Supplementary meterial for

Size fraction effect on phthalate esters accumulation, bioaccessibility and in vitro cytotoxicity of indoor/outdoor dust and risk assessment of human exposure


Wei Wang, Fu-Yong Wu, Min-Juan Huang, Yuan Kang, Kwai Chung Cheung, Ming Hung Wong*

State Key Laboratory in Marine Pollution – Croucher Institute for Environmental Sciences, Hong Kong Baptist University and City University of Hong Kong, Kowloon Tong, Hong Kong, PR China

* To whom correspondence should be addressed: E-mail: (M.H. Wong); Tel.: + 852 3411-7746, Fax: +852 3411-7743.

1. Materials and methods

1.1 Bioaccessibility of phthalate esters determination in dust

1.1.1 Determination methods of bioaccessibility

A 1-h empty time and 4-h small intestinal transit were selected. Gastric solution used for the test containing 0.25 g of pepsin (sigma-Aldrich, USA), 17.5 g of NaCl, 1 g of citrate, 1 g of malate, 0.85 g of lactic acid, and 1 mL of acetic acid in 2 L of deionized water was prepared, with pH adjusted to 1.5 with 6 M HCl. 2 g of dust was added into 40 mL of gastric solution and the mixture was shaken at 37 ˚C for 1 h. Then, the mixture was centrifuged at 7000 × g for 10 min and the supernatant as filtered through a 0.45 µm glass fiber filter. Then, 30 mL of the original gastric solution was added to suspend the pellets in the centrifuge tube.

The gastric solution was modified to small intestinal solution by adjusting the pH to 7.0 with NaHCO3 solution followed by adding 0.2 g of porcine bile extract (Sigma-Aldrich, USA) and 0.04 g of porcine pancreatin (Sigma-Aldrich, USA). After shaking for 4 h at 37 ˚C, the mixture was centrifuged at 7000 × g for 10 min and the supernatant was filtered through a 0.45 µm glass fiber filter. The filtrate (gastric and intestinal) was both extracted with 40 mL of n-hexane/acetone (3:1, v/v) for 10 min in a 250 mL separatory funnel. Extraction with 40 mL of n-hexane was carried out two more times. All the extracts were combined into one mixture extract, which was dried with 5 g of anhydrous sodium sulfate. The extract was reduced to 2 mL using a rotary evaporator and purified by Florisil clean-up methods [1]. The solution was then concentrated to 200 µL for phthalates analyses. Deuterated PAHs (acenaphthene-d10, phenanthrene-d10, chrysene-d12 and perylene-d12) were added as internal standards for quantification into all extracts.

1.1.2. Bioaccessibility calculation

The bioaccessbility (%BA) of phthalates (stomach and intestine) were calculated as the ratio of the amount of phthalates in liquid phase to the total phthalates in indoor dust [2]. The total phthalates for intestinal digestion were considered as equal to the total phthalates in dust samples subtracted by phthalates in gastric juice.

%BA= (BA extracted phthalates / total phthalates) × 100 (S1)

1.2. Cell culture and MTT assay

The CCRF-CEM (human acute lymphoblastic leukemia) cell line was purchased from the Chinese Academy of Sciences Cell Bank. Cells were maintained in RPMI-1640 culture medium (ATCC, USA) supplemented with 10% fetal bovine serum (FBS), 100 unites/mL penicillin, 100 µg/mL streptomycin and 100 µg/mL amphotericin B. Cells were cultured in a humidified atmosphere with 5% CO2 at 37˚C. Mitochondrial capacity to reduce MTT to formazan [3] was used to measure cell viability. When the culture growth reached between 80 and 100% of confluence, cells were seeded onto 96-well tissue culture microtiter plates at a density of 2 × 104 cells/100µL/well. After 24 h, the cell culture medium was removed after centrifuging at the low speed of 1000 rpm and replaced by 100 µL culture medium containing five different concentrations of each dust organic extract prepared by a 2-fold dilution for CCRF cell. In each well, the final concentration of DMSO was limited to 0.5%. After 24 h incubation, the culture medium was removed and the cells were washed twice with warm PBS. The cells were then incubated with serum free medium containing 0.5 mg MTT/mL at 37 ˚C. The medium was removed and replaced with 100 µL DMSO after 4 h. The colored solution was then detected at a wavelength of 540 nm and at 690 nm (reference length). 0.5% DMSO treated cells were used as the 100% viable control. Cell mortality was obtained by the following equation [S2]:

cell mortality= 1-(ODsample/ODDMSO), (S2)

where ODsample is the optical density of sample detection in MTT assay and ODDMSO is the optical density of DMSO control detection in MTT assay. LC50, which is the concentration of contaminants killing 50% of the cells, of each dust extract was calculated by using Trimmed Spearman-Karber method with the software provides by US EPA (Trimmed Spearman-Karber program).

