Article pubs.acs.org/est
Urinary Excretion of Phthalate Metabolites in School Children of China: Implication for Cumulative Risk Assessment of Phthalate Exposure Bin Wang,† Hexing Wang,† Wei Zhou,‡ Yue Chen,§ Ying Zhou,*,† and Qingwu Jiang† †
School of Public Health, Key Laboratory of Public Health Safety of Ministry of Education, Fudan University, Shanghai, 200032 China ‡ Department of Geriatrics, The Affiliated Taizhou Hospital of Wenzhou Medical University, Linhai City, Zhejiang, 317000, China § Department of Epidemiology and Community Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5 S Supporting Information *
ABSTRACT: We analyzed 13 metabolites of 9 phthalates in urine of 782 Chinese school children aged 8−11 years and estimated the daily intake for phthalates based on urinary metabolite levels. The daily intakes were compared with acceptable intake levels to calculate the hazard quotient (HQ) for single phthalate. Finally, the cumulative risk for each child was assessed by means of a hazard index (HI) which is the sum of HQs. Overall, 11 metabolites were found in at least 85% of the urine samples with the highest median concentration of 47.1 ng/mL (93.4 μg/g creatinine) for mono-nbutyl phthalate (MnBP). Monooctyl phthalate (MOP) and monoisononyl phthalate (MiNP) were not detectable. The cumulative risk assessment covering di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DnBP), di-isobutyl phthalate (DiBP), and butyl-benzyl phthalate (BBzP) demonstrated that 19.8% (volume model-based) and 40.3% (creatinine model-based) of the children exceeded 1 for the HI based on tolerable daily intake (TDI) values (considered as potential adverse antiandrogenic effect). Furthermore, at least 36% of the children from the manufacturing-intensive region had a HI higher than 1. The results indicate that Chinese children are widely exposed to phthalates and those from manufacturing-intensive regions are probably at a high risk of cumulative phthalate exposure.
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cumulative individual exposure to phthalates.3,13 The occurrence of phthalate metabolites has been determined in urine samples from a nationally representative population in the United States (U.S.).14 Several studies also have reported urinary phthalate metabolites in some European countries15−18 and Asian countries,19−22 indicating widespread human exposure to phthalates. The levels of exposure to phthalates have been shown to be higher in children than in adults.5,23−25 Human population studies have reported significant associations between fetal and perinatal exposure to some phthalates and antiandrogenic effects of male infants.26,27 Besides, delayed pubarche in girls is associated with phthalate exposure.28,29 In recent years, a number of studies have reported the associations of children’s exposure to phthalates with obesity, decreased thyroid function, asthma and other respiratory diseases, attention-deficit hyperactivity disorders, and negative behavioral development.30−34 High levels of DEHP metabolites were also found to be
INTRODUCTION Phthalates are a class of synthetic chemicals widely used in industrial and consumer products. High-molecular weight phthalates such as di(2-ethylhexyl) phthalate (DEHP), butylbenzyl phthalate (BBzP), di-isononyl phthalate (DiNP), and dioctyl phthalate (DOP) act primarily as plasticizers in the manufacture of polyvinyl chloride (PVC) building materials, floorings, food packaging, and medical devices. Low-molecular weight phthalates such as diethyl phthalate (DEP), dimethyl phthalate (DMP), di-n-butyl phthalate (DnBP), and di-isobutyl phthalate (DiBP) are mostly used in personal care products.1−4 Phthalates are not chemically bound to the products and can easily leach out into the surrounding environment.5 Thus, people are continuously exposed to phthalates through ingestion, inhalation, dermal absorption, and contact with medical devices as a result of widespread use.6−10 Since there is a ubiquitous existence of phthalates in the environment and daily consumer products, the potential for nonoccupational exposure to phthalates is high for individuals. In the human body, phthalates are rapidly metabolized to their respective monoesters, and some monoesters can be further metabolized and quickly excreted from the body.11,12 Therefore, urinary phthalate metabolites are used as biomarkers of © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1120
September 11, 2014 December 10, 2014 December 11, 2014 December 11, 2014 DOI: 10.1021/es504455a Environ. Sci. Technol. 2015, 49, 1120−1129
Article
Environmental Science & Technology significantly associated with autism35 and reduced IQ scores in children.36 Therefore, high priority should be given to exploring the phthalate exposures for children. China is a large market for the production and consumption of phthalates in the world. However, information about phthalate exposure in Chinese children is scarce. To our knowledge, only one study investigated the association between urinary phthalate metabolites levels and body mass index in Chinese children with a small sample size.30 In view of this, large-scale studies on levels and risk assessments of exposure to phthalates among Chinese children are urgently needed. In the present study, we carried out a multicenter study to determine the levels of 13 phthalate metabolites in first morning urine samples in a cohort of 782 Chinese school children and compared the estimated daily intakes with reference limit values. The aims of the study are to characterize the profiles of phthalate exposure and to assess the health risks of cumulative exposure to phthalates in Chinese children.
