0005 0.0113

(0.0047, 0.0292) (0.00400, 0.0258) (  0.0101, 0.0111) (  0.0011, 0.0237)

0.0066 0.0074 0.9229 0.0745

Ozone First trimester Second trimester Third trimester All trimester

0.0010  0.0069  0.0004  0.0066

(  0.0022, (  0.0098, (  0.0037, (  0.0121,

0.0042)  0.0039) 0.0030)  0.0011)

Particulate matter (PM10) First trimester 0.0002 Second trimester  0.0001 Third trimester  0.0004 All trimester  0.0003

(  0.0006, (  0.0010, (  0.0011, (  0.0013,

0.0009) 0.0008) 0.0003) 0.0007)

0.0091) 0.0059) 0.0068) 0.0101)

o .0001 0.0022 0.0163 o .0001

0.5274 o .0001 0.8330 0.0192 0.6662 0.9050 0.2572 0.5951

a Using multiple linear regression, adjusting for family income, maternal education, maternal smoking, alcohol drinking during pregnancy, maternal age, child gender, parity, gestational age, and birth season.

manganese existed. The same trend of association occurred for CO and SO2, with the exception that total gestational period for SO2 had borderline association with cord blood manganese. A negative association was found between O3 and manganese for the second trimester and for the total gestational period. No significant relationship was observed between PM10 and fetal cord blood manganese concentration. In order to clarify the interaction between several ambient air pollutants, multiple linear regression using two-pollutant model was done for the total period of gestation, adjusting for potential confounders. It was found that NO2 remained a significant predictor for cord blood manganese after adjusting for any other pollutant. CO, SO2, and ozone became insignificant when NO2 was added into the model (Table 4). After adjusting for potential confounding factors, cord blood manganese was highly associated with ambient NO2 levels during the whole period of gestation, i.e., 43.5 mg/L for lowest quartile of NO2 (range 13.1–16.5 ppb), 45.8 mg/L for the second lowest quartile (16.5–17.8 ppb), 47.6 mg/L for the third lowest quartile (17.8– 23.6 ppb), and 49.8 mg/L for the highest quartile (23.6–26.1 ppb) (Fig. 2). There was a 6.3 mg/L difference in cord blood manganese between children born to mothers exposed to the highest and the lowest quartiles of NO2 during gestation. If unexposed to NO2 (0 ppb), the cord blood manganese level was extrapolated to 34 mg/L.

4. Discussion To the best of our knowledge, this is the first study assessing the relationship between prenatal exposure to ambient air pollutants and the concentration of manganese in fetal cord blood. Cord blood manganese was found to be strongly associated with the average NO2 concentration in ambient air during gestation, especially during the earlier stages of pregnancy. This finding strongly suggested significant contribution of traffic emission to fetal exposure to manganese.

Y.-Y. Lin et al. / Environmental Research 112 (2012) 1–7

Adjustment of Cord Blood Mn, µg / L

54 52 50 48 46 44 42 40 group 1*

group 2

group 3

group 4

Ambient air NO2 level Fig. 2. Cord blood manganese levels in mothers exposed to different levels of NO2 during gestation. * Ambient air NO2 level: group 1: o 25th percentile, group 2: 25th–50th percentile, group 3: 50th to 75th percentile, group 4: 475th percentile. Note: After adjusting for family income, maternal education, maternal smoking, alcohol drinking during pregnancy, maternal age, child gender, parity, gestational age, and birth season, we used a stratified analysis to compare different NO2 levels with the adjusted cord blood manganese concentration (95% CIs).

We have previously reported the relationship between petrol station density and fetal Mn level in cord blood (Lin et al., 2011). This current study is an extension to the previous study, while documenting more clearly the role of traffic emission on fetal exposure to Mn. It is true that NOx emission was correlated to density of petrol stations in Lin et al. (2011). However, the correlation was not perfect. Namely, the correlation coefficient was not 1.0, leaving an uncertainty whether traffic emission after automobile combustion causes the exposure to Mn, or emission from the petrol stations before combustion causes it. In this current study, we are more convinced that emission from the automobile is the source of Mn exposure in fetus. As in Lin et al., cord Mn in fetuses living in homes with density of petrol stations within 10 km of 4147 was only 1.17 folds that of those in homes with density of o26. Therefore, Lin et al. (2011) showed that density of petrol stations could be used as a surrogate of Mn exposure in a qualitative approach. However, the results of this current study allow us to quantitatively predict gestational exposure to Mn by using NO2 levels from the neighboring monitoring stations. They also allow the estimation of fetal cord blood Mn level while being completely unexposed to trafficemitted NO2. We believe this greatly enhanced our knowledge of the sources of fetal Mn exposure. In this study, exposure data were obtained from air pollution monitoring stations and not from individually-measured concentrations. Because the mothers’ residencies were not located exactly at the monitoring stations, the kriging method of spatial interpolation was applied in this study. The advantage of the kriging method is that it provides the best linear unbiased estimation; its standard error allows for the quantification of uncertainty. Unlike proximity models, the kriging method uses real pollution measurements in the computation of exposure estimates (Liao et al., 2006). During the processes of validation, the predicted value of one pollutant from a given monitoring station was estimated from measurements of the other 68 stations by the kriging method, and was compared to the measured pollutant level of this monitoring station (Appendix 1). This study took advantage of air pollutant measurements from existing monitoring stations encoded with GIS to characterize