1.3 Daily Intake (DI) calculation

Parameters and sources used to estimated human exposure to phthalates though indoor dust

From Dust Ingestion (S3)

Concentration of Cdust are the median values of dust determined in the present study; values of f1 for infants, teenagers, and adults are based on 21 of 24 h in a day (0.88), while the values for toddlers and children are based on 19 of 24 h in a day (0.79) [5]; values of f2 are 0.02 g/day for infants, 0.1 g/day for toddlers and 0.05 g/day for others [5,6,7]. M1 for infants, toddlers, children, teenagers and adults, in China, are 5, 19, 29, 53 and 63 kg, respectively, from the provincial government values (2005) of Shanghai [7].

From dermal absorption (S4)

Values of A, including hands, legs, and arms representing 25% of the total skin are respective 801, 2564, 3067, 3692 and 4615 cm2/day for infants, toddlers, children, teenagers and adults in both countries [5,6,7]; M2 is 0.096 mg/cm2; f3 values of infants, toddlers, children, and teenagers are the mean value of child, which is two times to that of adult [8], DMP (child: 0.000955), DEP (0.002051), DiBP (0.001202), DBP (0.001556), BzBP (0.000707) and DEHP (0.000106).

1.4 Risk assessment

1.4.1. Average Daily Dose (ADD) Calculation

The following equations were used to estimate the human health risks associated with inhalation, dermal contact and non-dietary ingestion of phthalates in dust.




IngR= ingestion rate of indoor dust (mg day-1). For adults and children, the high dust ingestion rates of 110 mg day-1 and 200 mg day-1, respectively, were considered. In addition, the moderate ingestion rate of 0.56 mg day-1 and 50 mg day-1 were considered for adults and children, respectively [9]. The exposure frequency (EF) for adults was assumed to be 365 days year-1, due to the long time human spent in indoor. ED= exposure duration (year). ED for adults and children in this study was assumed to be 70 years and 6 years respectively. AT= average time (days), for 70 × 365 and 6 × 365 for adults and children. IRinhalation is the inhalation rate (children: 10 m3/day; adults: 20 m3/day) [10]. SA is the dermal exposure area (children: 2800 cm2; adults: 3300 cm2) [11]. AF is the dermal adherence factor (children: 0.2 mg/cm2; adults: 0.2 mg/cm2) [11]. ABS is the dermal adsorption fraction (children: 0.1; adult: 0.1) [11]. PEF is the particle emission factor (1.36×109 m3 /kg) [11]. BW is the average body weight (kg). The standard values of 61.5 kg for adults [12] and 15 kg [9] for children were used. Human health risks associated with non-dietary ingestion of bioaccessible phthalates in indoor dust were estimated according to the following equation: ADDbio=BA% × ADDingest. (S8)

1.4.2. Hazard quotient (HQ)

After the ADD for the three exposure pathways (ADDingestion, ADDdermal, ADDinhaltion) was calculated, a hazard quotient (HQ) based on non-cancer toxic risk can then be estimated by dividing the average daily dose to a specific reference dose (RfD)


The reference dose (RfD) (mg kg-1 day-1) is an estimation of maximum permissible risk on human populations through daily exposure, taking a sensitive group (children) during a lifetime into consideration. In general, there are three RfDs for three exposure pathways; RfDo (mg kg-1 day-1) for ingestion, RfDo (mg kg-1 day-1) × gastrointestinal absorption factor (GIABS) for dermal contact and RfCi (mg m-3) for inhalation. The threshold of RfD value can be use to indicate whether there is adverse health effect during a lifetime. The reference dose of DEHP and DBP and DEP is 20, 100, 800 µg kg-1 d-1[13].

If an average daily dose (ADD) value is lower than the RfD, it is unlikely that there will be any adverse health effect, whereas if the ADD value is higher than the RfD, it is likely that the factor is question will cause adverse human health effects. The calculation was based on the Risk Assessment Guidance for Superfund, Vol. I: Human Health Evaluation Manual [14]. The ratio of average daily dose to the reference dose can be used to estimate the non-cancer risk on humans; where HQ ≤ 1 indicates no adverse health effects and HQ 1 indicates likely adverse health effects.