onidase from Helix pomatia were purchased from the SigmaAldrich (St. Louis, MO, USA). The analyses of phthalate metabolites in urine were performed by an Acquity UPLC system coupled to a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA) as previously described.30,38 After an aliquot (1.0 mL) of urine sample was enzymatically hydrolyzed, the phthalate metabolites were separated from other urine components by solid phase extraction (SPE) with an Oasis MAX mixed-mode SPE cartridge (combining reversed-phase and anion-exchange mechanisms, Waters, Milford, MA, USA). Chromatographic separation was achieved using a C18 column (Acquity UPLC BEH C18, 100 mm × 2.1 mm × 1.7 μm). The phthalate metabolites were detected in negative ion mode with a mobile phase of acetonitrile/water containing 0.1% acetate acid. Multiple-reaction monitoring (MRM) mode was used for the quantitative analysis of these compounds based on the internal standard curve method. The limits of detection (LOD) of phthalate metabolites ranged from 0.11 to 1.81 ng/mL. Detailed information regarding sample pretreatment and instrumental analysis is presented in the Supporting Information and Table S2. Creatinine adjustment was used to correct for urine dilution. Urinary creatinine concentrations were analyzed with an enzymatic method on an Architect C8000 automatic biochemical analyzer (ARCHITECT C8000, Abbott Laboratories, Illinois, USA). Quality Assurance/Quality Control. For each batch of 20 samples analyzed, 2 procedural blanks and 4 matrix spiked samples at two different spiking concentrations (10 and 50 ng/ mL) were processed. The average recoveries and relative standard deviation (RSD) of phthalate metabolites ranged from 70.3% to 124% and from 9.2% to 19.1%, respectively. New calibration standards were prepared for every batch of samples analyzed, with six concentration levels ranging from 0.2 to 100 ng/mL. The correlation coefficient of calibration curves was above 0.995. As a check for the drift of chromatographic retention time and cross contamination between urine samples, a midpoint calibration standard was injected after every 10 samples. QA/QC results are listed in Table S3, Supporting Information. Daily Intake Estimation. On the basis of the urinary phthalate metabolite concentrations, daily intake (DI) of parent phthalates was estimated for each child. We applied two calculation models that were based on urine volume and urine creatinine excretion, respectively. The volume model-based intake was calculated by the following equation:39
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MATERIALS AND METHODS Study Population and Sample Collection. The subjects were selected at random from our cohorts of school children in three regions of Yangtze River Delta in China: Minhang District (Shanghai City), Haimen City (Jiangsu Province), and Yuhuan County (Zhejiang Province). Minhang is a developed district close to the center of the Shanghai City; Haimen is a county-level city, and Yuhuan is an island county known as one of the major manufacturing industry areas in China. The study design has been described in detail previously.37 Briefly, one elementary school was randomly selected from each of the three sites. In each selected school, all the children between third and fifth grades were chosen with the age ranging from 8 to 11 years old. The first morning urine was collected by each participant on the same day when an annual regular physical examination was carried out. A total of 782 first morning voids were collected during the period from March to May 2012. The urine samples were collected in 50 mL polypropylene tubes and transported on ice to the laboratory and then aliquoted and stored at −80 °C until analysis. The distributions of participants by age, sex, body weight, and height in the three sampling regions are listed in Table S1 (Supporting Information). Assent or informed consent was obtained from children and their legal guardians. The study was approved by the Ethical Review Board of Fudan University School of Public Health. Target Compounds and Analysis. Urine samples were analyzed for 13 phthalate metabolites: DEHP metabolites: mono (2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono[(2-carboxymethyl) hexyl] phthalate (MCMHP); DnBP metabolite: mono-n-butyl phthalate (MnBP); DiBP metabolite: monoisobutyl phthalate (MiBP); DEP metabolite: monoethyl phthalate (MEP); DMP metabolite: monomethyl phthalate (MMP); dicyclohexyl phthalate (DCHP) metabolite: monocyclohexyl phthalate (MCHP); BBzP metabolite: monobenzyl phthalate (MBzP); DiNP metabolite: mono-3-methyl-5-dimethylhexyl phthalate (isononyl, MiNP); DOP metabolite: monooctyl phthalate(MOP). Thirteen metabolites, 6 isotopically labeled internal standards (13C4-MMP, 13C4-MEP, 13C4-MnBP, 13C4-MECPP, 13C4MEHP, 13C4-MBzP), and 13C4-4-methylumbelliferone were purchased from the Cambridge Isotope Laboratories (Andover, MA, USA). 4-Methylumbelliferone glucuronide and β-glucur-
DI(μg/kg bw/day) =
UC m(μg/L) × UV (L/day) × MWP fue × bw (kg) × MWm
where UCm is the urinary phthalate metabolite concentration, UV is the daily excretion volume of urine that was calculated from the normal reference daily urine excretion rate of 22.4 mL/kg body weight and the child’s body weight,40 bw is the body weight, MWP and MWm are the respective molecular weights of parent phthalate and its metabolites, f ue is the molar fraction of the urinary metabolite excreted in relation to the oral intake of phthalate from previous studies, and f ue values for individual phthalate and corresponding metabolite are listed in Table S4, Supporting Information. According to the latest knowledge of f ue values for 4 metabolites of DEHP (MEHP, MEOHP, MECPP, MEHHP),41 the total daily intake of DEHP 1121
DOI: 10.1021/es504455a Environ. Sci. Technol. 2015, 49, 1120−1129
Article
Environmental Science & Technology
Table 1. Urinary Concentrations of Phthalate Metabolites (in ng/mL and in μg/g Creatinine) in Chinese Children Aged 8−11 years (n = 782)a percentiles phthalate
metabolite
%>LOD
GM (95%CI)
25th
50th
75th
95th
range
98.6
7.9 (7.2−8.7) 16.5 (15.3−17.9)b 38.1 (35.5−40.7) 79.7 (75.2−84.5) 45.1 (41.7−49.0) 94.4 (87.1−102) 21.7 (19.5−24.5) 45.4 (41.0−50.2) 0.71 (0.67−0.76) 1.49 (1.40−1.60) 0.27 (0.26−0.29) 0.57(0.53−0.61) 5.4 (5.1−5.8) 11.4 (10.6−12.2) 17.4 (16.3−18.6) 36.4 (34.4−38.5) 11.6 (10.8−12.5) 24.3 (22.7−25.9) 20.2 (18.8−21.7) 42.3 (39.9−44.8) 11.6 (10.8−12.4) 24.3 (22.9−25.7) 65.4 (61.7−69.5) 137 (129−144)
3.8 9.3 19.7 45.9 20.9 48.0 7.2 16.4 0.4 0.8 0.3 0.3 3.6 6.2 9.8 21.6 5.8 12.3 10.1 24.3 5.8 14.0 34.4 80.8
8.4 15.4 38.5 75.1 47.1 93.4 18.7 40.5 0.8 1.5 0.3 0.6 5.9 11.7 17.6 36.0 11.8 23.6 20.7 40.3 11.8 23.0 64.7 133
16.8 29.1 72.1 133 105 200 60.2 104 1.3 2.8 0.3 1.0 9.5 21.2 31.2 58.5 22.2 43.3 38.1 69.2 22.3 40.0 114 220
50.2 101 190.3 333 351 554 355 600 2.6 6.9 1.0 2.8 21.3 56.5 77.6 151 63.5 119 108 172 60.6 95.7 326 536
Urinary Excretion of Phthalate Metabolites in School Children of China: Implication for Cumulative Risk Assessment of Phthalate Exposure Bin Wang,† Hexing Wang,† Wei Zhou,‡ Yue Chen,§ Ying Zhou,*,† and Qingwu Jiang† †
School of Public Health, Key Laboratory of Public Health Safety of Ministry of Education, Fudan University, Shanghai, 200032 China ‡ Department of Geriatrics, The Affiliated Taizhou Hospital of Wenzhou Medical University, Linhai City, Zhejiang, 317000, China § Department of Epidemiology and Community Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5 S Supporting Information *
ABSTRACT: We analyzed 13 metabolites of 9 phthalates in urine of 782 Chinese school children aged 8−11 years and estimated the daily intake for phthalates based on urinary metabolite levels. The daily intakes were compared with acceptable intake levels to calculate the hazard quotient (HQ) for single phthalate. Finally, the cumulative risk for each child was assessed by means of a hazard index (HI) which is the sum of HQs. Overall, 11 metabolites were found in at least 85% of the urine samples with the highest median concentration of 47.1 ng/mL (93.4 μg/g creatinine) for mono-nbutyl phthalate (MnBP). Monooctyl phthalate (MOP) and monoisononyl phthalate (MiNP) were not detectable. The cumulative risk assessment covering di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DnBP), di-isobutyl phthalate (DiBP), and butyl-benzyl phthalate (BBzP) demonstrated that 19.8% (volume model-based) and 40.3% (creatinine model-based) of the children exceeded 1 for the HI based on tolerable daily intake (TDI) values (considered as potential adverse antiandrogenic effect). Furthermore, at least 36% of the children from the manufacturing-intensive region had a HI higher than 1. The results indicate that Chinese children are widely exposed to phthalates and those from manufacturing-intensive regions are probably at a high risk of cumulative phthalate exposure.