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individual potential exposure. Potential errors could have been introduced to the predicted values of air pollutants. However, such errors were likely random errors, and more likely would push the observed relationship toward the null hypothesis, i.e., no observed relationship between air pollutants and cord blood manganese level. Five criteria air pollutants, CO, NOx (NO and NO2), O3, SO2, and PM10 were selected in this study. Concentrations of NO2 and CO in ambient air are commonly used as measurements of trafficrelated air pollution (Lee et al., 2008; Weng et al., 2008). On the other hand, SO2 and PM10 are considered to be related to combustion from stationary sources or industries (Guo et al., 1999). Though PM10 were commonly seen and monitored in most of the sampling areas, it would have been useful to determine the influence of fine and coarse particles. However, PM2.5 had not been monitored in most of the stations until year 2005. Since our study participants were recruited in 2004–2005, such information was not available. A recent publication by our group found that PM2.5 accounted for 60–70% of PM10 by weight in northern Taiwan (Chen et al., 2011). However, this proportion may vary among geographical locations. Since PM2.5 was included into the monitoring stations, further study to include data on PM2.5 is warranted. Although in one-pollutant models (Table 3), fetal cord blood Mn was associated with ambient exposure to CO and SO2, adding NO2 in the two-pollutant model analysis essentially nullified the effects of CO and SO2, indicating that CO and SO2 was in fact unrelated to fetal cord blood Mn (Table 4). Our finding that NO2 exposure was most related to cord manganese level indicated that maternal exposure to traffic emission contributed significantly to the fetal exposure to manganese. Manganese is an essential mineral nutrient in humans and other animals and is required for normal amino acid, lipid, protein, and carbohydrate metabolism (Wood, 2009). Manganese is a normal component of human tissues and fluids; levels of manganese are highest in the liver, pancreas, and kidney, and ˇ ´ and Lucchini, 2007). In the general lowest in bones and fats (Saric population, food is the main source of manganese (ATSDR, 2008). Diet duplicate study on Mn in food is lacking in Taiwan, but 2.7371.28 mg/kg was reported in Australia (Gulson et al., 2006), indicating significant source of Mn from dietary intake. Levels of airborne manganese are higher in areas with higher traffic densities (Boudia et al., 2006; Loranger and Zayed, 1997). Studies on ambient levels of Mn had been scarce in Taiwan. In Taiwan in 2007–2008, ambient manganese levels were detectable at concentrations of 11.4, 21.2, 15.0, and 11.3 ng/m3 in northern, central, southern, and eastern Taiwan, respectively (Taiwan EPA, 2009), which were all below the US EPA reference concentration (Rfc) of 0.050 mg/m3. Airborne manganese could have been the source of exposure in the pregnant mothers and, in turn, their fetuses. In southwest Quebec, a study on blood manganese levels in pregnant women and cord blood found higher manganese in cord blood as compared to mothers’ in the third trimester, suggesting an active transport mechanism through the placenta to the fetus (Takser et al., 2004). In our study, it is likely that the cord blood manganese came mainly from the mothers’ blood. Therefore, the correlation between cord blood manganese and the ambient NO2 was likely caused by the mothers’ exposure to traffic emissions. One would think that the cord blood manganese was more related to maternal exposure to air pollutants toward the end of gestation simply due to time proximity. However, our findings showed that cord blood manganese was most significantly associated with NO2 exposure in earlier stages of pregnancy. Some studies have shown that women tended to spend more time at home during the later stages of pregnancy (Nethery et al., 2009). This might have reduced the mothers’ exposure to ambient pollutants in the