1.4.3. Equation of cancer risk assessment on human via ingestion, dermal contact and inhalation

The procedure for conducting cancer risk assessment on humans was similar for non-cancer risk assessment. However, the average daily dose (ADD) was changed to life average daily dose (LADD) and instead of dividing the ADD by the reference dose (RfD), LADD was multiplied by a slope factor (SF). Generally, there are three SFs: SFO (mg kg day-1) for ingestion, SFO (mg kg-1 day-1) -1 × gastrointestinal absorption factor (GIABS);

The equations for estimating cancer risk through ingestion, dermal contact and inhalation of dust are listed as follows [11]:

LADD for ingestion× SFO (S10)

LADD for dermal contact × SFO×GIABS (S11)

LADD for inhalation × IUR (S12)

Where LADD = Life average daily dose; SFO = oral slope factor (mg kg-1day-1)-1, GIABS = gastrointestinal absorption factor, and IUR = inhalation unit risk (mg m-3)-1. Here, the SFO for DEHP is 1.4 × 10-2 (mg kg-1day-1)-1 and the GIABS is 1 [13].

Note: LADD is equal to ADD with a period of life span of 70 years.

The LADD (mg kg-1 day-1) was estimated through the human life span of 70 years and SF is the human cancer risk per unit (mg kg-1 day-1) dose derived from either animal bioassays or human data. According to Human Health Evaluation Manual, cancer risk can be summed by similar substances and across different exposure pathways, as long as the risks are for the same individuals [11]. Hence, there is an estimation of combined cancer risk values of DEHP through ingestion, dermal contact and inhalation in this study by adding the results of equation (S10)-(S12). Qualitative descriptions of life time cancer risk are as follow: very low when the estimated value is equal to or less than one in a million, low when it is greater than one in a million to less than one in ten thousands, moderate when it is one in ten thousand to less than one in ten and vey high when the value equals to or outweighs one in ten.

Table S1
Characteristics of sampling households.
Parameter / Values (Number of samples in each category)
Site location / GZ: Tianhe (n = 4), Liwan (n = 4), Yuexiu (n = 4)
Haizhu (n = 4) and Panyu (n = 4)
HK: Ap Lei Chow (n = 1), Shek Tong Tsui (n = 2), Yau Ma Tei (n = 3),
Kwun Tong (n = 2), Sha Tin (n = 2), Sham Shui Po (n = 3),
Kowloon City (n = 4), Tuen Mun (n =1) and Tin Shui Wai (n = 2)
Cooking times per day / 0 (12), 1 (16), 2 (9), 3 (4)
House is facing a busy traffic street? / Yes (5), No (35)
Age of building / 1-5 years (6) 6-10 years (12) 11-20 years (22)
Size (m2) / 0-50 (9), 50-90 (24), 100-120 (7)
Floor cover / Carpet (5), Hardwood (7), Tile (40)
Smoking / No (38), Yes (2)
Number of occupants / 1 (6), 2 (12), 3 (12), 4-6 (12)
Floor level / 1 (0), 2 (4), 3 (12), 4 (0), 5 (10), >5 (14)
Vacuuming frequency / Twice a week (12), Once a week (28)
Air conditioning? / Yes (40), No (0)
Windows opened? / Every day (36), 2-3 times a week (4), Occasionally (0)
Last paint / 1 year ago (1), 1-5 years ago (22), > 5 years ago (17)
Pets / Yes (2), No (38)

Table S2 Estimated total human daily intakes of phthalates from urinary metabolites (ng/kg/day)

infant / 400 / 700 / 3900 / 3900 / 20000
toddler / 400 / 700 / 3900 / 3900 / 20000
children / 400 / 700 / 3900 / 3900 / 20000
teenager / 1300 / 3900 / 6300 / 6300 / 3700
adults / 1300 / 3900 / 6300 / 6300 / 3700

Data cited from [7].

Table S3 Data used to estimate contribution of different human exposure routes of Phthalates to total daily intake (ng/kg/day)

indoor air
infant / 100 / 70 / 1700 / 800 / 600
toddler / 40 / 30 / 700 / 300 / 200
children / 40 / 30 / 600 / 300 / 200
teenager / 30 / 20 / 500 / 200 / 200
adults / 20 / 20 / 400 / 200 / 100
outdoor air
infant / 0.2 / 0.8 / 8 / 9 / 20
toddler / 0.1 / 0.6 / 6 / 7 / 20
children / 0.1 / 0.6 / 6 / 7 / 20
teenager / 0.05 / 0.2 / 2 / 3 / 7
adults / 0.04 / 0.2 / 2 / 2 / 5

Data cited from [7].