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cumulative individual exposure to phthalates.3,13 The occurrence of phthalate metabolites has been determined in urine samples from a nationally representative population in the United States (U.S.).14 Several studies also have reported urinary phthalate metabolites in some European countries15−18 and Asian countries,19−22 indicating widespread human exposure to phthalates. The levels of exposure to phthalates have been shown to be higher in children than in adults.5,23−25 Human population studies have reported significant associations between fetal and perinatal exposure to some phthalates and antiandrogenic effects of male infants.26,27 Besides, delayed pubarche in girls is associated with phthalate exposure.28,29 In recent years, a number of studies have reported the associations of children’s exposure to phthalates with obesity, decreased thyroid function, asthma and other respiratory diseases, attention-deficit hyperactivity disorders, and negative behavioral development.30−34 High levels of DEHP metabolites were also found to be
INTRODUCTION Phthalates are a class of synthetic chemicals widely used in industrial and consumer products. High-molecular weight phthalates such as di(2-ethylhexyl) phthalate (DEHP), butylbenzyl phthalate (BBzP), di-isononyl phthalate (DiNP), and dioctyl phthalate (DOP) act primarily as plasticizers in the manufacture of polyvinyl chloride (PVC) building materials, floorings, food packaging, and medical devices. Low-molecular weight phthalates such as diethyl phthalate (DEP), dimethyl phthalate (DMP), di-n-butyl phthalate (DnBP), and di-isobutyl phthalate (DiBP) are mostly used in personal care products.1−4 Phthalates are not chemically bound to the products and can easily leach out into the surrounding environment.5 Thus, people are continuously exposed to phthalates through ingestion, inhalation, dermal absorption, and contact with medical devices as a result of widespread use.6−10 Since there is a ubiquitous existence of phthalates in the environment and daily consumer products, the potential for nonoccupational exposure to phthalates is high for individuals. In the human body, phthalates are rapidly metabolized to their respective monoesters, and some monoesters can be further metabolized and quickly excreted from the body.11,12 Therefore, urinary phthalate metabolites are used as biomarkers of © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1120
September 11, 2014 December 10, 2014 December 11, 2014 December 11, 2014 DOI: 10.1021/es504455a Environ. Sci. Technol. 2015, 49, 1120−1129
Article
Environmental Science & Technology significantly associated with autism35 and reduced IQ scores in children.36 Therefore, high priority should be given to exploring the phthalate exposures for children. China is a large market for the production and consumption of phthalates in the world. However, information about phthalate exposure in Chinese children is scarce. To our knowledge, only one study investigated the association between urinary phthalate metabolites levels and body mass index in Chinese children with a small sample size.30 In view of this, large-scale studies on levels and risk assessments of exposure to phthalates among Chinese children are urgently needed. In the present study, we carried out a multicenter study to determine the levels of 13 phthalate metabolites in first morning urine samples in a cohort of 782 Chinese school children and compared the estimated daily intakes with reference limit values. The aims of the study are to characterize the profiles of phthalate exposure and to assess the health risks of cumulative exposure to phthalates in Chinese children.