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second and third trimesters. In addition, the main excretion route of manganese is through the bile and thereafter feces. It is likely that in fetuses, such excretion route is not as efficient as in adults and thus accumulation becomes more prominent than that in their mothers. The fetal cord blood manganese concentration in this study (47 mg/L, geometric mean) was similar to reports from other countries such as 45 mg/L from Montreal (mean), 42 mg/L from Paris (mean), and 40 mg/L from Ottawa County (mean) (Smargiassi et al., 2002; Zota et al., 2009). In this study, after adjusting for potential confounding factors, cord blood manganese was highly associated with ambient NO2 levels during the whole period of gestation, i.e., 43.5, 45.8, 47.6, and 49.8 mg/L for the lowest to highest quartiles (Fig. 2). Although lowest observable adverse effect level (LOAEL) of manganese has not been concluded in fetal cord blood, future studies are warranted to determine whether exposure to traffic emission causes a higher percentage of fetuses to exposure levels higher than the LOAEL, and whether such exposure causes adverse effects. Despite a strong suggestion of contribution from traffic emission to fetal cord blood manganese, it is apparent that traffic emission is not the only, nor the most important source of manganese. Extrapolating from the relationship between ambient NO2 and fetal manganese concentrations, even at a level of zero ambient NO2, the cord blood manganese level was higher than 30 mg/L. This finding indicates the presence of cord blood manganese not explained by traffic exposure. Studies of the other sources of manganese in the general population in Taiwan are lacking. As for occupational sources, workers in ferromanganese smelting industry were reported to expose to Mn (Wang et al., 1989). None of our parents belong to this industry, or to metalrelated industry. We therefore consider occupational contribution minimal in this birth cohort. Food is a well-known source of ˇ ´ and Lucchini, 2007). Wheat, rice, nuts, tea, manganese (Saric legumes, pineapples, whole grains, and milk products are important sources of manganese from food. In Taiwan, consumption of wheat, rice, nuts, pineapple, and milk products in the general population is rather common. Therefore, food sources likely contribute to the fetal blood Mn level of 430 mg/L at extrapolated zero ambient exposure to NO2. On the other hand, a clear dose relationship between NO2 and cord blood manganese suggests that the contribution of manganese from traffic emissions cannot be ignored and deserves further attention in the context of public health. Measurement of ambient levels of Mn is lacking in Taiwan. However, in gasoline, diesel, and the exhausts of automobiles using these fuels in Taiwan, Mn has been detectable (Taiwan EPA). In Australia, blood Mn in young children was found to be unassociated with ‘‘traffic exposure,’’ as measured by proximity to roadway and traffic flow rate (Gulson et al., 2006). This is different from our findings. However, several differences in these two studies might have contributed to the different findings. The exposure to traffic emission was probably lower in Australia, as indicated by lower exposure to CO and NO2 (Guo et al., 1999; Hansen et al., 2006). Populations from urban, suburban, and rural areas were included in this study, which allow for larger variation in exposure to air pollutants. In addition, this current study recruited a total of 1343 infant-mother pairs, allowing for stronger statistical power for hypothesis testing. The strengths of this study include, first of all, a rather broad coverage of populations from various areas in Taiwan, allowing for a wide range of exposure to air pollutants. Also, the study considered several criteria air pollutants and utilized two-pollutant analysis approaches to identify the most likely pollutants contributing to fetal cord Mn. Furthermore, a rather large sample size in this study allowed for categorization into exposure

gradients and thus for documentation of dose–response relationships between air pollutant exposure and fetal cord Mn. Finally, personalized data were obtained to control for several potential factors reported in previous studies, including maternal age, education, and cigarette smoking during pregnancy (Smargiassi et al., 2002; Takser et al., 2004). This study has several limitations. It used the mother’s address at childbirth to represent the geographic location at which the mother spent her time during pregnancy. Although this method has the advantage of individualizing exposure to traffic, it might suffer from potential misclassification resulting from the mother’s move during pregnancy. A recent study of the use of maternal residence at delivery as a way of determining environmental exposure showed that mobility during pregnancy is likely to be prevalent enough to introduce exposure misclassification (Fell et al., 2004). However, such misclassification would most likely be non-differential and push the observed relationship toward the null hypothesis (Ritz and Wilhelm, 2008; Ritz et al., 2007). Also, this study did not examine personal manganese exposure from indoor sources such as cooking and indoor ventilation. In addition, occupational exposure was not thoroughly assessed even though none of the parents were working in metal-related industries. Finally, a detailed dietary history analysis of food duplicate was not conducted. Thus, the contribution of individual constituents of nutrition to Mn is unclear. However, due to ubiquitous availability of all food categories in Taiwan, air pollutant exposure and dietary exposures to Mn are probably unrelated. Therefore, in examining the hypothesis of the relationship between cord blood Mn and ambient exposure to air pollutants, dietary factors can be considered as a source of random error. Such error would have pushed the observed relationship toward the null hypothesis.

5. Conclusion After adjusting for potential confounders, this study found that the level of manganese in fetal cord blood was associated with the NO2 concentration in ambient air, especially during the early stages of pregnancy. This finding suggests that traffic emission contributes to fetal exposure to manganese. Further study exploring whether traffic-related manganese exposure causes adverse fetal health effects is warranted.

Acknowledgments This study was partially supported by the National Science Council, Taiwan, R.O.C (NSC96-2314-B-002-113-MY2 and NSC100-2621-M-002-009), and partially supported by the Global Research Lab (K21004000001-10A0500-00710) through the National Research Foundation of Korea, which was funded by the Ministry of Education, Science and Technology.

Appendix A. Supporting materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.envres.2011.11.006.

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