Title: 1
Phthalate exposure in pregnant women and their children in central Taiwan 2
Authors names and affiliations: 3
Susana Lin 4
E-mail: susanalin@nhri.org.tw 5
Affiliation: Division of Environmental Health and Occupational Medicine, National 6
Health Research Institutes, Taiwan 7
Hsiu-Ying Ku 8
E-mail: shiuo@nhri.org.tw 9
Affiliation: Graduate Institute of Life Science, National Defense Medical Center 10
Pen-Hua Su 11
E-mail: jen@csh.org.tw 12
Affiliation 1: Department of Pediatrics, Chung Shan Medical University Hospital, 13
Taichung, Taiwan. 14
Affiliation 2: School of Medicine, Chung Shan Medical University, Taichung, Taiwan. 15
Jein-Wen Chen 16
E-mail: jwchen@nhri.org.tw 17
Affiliation: Division of Environmental Health and Occupational Medicine, National 18
Health Research Institutes, Taiwan 19
Po-Ching Huang 20
E-mail: pchuang@nhri.org.tw 21
Affiliation: Division of Environmental Health and Occupational Medicine, National 22
Health Research Institutes, Taiwan 23
Jürgen Angerer 24
E-mail: juergen.angerer@ipasum.imed.uni-erlangen.de 25
2 Affiliation: Institute ad Outpatient Clinic of Occupational, Social and Environmental 26
Medicine, University of Erlangen-Nurenberg 27
Shu-Li Wang 28
E-mail: slwang@nhri.org.tw 29
Affiliation 1: Division of Environmental Health and Occupational Medicine, National 30
Health Research Institutes, Taiwan 31
Affiliation 2: School of Medicine, Chung Shan Medical University, Taichung, Taiwan 32
Corresponding author: 33
Shu-Li Wang 34
National Health Research Institutes 35
Division of Evironmental Health and Occupational Medicine 36
No35, Keyan Road, Zhunan Town, Miaoli County 35035, Taiwan. 37 E-mail: slwang@nhri.org.tw 38 Phone: +886-37-246166-36519 39 Fax: +886-37-587406 40 41
Abstract 42
43
Phthalate exposure has been found to be associated with endocrine disruption, 44
respiratory effects, and reproductive and developmental toxicity. The intensive use of plastics 45
may be increasing the exposure to phthalates in the Taiwanese population, particularly for 46
young children. 47
We studied phthalate metabolites in pregnant women and their newborns in a general 48
population in Central Taiwan. A total of 430 pregnant women agreed to participate and one 49
hundred maternal urine samples and thirty paired cord blood and milk samples were 50
randomly selected from those participants. Eleven phthalate metabolites (MEHP, 51
5OH-MEHP, 2cx-MEHP, 5cx-MEPP, 5oxo-MEHP, MiBP, MnBP, MBzP, OH-MiNP, 52
oxo-MiNP, and cx-MiNP) representing exposure to five commonly used phthalates (DEHP, 53
DiBP, DnBP, BBP, DiNP) were measured in the urine of pregnant women, cord serum and 54
breast milk after delivery, and in the urine of their children. Exposure was estimated based on 55
excretion factors and correlation among metabolites of the same parental compound. Thirty 56
and fifty-nine urinary samples from 2 and 5 years-old children, respectively, were randomly 57
selected from the 185 children who were followed successfully. 58
The total urinary phthalate metabolite concentration (geometric mean, μg/L) was 59
found to be higher in 2-year-olds (398.6) and 5-year-olds (333.7) than in pregnant women 60
4 (205.2). Metabolites in urine are mainly from DEHP. The proportion of DiNP metabolites 61
was higher in children’s urine (4.39 and 8.31%, ages 2 and 5) than in that of adults (0.83%) 62
(p<0.01). When compared with urinary levels, phthalate metabolite levels were low in cord 63
blood (37.45) and milk (14.90). DEHP metabolite levels in women’s urine and their 64
corresponding cord blood were significantly correlated. When compared to other populations 65
in the world, DEHP derived metabolites in maternal urine in Taiwan were higher, while 66
phthalate metabolite levels in milk and cord blood were similar. The levels of phthalate 67
metabolites in milk and cord blood were comparable to those found in other populations. 68
Further studies of the effects on health related to DEHP and DiNP exposure are necessary. 69
Keywords: 70
Phthalate; environmental exposure; Taiwan; cord blood 71
72 73
Introduction 74
Phthalates are chemicals widely used in commercial products such as plastic softeners 75
and solvents in personal care products, lubricants and insect repellents (Fay et al., 1999; Koo 76