onidase from Helix pomatia were purchased from the SigmaAldrich (St. Louis, MO, USA). The analyses of phthalate metabolites in urine were performed by an Acquity UPLC system coupled to a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA) as previously described.30,38 After an aliquot (1.0 mL) of urine sample was enzymatically hydrolyzed, the phthalate metabolites were separated from other urine components by solid phase extraction (SPE) with an Oasis MAX mixed-mode SPE cartridge (combining reversed-phase and anion-exchange mechanisms, Waters, Milford, MA, USA). Chromatographic separation was achieved using a C18 column (Acquity UPLC BEH C18, 100 mm × 2.1 mm × 1.7 μm). The phthalate metabolites were detected in negative ion mode with a mobile phase of acetonitrile/water containing 0.1% acetate acid. Multiple-reaction monitoring (MRM) mode was used for the quantitative analysis of these compounds based on the internal standard curve method. The limits of detection (LOD) of phthalate metabolites ranged from 0.11 to 1.81 ng/mL. Detailed information regarding sample pretreatment and instrumental analysis is presented in the Supporting Information and Table S2. Creatinine adjustment was used to correct for urine dilution. Urinary creatinine concentrations were analyzed with an enzymatic method on an Architect C8000 automatic biochemical analyzer (ARCHITECT C8000, Abbott Laboratories, Illinois, USA). Quality Assurance/Quality Control. For each batch of 20 samples analyzed, 2 procedural blanks and 4 matrix spiked samples at two different spiking concentrations (10 and 50 ng/ mL) were processed. The average recoveries and relative standard deviation (RSD) of phthalate metabolites ranged from 70.3% to 124% and from 9.2% to 19.1%, respectively. New calibration standards were prepared for every batch of samples analyzed, with six concentration levels ranging from 0.2 to 100 ng/mL. The correlation coefficient of calibration curves was above 0.995. As a check for the drift of chromatographic retention time and cross contamination between urine samples, a midpoint calibration standard was injected after every 10 samples. QA/QC results are listed in Table S3, Supporting Information. Daily Intake Estimation. On the basis of the urinary phthalate metabolite concentrations, daily intake (DI) of parent phthalates was estimated for each child. We applied two calculation models that were based on urine volume and urine creatinine excretion, respectively. The volume model-based intake was calculated by the following equation:39
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MATERIALS AND METHODS Study Population and Sample Collection. The subjects were selected at random from our cohorts of school children in three regions of Yangtze River Delta in China: Minhang District (Shanghai City), Haimen City (Jiangsu Province), and Yuhuan County (Zhejiang Province). Minhang is a developed district close to the center of the Shanghai City; Haimen is a county-level city, and Yuhuan is an island county known as one of the major manufacturing industry areas in China. The study design has been described in detail previously.37 Briefly, one elementary school was randomly selected from each of the three sites. In each selected school, all the children between third and fifth grades were chosen with the age ranging from 8 to 11 years old. The first morning urine was collected by each participant on the same day when an annual regular physical examination