and Lee, 2004; Lee et al., 2005). Potential sources of exposure for di(2-ethylhexyl)phthalate 77
(DEHP) include polyvinylchloride containing medical devices, food packaging, plastic toys, 78
furniture, and car upholstery. Di-n-butyl phtyalate (DnBP) is present in medicines, cosmetics, 79
cellulose acetate plastics, latex adhesives, nail polish and other cosmetic products; butyl 80
benzyl phthalate (BBP) is found in vinyl flooring, adhesives, sealants, food packaging, 81
furniture upholstery, vinyl tile, carpet tiles, artificial leather, and di-isononyl phthalate (DiNP) 82
is widely used in children’s toys (Sathyanarayana, 2008). Recent studies suggest that the 83
intensive use of plastic material in Taiwan may be increasing the exposure to DEHP in the 84
Taiwanese population (Chen et al., 2008). 85
According to some epidemiological studies, phthalate exposure is associated with 86
adverse health outcomes, such as shorter anogenital distances at birth (Swan, 2006), 87
respiratory effects (Hoppin et al.,2004; Jaakkola et al., 1999; Jaakkola et al., 2000), and 88
increased waist circumference and insulin resistance (Stahlhut et al., 2007). Exposure to 89
MnBP, mono-benzyl phthalate (MBzP), and Mono-2-ethylhexyl phthalate (MEHP) is 90
associated with an overall pattern of decline in sperm motility (Duty et al., 2004). 91
Pregnant and lactating women represent a population of special concern because of 92
the potential impact of their exposure to phthalates on the fetus and nursing infant. I Exposure 93
data for children under age 6 are scarce (Jahnke et al., 2005; McKee, 2004; NTP-CERHR, 94
2003a). Metabolites of DEHP, DBP and BBP have been monitored in children aged 2-6 95
(Koch et al., 2004; Koch et al., 2005). A decrease in the anogenital distance in male infants 96
has been found to be associated with phthalate exposure, as determined by urinary MEP, 97
MBP, MBzP and MiBP levels (Swan et al 2005). In another study, MBP in maternal urine 98
6 and amniotic fluid was found to be associated with a shorter anogenital distance only in 99
female infants (Huang et al., 2009). In pregnant women, urinary MBP is negatively correlated 100
with thyroxine, free thyroxine and FT4 levels (Huang et al., 2007). 101
Many experimental studies using different laboratory animals (primarily rats) have 102
examined the reproductive toxicity, developmental toxicity, endocrine disruption, and 103
genotoxicity that might be induced by phthalic acids. For example, anti-androgenic effects 104
including delayed puberty in F0, decreased sperm production and fecundity in F1,
105
malformations in F1 reproductive organs, and decreased F2 litter size, were reported for DBP
106
(NTP-CERHR, 2003b). DBP’s metabolite, MBP, is responsible for the toxic effects 107
associated with DBP exposure. These include increased prenatal mortality, decreased fetal 108
weight, cleft palate, fused sternebrae, reduced anogenital distance in males, cryptorchidism, 109
hypospadias, and agenesis of the epididymides or seminal vesicles (NTP-CERHR, 2003b). 110
High doses of DiNP caused an increase in liver weight, peroxisomal proliferation, skeletal 111
variations and renal toxicity in a one-generation and a two-generation toxicity study 112
(Moorman et al., 2000; NTP-CERHR, 2003a). In rats, BBP exposure was associated with 113
decreased testicular weight, reduced ano-genital distance, increased incidence of nipple 114
retention and decreased birth weight in both sexes of the first filial generation (Gray et al., 115
2000; Parks et al., 2000). Treatment with DEHP was also associated with altered ano-genital 116
distance and nipple retention (NTP-CERHR, 2005). 117
In vitro studies help in understanding the possible mechanisms of toxicity. Phthalates 118
and their metabolites can bind to several nuclear receptors and act as endocrine disruptors or 119
metabolic disruptors (Desvergne et al., 2009). In a series of reporter gene assays, DBP, MBP 120
and DEHP have been found to have both anti-androgenic and androgenic activities at 121
different concentrations. These compounds also showed thyroid receptor (TR) antagonistic 122
activity. Only DBP was reported to have estrogenic activity (Shen et al., 2009). BBP has an 123
affinity for binding to estrogen receptors (ER) (Blair et al., 2000; Hashimoto et al., 2000; 124
Matthews et al., 2000; Zacharewski et al., 1998), and activates ER-mediated transcription 125
(Coldham et al., 1997; Harris et al., 1997; Hashimoto et al., 2000; Nishihara et al., 2000; 126
Zacharewski et al., 1998). DEHP has a weak agonistic activity at aryl hydrocarbon receptors 127
(AhR) (Kruger et al., 2008), constitutive androstane receptors (CAR, Nr1i3) (Eveillard et al., 128
2009), and Pregnane X nuclear receptors (PXR, Nr1i2) (Cooper et al., 2008; Hurst and 129
Waxman, 2004). Phthalates, especially MEHP, interfere with steroid production, particularly 130
estradiol production and aromatase expression. A possible mechanism for this interference is 131
through mediation at peroxisome proliferator-activated receptors (PPAR) (Lovekamp-Swan 132
et al., 2003; Lovekamp and Davis, 2001). MEHP is a true ligand for all three PPAR isotypes 133
and a selective modulator of PPAR gamma (Desvergne et al., 2009). BBP does not activate 134
progesterone receptor-mediated transcription (Tran et al., 1996) or AR-mediated transcription 135
(Sohoni and Sumpter, 1998). While BBP alone did not show a significant agonistic AhR 136
8 effect, it enhanced TCDD induced AhR activity in a dose-dependent manner (Kruger et al., 137
2008). BBP exposure in female rats is also associated with a significant increase in liver 138
Ethoxyresorufin-O-deethylation (EROD) activity (Singletary et al., 1997). BBP also induces 139
human breast cancer cell proliferation (Harris et al., 1997; Korner et al., 1998; Soto et al., 140
1997). 141
We monitored eleven phthalate metabolites (MEHP, 5OH-MEHP, 2cx-MEHP, 142
5cx-MEPP, 5oxo-MEHP, MiBP, MnBP, MBzP, OH-MiNP, oxo-MiNP, and cx-MiNP) in 143
pregnant women (urine, serum and milk), their newborns (cord blood) and prospectively in 144
their children at ages 2-3 and 5-6 (urine) from a general population in Central Taiwan. These 145
eleven metabolites were derived from exposure to five commonly used phthalates: DEHP 146
(MEHP, 5OH-MEHP, 2cx-MEHP, 5cx-MEPP, and 5oxo-MEHP), DiBP (MiBP), DnBP 147
(MnBP), BBP (MnBP and MBzP), and DiNP (OH-MiNP, oxo-MiNP, and cx-MiNP). 148
Exposure to phthalic acids was estimated based on the 95% confidence interval for the level 149
of each measured urinary metabolite, and excretion fraction published in the literature. 150
Correlations among metabolites of the same parental compounds and among different types 151
of samples from pregnant women and their corresponding children were also tested. 152
153
Methods 154
Participants, specimen and data collection
156
The subjects were pregnant women from Central Taiwan, aged between 25 and 35 157
and without clinical complications. We invited all pregnant women visiting the local medical 158
center between December 2000 and November 2001 to participate in this study. A total of 159
610 women were approached, and 430 (participation rate: 75%) agreed to be interviewed. To 160
the best of our knowledge, those who refused to participate did not differ in age or social 161
status from those who did participate. All of the participants completed a questionnaire 162
concerning maternal age, parity, baby’s weight, educational level, disease history, dietary and 163
smoking habits, and breast-feeding history. Maternal urine was collected from subjects 164
during the third trimester of pregnancy (28-36 weeks), and umbilical-cord serum was 165
collected upon delivery. Women who agreed to collect samples of breast milk were trained in 166
collection procedures in order to minimize the risk of contamination. A total of 175 167
participants provided adequate breast-milk (>60 ml) samples. Newborns were followed again 168
when they were 2-3 years old (185 subjects, in 2003-2004) and 5-6 years old (185 subjects, in 169
2006-2007). For newborns and children aged 2-3, spot urine samples were collected in a 170
pediatric urine bag with the assistance of a parent at the hospital. For children aged 5-6, urine 171
samples were collected in a glass beaker. Immediately after collection, urine samples were 172
transferred into amber glass bottles and stored at -20°C for analyses of phthalate monoesters 173
and creatinine. One hundred maternal urine samples, fifty-nine urine samples from children 174