was carried out. A total of 782 first morning voids were collected during the period from March to May 2012. The urine samples were collected in 50 mL polypropylene tubes and transported on ice to the laboratory and then aliquoted and stored at −80 °C until analysis. The distributions of participants by age, sex, body weight, and height in the three sampling regions are listed in Table S1 (Supporting Information). Assent or informed consent was obtained from children and their legal guardians. The study was approved by the Ethical Review Board of Fudan University School of Public Health. Target Compounds and Analysis. Urine samples were analyzed for 13 phthalate metabolites: DEHP metabolites: mono (2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono[(2-carboxymethyl) hexyl] phthalate (MCMHP); DnBP metabolite: mono-n-butyl phthalate (MnBP); DiBP metabolite: monoisobutyl phthalate (MiBP); DEP metabolite: monoethyl phthalate (MEP); DMP metabolite: monomethyl phthalate (MMP); dicyclohexyl phthalate (DCHP) metabolite: monocyclohexyl phthalate (MCHP); BBzP metabolite: monobenzyl phthalate (MBzP); DiNP metabolite: mono-3-methyl-5-dimethylhexyl phthalate (isononyl, MiNP); DOP metabolite: monooctyl phthalate(MOP). Thirteen metabolites, 6 isotopically labeled internal standards (13C4-MMP, 13C4-MEP, 13C4-MnBP, 13C4-MECPP, 13C4MEHP, 13C4-MBzP), and 13C4-4-methylumbelliferone were purchased from the Cambridge Isotope Laboratories (Andover, MA, USA). 4-Methylumbelliferone glucuronide and β-glucur-
DI(μg/kg bw/day) =
UC m(μg/L) × UV (L/day) × MWP fue × bw (kg) × MWm
where UCm is the urinary phthalate metabolite concentration, UV is the daily excretion volume of urine that was calculated from the normal reference daily urine excretion rate of 22.4 mL/kg body weight and the child’s body weight,40 bw is the body weight, MWP and MWm are the respective molecular weights of parent phthalate and its metabolites, f ue is the molar fraction of the urinary metabolite excreted in relation to the oral intake of phthalate from previous studies, and f ue values for individual phthalate and corresponding metabolite are listed in Table S4, Supporting Information. According to the latest knowledge of f ue values for 4 metabolites of DEHP (MEHP, MEOHP, MECPP, MEHHP),41 the total daily intake of DEHP 1121
DOI: 10.1021/es504455a Environ. Sci. Technol. 2015, 49, 1120−1129
Article
Environmental Science & Technology
Table 1. Urinary Concentrations of Phthalate Metabolites (in ng/mL and in μg/g Creatinine) in Chinese Children Aged 8−11 years (n = 782)a percentiles phthalate
metabolite
%>LOD
GM (95%CI)
25th
50th
75th
95th
range
98.6
7.9 (7.2−8.7) 16.5 (15.3−17.9)b 38.1 (35.5−40.7) 79.7 (75.2−84.5) 45.1 (41.7−49.0) 94.4 (87.1−102) 21.7 (19.5−24.5) 45.4 (41.0−50.2) 0.71 (0.67−0.76) 1.49 (1.40−1.60) 0.27 (0.26−0.29) 0.57(0.53−0.61) 5.4 (5.1−5.8) 11.4 (10.6−12.2) 17.4 (16.3−18.6) 36.4 (34.4−38.5) 11.6 (10.8−12.5) 24.3 (22.7−25.9) 20.2 (18.8−21.7) 42.3 (39.9−44.8) 11.6 (10.8−12.4) 24.3 (22.9−25.7) 65.4 (61.7−69.5) 137 (129−144)
3.8 9.3 19.7 45.9 20.9 48.0 7.2 16.4 0.4 0.8 0.3 0.3 3.6 6.2 9.8 21.6 5.8 12.3 10.1 24.3 5.8 14.0 34.4 80.8
8.4 15.4 38.5 75.1 47.1 93.4 18.7 40.5 0.8 1.5 0.3 0.6 5.9 11.7 17.6 36.0 11.8 23.6 20.7 40.3 11.8 23.0 64.7 133
16.8 29.1 72.1 133 105 200 60.2 104 1.3 2.8 0.3 1.0 9.5 21.2 31.2 58.5 22.2 43.3 38.1 69.2 22.3 40.0 114 220
50.2 101 190.3 333 351 554 355 600 2.6 6.9 1.0 2.8 21.3 56.5 77.6 151 63.5 119 108 172 60.6 95.7 326 536