10 aged 5-6 , 30 urine samples from children aged 2-3 , and 30 paired cord blood samples and 175
milk samples were randomly retrieved and sent for analysis. The difference in the number of 176
children’s samples was due to freshness and budget limitations, and the fact that one of the 177
samples had insufficient volume for creatinine analysis. 178
179
Analysis of phthalate metabolites
180
The concentrations of eleven phthalate metabolites (MEHP, 5OH-MEHP, 2cx-MEHP, 181
5cx-MEPP, 5oxo-MEHP, MiBP, MnBP, MBzP, OH-MiNP, oxo-MiNP, and cx-MiNP) in 182
urine, cord serum and breast milk were determined with LC-MS-MS methods as described in 183
previous publications (Koch et al., 2003; Preuss et al., 2005) by Dr Jürgen Angerer’s lab at 184
the University of Erlangen, Germany. Metabolite concentrations are expressed as “μg/L” or 185
“μg/g creatinine”. “Total metabolites” refers to the sum of metabolites calculated by adding 186
the concentrations of all metabolites. 187
188
Determination of creatinine levels in urine
189
Urinary creatinine levels were measured by Kaohsiung Medical University Chung-Ho 190
Memorial Hospital, using the spectrophotometric method, with picric acid as the reactive 191
agent, and read at 520nm. 192
Statistical methods
194
We verified the distribution of data for phthalate metabolites for normality. As the 195
data were generally skewed slightly to the right, log transformations of phthalate metabolite 196
values and geometric means were applied in parametric statistical tests. Metabolite levels 197
under the limits of detection (<LOD) were recorded as half the LOD value. Samples from 198
male and female children were considered both separately and together to determine gender 199
differences. Pearson’s correlation tests (r = Pearson correlation coefficient) were used to 200
check for correlations among values of metabolites from the same parental compound in 201
different matrices. The metabolite profile of the urine samples from each subject was 202
expressed as a percentage. The geometric means of the groups were used for parametric tests 203
and medians were used for non parametric tests. The Statistical Package for Social Science 204
(version 15.0; SPSS, Chicago, IL, USA) was used for statistical analysis. 205
206
Estimation of parental compound levels
207
The amount of parental phthalate to which each subject might have been exposed was 208
estimated using excretion fractions reported in the literature (Wittassek et al., 2007). We 209
calculated the range of possible original parental compound levels, based on the 95% 210
confidence interval for the level of each urinary metabolite. 211
12 (1) Parental compound = metabolite concentration × excretion fraction
213 214
We also calculated the daily intake (Estimated Daily Intake, EDI) for these parental 215
compounds, taking into account an average body weight of 55 kg for Taiwanese women, 16.5 216
kg for children aged 2-3, and 20 kg for children aged 5-6 (DOH 2000). A daily urine 217
excretion of 0.8-2.2L was calculated for pregnant women, 0.6L for children aged 2-3 and 0.7 218
L for children aged 5-6 (Fleisher et al., 2002). 219
220
(2) Estimated daily intake = estimated parental compound concentration × daily urine 221
excretion × average body weight 222
223
As an example, to estimate the maximum exposure to BBP for children aged 5 and 6, 224
we used the MnBP concentration in their urine (GM max= 4.86), and an excretion rate of 225
73%, as reported by Wittassek et al 2007; therefore, the parental compounds from which this 226
metabolite originated should be 4.86×100÷73＝6.66μg/L. For the estimation of daily intake, 227
a daily urinary excretion of 0.7L urine/day for children this age was multiplied by the 228
estimated parental compound concentration and the result divided by the average body 229
weight of 20 kg. The estimated daily intake in this example would be 6.66×0.7=0.23 μg/kg 230
bw/day. 231
Results 233
234
General characteristics of the population
235
Table 1 shows the general characteristics of participating subjects, including maternal 236
age, maternal education level, and breast feeding patterns. The average age of the mothers 237
was 29, and the average pre-pregnant BMI was 21. Forty-one percent of the breast-feeding 238
mothers were taking supplements: vitamins, calcium, folic acid or Chinese herbs. Forty-six 239
percent of the infant subjects were male. Mean body weights at birth for the 2-3-year-old 240
cohort and the 5-6-year-old cohort were 3290±460g and 3240±450g, respectively. 241
242
Urinary metabolite levels
243
Urinary phthalate metabolite levels in children at ages 2 and 5 and pregnant women 244
were compared using values with and without creatinine adjustment (Table 2). Generally, 245
metabolite levels were higher in children than in pregnant women. This could be observed 246
whether creatinine adjustment was applied or not. Total phthalate metabolite concentrations 247
without creatinine adjustment were found to be higher in 2-year-old children (GM = 248
398.6μg/L, 282.6–562.3) and 5-year-old children (333.7μg/L, 251.8–442.2) than in pregnant 249
women (205.2μg/L, 172.7–243.8). When creatinine adjustment was applied, children 250
appeared to have even higher phthalate metabolite concentrations; however, we should take 251
14 into account the fact that the level of creatinine in pregnant women’s urine was higher than 252
that in children (PW: 76.60μg/L; 5Y: 59.53μg/L; and 2Y: 62.28μg/L). 253
Analysis of the proportion of metabolites in urine helps identify the parental 254
compound and source of exposure. Phthalate metabolites in urine were mainly those from 255
DEHP, followed by metabolites from DnBP or BBP and those from DiNP. The sums of 256
urinary DEHP metabolites, with a GM of 102.2 μg/L for pregnant women, 152.3μg/L for 257
5-year old children and 200.3μg/L for 2-year old children, were proportionally higher. The 258
second most abundant metabolite was MnBP (Pw: 72.29μg/L; 5y: 75.09μg/L; 2y: 259
100.44μg/L). It was followed by MiBP (Pw: 12.49μg/L; 5y: 25.24μg/L; 2y: 17.21μg/L), and 260
MBzP (Pw: 0.96μg/L; 5y: 3.61μg/L; 2y: 3.40μg/L). The ratio of DiNP metabolites/Total 261
phthalate metabolites observed in children’s urine samples was higher than that in adult 262
samples. The total DiNP metabolite concentration was 1.71μg/L for pregnant women, 263
27.73μg/L for 5-year old children and 17.46μg/L for 2-year old children. The proportion of 264
each metabolite over total phthalate metabolite is shown in Table 3. No gender difference 265
was found at any age, either for the total concentration or for any particular metabolite. 266
267
Phthalate metabolites in cord sera and milk samples
268
Only MEHP, MiNP and MnBP were detected in some of the breast milk samples 269
(Table 4). MEHP was detected in 73% of the samples. When compared to urine, the 270
concentrations of phthalate metabolites were much lower in milk and cord blood samples. 271
The compositions of the phthalate metabolites found in these matrices were also very 272
different. The most abundant metabolite in cord blood serum samples was MEHP, rather than 273
oxidized metabolites of DEHP (Table 4). 274
275
Correlational studies
276
We found that DEHP metabolite concentrations in urine samples of pregnant women 277
(without creatinine adjustment) and their corresponding cord blood samples were 278
significantly correlated for two of the metabolites, 5cxMEPP (r=0.53, p<0.01) and 279
2cxMMHP (r=0.44, p<0.01) (Table 5). This correlation was still observed when the 280
creatinine adjustment was applied to the urine samples (data not shown). Oxo-MiNP 281
concentrations in urinary samples of children at both ages were well correlated (r =0.41, 282
p<0.05). 283
Concentrations of different metabolites derived from DEHP and DiNP within the 284
same sample were well correlated as were the sum of metabolites from the same parental 285
compound (Pearson’s correlation between 0.7 and 0.988). DBzP-derived metabolites are 286
MBzP and MnBP. We observed no correlation between the concentrations of these two 287
metabolites (data not shown). DnBP is another source for MnBP. This lack of DBzP 288
derivation suggests that DnBP is the main contributing source for the formation of MnBP. 289
16 290
Estimation of parental compound exposure
291
The estimated parental contribution to actual phthalate metabolite levels in urinary 292
samples and the estimated daily intake of these compounds suggested that children were 293
more exposed to phthalate than were pregnant women. Children aged 2-3 seem to have been 294
exposed to more total phthalate, particularly to DEHP and DnBP at that younger age than 295
when they were 5-6 years old. An estimated daily intake of each parental compound and a 296
tolerable daily intake (TDI) reference are shown in Table 6. The exposure of the participants 297
in this project did not exceed tolerable daily intake as determined by the European Food 298 Safety Authority (2005). 299 300 Discussion 301
Compared to a CDC study of a population aged 6 and older in the USA (EPA, 2005), 302
the phthalate metabolite levels in the current study (as a geometric mean) were higher for 303
MEHP, 5OH MEHP, 5oxoMEHP, 5cxMEPP and MnBP, and lower for MBzP. When 304
compared to a German study of nursery school children aged 2-6 (Koch et al., 2004), the 305
level of MEHP in our study (as a median) was higher, while the levels of 5OH MEHP, 5oxo 306
MEHP, MnBP and MBzP were lower. When compared to levels in children aged 3-5 in the 307
GerES IV study (Becker et al 2009), only the MEHP level in our study was higher. The levels 308
of 5OH MEHP, 5oxo MEHP and MnBP in our study were lower than those in a Japanese 309
study performed exclusively on pregnant women (Suzuki et al., 2009). As stated in previous 310
reports about human DEHP metabolism, the urinary DEHP metabolites are oxidized products, 311
rather than monoesters, and the simple monoester MEHP was the dominant metabolite in 312
blood serum (Wittassek et al., 2007). The MEHP concentration was also found to be higher 313
than that of other oxidized metabolites in our cord serum samples (Table 4). Although 314
phthalate metabolite levels in milk and cord sera were low, cord blood metabolite levels were 315
well correlated with maternal urinary metabolite levels (Table 5); therefore, maternal urinary 316
metabolite levels may be useful in prenatal exposure studies. 317
The phthalate metabolites detected in breast milk in this study were mainly MEHP 318
and MnBP (Table 4). The levels of MEHP in milk were lower than those reported from the 319
USA (Calafat et al 2004), Denmark (Mortensen et al 2005, Main et al 2006), Finland (Main 320
et al 2006) and Italy (Latini et al 2009). Levels of MnBP were second highest in the studies in 321
Finland and Italy Concentrations of phthalate metabolites in milk, blood and serum were 322
close to the limit of detection; therefore, phthalate metabolites in urine maybe more 323
informative than those in milk or serum (Hogberg et al., 2008). 324
We found no correlation among levels of metabolites in different matrices from the 325
same person, i.e., urine sample, milk sample, and cord blood. This observation is in 326
agreement with previous studies, where authors concluded that phthalate concentrations in 327
18 urine were higher than those in serum, milk or saliva, and did not reflect the concentrations of 328
oxidative metabolites in other body fluids, especially milk (Hines et al., 2009, Hogberg et al., 329
2008). The composition of phthalate metabolites was very different in urine and milk samples. 330
Although it is not clear how phthalate metabolites are secreted into different body fluids or 331
travel across the placenta, they probably have different rates of secretion. An in vitro 332
experiment where the placenta was perfused with different phthalate monoesters showed 333
differential diffusion rates across the placenta for different phthalate metabolites (Mose et al 334
2007). This suggested that physical-chemical properties of the compounds may influence 335
the tissue distribution of the metabolites. This may also explain the lack of correlation among 336
different matrices in the same subject. In general, DEHP and DiNP derived metabolites better 337
represented the overall phthalate exposure as they were the most important contributors in 338
pregnant women and children, respectively. 339
The phthalate exposure profile suggests that the major health risks that might be 340
associated with this population could be anti-androgenic activity and tyrosine receptor 341
antagonistic activity related to MBP, DEHP, and DBP exposure. Mixtures of phthalate esters 342
exhibit cumulative, largely dose-additive effects on male rat reproductive tract development 343
when administered during sexual differentiation in utero (Howdeshell, Furr et al., 2008). 344
DBP+DEHP increased the incidence of many reproductive malformations including 345
epididymal agenesis and reduced androgen-dependent organ weights (Howdeshell et al 2007). 346
Mixtures including BBP, DBP, DEHP, DiBP and DPP reduced testosterone production in a 347
dose-additive manner (Howdeshell, Wilson et al 2008). 348
DiNP is widely used in toys and the proportions of its metabolites differed among 349
urine samples from 2-3 year-olds, 5-6 year-olds, and mothers. Our levels tended to be lower 350
than those in Japanese (Suzuki et al., 2009), US (CDC, 2005; Swan et al., 2005), and German 351
populations (Becker et al., 2009). Higher exposure to DiNP in children could be associated 352
with renal toxicity or problems in skeletal development. 353
MnBP and MBzP are breakdown products of BBP. In an experiment where stable 354
isotope-labelled BBP was administered to eight volunteers in a single dose, 73% was 355
excreted as MBzP and 6% as MnBP on a molar basis (Anderson et al., 2001). We found that 356
the concentration of MnBP was much larger than that of MBzP in our urine samples. Because 357
MnBP can also be derived from DnBP, our results suggest that DnBP is probably the 358
principal contributor of MnBP in this population. 359
Although estimated daily intake values were below the tolerable daily intake 360
according to CSTEE 1998 (Koch et al, 2003) and EFSA (2005), MiBP and MnBP levels in 361
maternal urine were higher than those reported for mothers who gave birth to newborns with 362
shorter ano-genital distances (Swan, 2006; Swan et al., 2005), indicating a potential risk for 363
anti-androgenic effects in this population. 364
20 Higher exposures to DEHP in general and to DiNP in children seem to characterize 365
the phthalate exposure in this population. Although the daily intake values estimated from 366
urinary metabolites were within the tolerable daily intake levels as published by the European 367
Food Safety Authority (EFSA, 2005), it is worth noting that phthalate acids are currently 368
banned for use in toys and school supplies and, therefore, TDI is no longer applicable. 369
Further studies will look deeper into the health effects reported as a result of exposure to 370
DEHP and DiNP, e.g., gender-related behaviour, obesity, liver function, bone density, and 371
allergy, in the population we have been following for several years. 372
373
Conclusions 374
Eleven phthalate metabolites derived from exposure to five commonly used phthalates 375
were monitored. Higher exposures to DEHP in general, to DiNP for children, and lower 376
exposure to BBP are the characteristics of phthalate exposure for pregnant women and their 377
children in Central Taiwan. 378
We found no association between metabolite levels in the mothers and in their 379
children at ages 2 or 5. This may be due to differences in types of ingested food and habits. 380
Metabolite levels in different matrices from the same person, i.e., urine sample, milk sample, 381
and cord blood were not associated. Metabolites derived from the same parental compound, 382
as in the cases of DEHP and DiNP, were well correlated. Although the metabolites derived 383
from a particular phthalic acid are inter-correlated, the additive effects and variability of the 384
secretion fraction suggest that the measurement of individual metabolites is necessary in 385
order to estimate the total exposure. 386
Compared to other populations in the world, the phthalate metabolite levels found in 387
this study were higher for DEHP derived metabolites in urine, whether or not the data was 388
adjusted by creatinine. The levels of phthalate metabolites in milk and cord blood were 389
comparable to those found in other populations. Further follow up studies on health effects 390
related to DEHP and DiNP exposure are necessary. 391
22 Acknowledgements
392
We greatly appreciate the excellent assistance of Ms. Hsiao-Yen Chen for the various 393 specimen collections. 394 395 Grant 396
This work was supported by National Health Research Institutes, Taiwan (EO-97-PP-05 and 397
EO-98-PP-03) 398
399 400
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