Distribution and Pattern Profiles of Chlorinated Paraffins in Human

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Letter

Distribution and pattern profiles of chlorinated paraffins in human placenta of Henan Province, China Ying Wang, Wei Gao, Yawei Wang, and Guibin Jiang Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.7b00499 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Environmental Science & Technology Letters

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Distribution and Pattern Profiles of Chlorinated Paraffins

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in Human Placenta of Henan Province, China

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Ying Wang a,b, Wei Gaoa,c, Yawei Wang a,c,d*, Guibin Jianga,d

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a

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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18

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Shuangqing Road, Haidian District, Beijing, 100085

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b

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Zhou, 341000, Jiang Xi

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

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c

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d

College of Chemistry and Chemical Engineering, Gannan Normal University, Gan

University of Chinese Academy of Science, Beijing 100049, China Institute of Environment and Health, Jianghan University, Wuhan 430056, China

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*Corresponding author

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Dr. Yawei Wang

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State Key Laboratory of Environmental Chemistry and Ecotoxicology

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Research Center for Eco-Environmental Sciences

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Chinese Academy of Sciences

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P.O. Box 2871, Beijing 100085, China

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Tel: +8610-6284-9124

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Fax: +8610-6284-9339

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E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract:

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Due to the high production volume, wide industrial applications and

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environmental persistence, chlorinated paraffins (CPs) have been observed in various

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environmental matrices. However, data are scarce regarding to human exposure to

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CPs. In this study,54 human placentas were collected and analyzed by gas

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chromatography quadrupole time-of-flight mass spectrometry (GC-QTOF-HRMS).

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Short chain CPs (SCCPs) were detected in all samples, with the concentrations

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ranging from 98.5 to 3771 ng/g lw (lipid weight). Medium chain CPs (MCCPs) were

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detected in 38 of the samples, with the concentrations being in the range of 80.8 to

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954 ng/g lw. SCCPs with 6-7 chlorines were predominant in human placentas and

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were mostly within the 10-11 carbon chain groups. In most samples, C15H25Cl7 were

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found to be the most abundant MCCP congener groups. Pearson correlation analysis

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indicated that there was a significant positive correlation between the ΣSCCPs and

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ΣMCCPs concentrations in the placenta samples, whereas the concentrations of the

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ΣSCCPs/ΣMCCPs had no significant correlations with the gravida's age, weight or the

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baby's weight. To the best of our knowledge, this report describes the first

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investigation of CPs in the human placenta, which provides data for future evaluation

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of human exposure to CPs.

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Keywords: short-chain chlorinated paraffins; medium-chain chlorinated paraffins;

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human placenta; exposure level

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Introduction

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Chlorinated paraffins (CPs) are a group of chlorinated n-alkanes with thousands of

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isomers. According to the length of the carbon chain, CPs are classified into three

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groups: short-chain CPs (SCCPs, C10-13), medium-chain CPs (MCCPs, C14-17) and

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long-chain CPs (LCCPs, C18-30)1. CPs have been widely applied in, for instance,

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paints, sealants, metal-working lubricants, flame retardants, and plasticizers in rubbers.

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CPs can be released into the environment during their production, shipping, usage and

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disposal2. Human beings are exposed to CPs throughout their lives. CPs may pose risk

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to human health as well as ecosystems, due to their persistence, long-range air

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transport, bioaccumulation through the food web and toxicological properties. Several

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countries (e.g., Canada, the United States of America, Norway, the European Union,

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and others) have limited the production and use of CPs3. In 2017, the Eighth

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Conference of the Parties of Stockholm Convention listed SCCPs into Annex A to

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eliminate its production and usage worldwide. As the largest producer of CPs in the

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world, the annual production volumes have increased rapidly to about 1050000 tonnes

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in 20134. However, to the best of our knowledge, there are no restrictive regulations

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on the production and use of CPs in China to date3.

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Previous studies have shown that SCCPs have potential ecological toxicity, which

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may affect the organism's glycolysis, amino acid and fatty acid metabolism of human

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hepatoma HepG2 cells5, and cause hepatic enzyme induction and thyroid

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hyperactivity and further lead to carcinogenicity in mammals6. In general, highly

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chlorinated SCCPs and MCCPs have the potential to be biomagnified via the aquatic

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food chain7.

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To assess the human exposure to contamination, blood, urine, breast milk and the

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placenta are often analyzed. Thereinto, analysis of the human placenta has been 4


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proposed as an ideal opportunity to study the exposure of the mother-infant pair to

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environmental pollutants8. Placenta is the important organ that connects developing

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fetus to nutrients intake, oxygen supply and waste exchange via the mother's blood

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supply. In addition, the placenta acts as a barrier to some foreign toxic compounds,

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which are stored in the placental tissue. However, many xenobiotics can also penetrate

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the placental barrier, reach the umbilical cord blood and influence the health of the

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fetus. Therefore, the chemical concentration in the human placenta may indicate the

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maternal exposure-level during the entire pregnancy and enable the assessment of

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potential health risks to offspring to a certain degree. Previous investigations have

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discussed

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polychlorinated phenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and others

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in the human placenta9-12. As a group of new POPs, the physicochemical properties of

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CPs are similar to other POPs (e.g., PCBs and OCPs) and they have been found in

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various environmental mediums (e.g., air, water, soil, aquatic organisms, and others)

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at high levels. However, to the best of our knowledge, no investigation regarding the

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exposure of the human placenta to CPs has been conducted.

the

exposure

level

of

organic

chlorinated

pesticides

(OCPs),

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In this study, human placenta samples were collected from a hospital in central

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China to analyze the levels of SCCPs and MCCPs simultaneously using gas

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chromatography quadrupole time-of-flight mass spectrometry (GC-QTOF-HRMS).

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The objectives of this study were to examine the presence of CPs in human placenta,

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the correlations among different CP homologs, and the potential links between the

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demographic characteristics of the mothers and the newborns.

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Materials and Methods

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Sample Collection and Pretreatment

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A total of 54 samples of human placentas were collected from a hospital in the 5



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Henan province of China in 2016 after receiving the approval of the Research Center

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for Eco-Environmental Sciences ethics committee and the consent of donors. All

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samples were placed in a sterile plastic bag at the hospital and frozen at -20℃

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immediately until analysis. Most of collected samples were the whole placenta, and

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only few samples were collected as a piece from the placentas. The same part of all

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the placentas was used for the final analysis. The donor's information, including age,

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weight and occupation, and the newborn baby's weight were recorded during

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sampling. This detailed information is shown in Table S1.

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Before extraction, the excess blood and the connective tissues from the placenta

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were removed. The placenta tissue was subsequently cut into small pieces,

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freeze-dried, homogenized in a glass mortar, and packed tightly with aluminum foil

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and a valve bag.

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The extraction and purification of CPs in the human placenta were performed by

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matrix solid-phase dispersion (MSPD) method which was reported in our previous

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paper13. Briefly, 1 g of freeze-dried placental tissue, 1 g of silica gel, 5 g of anhydrous

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sodium sulfate and 10 ng of internal standard (13C10-1,5,5,6,6,10-hexachlorodecane,

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purchased from Cambridge Isotope Laboratories, USA) were blended into a glass

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mortar. Using a glass pestle, the mixture was then thoroughly ground to a fine powder.

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The MSPD glass column (15 mm id and 30 cm length) with Poly Tetra Fluoro

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Ethylene (PTFE) cocks was packed from bottom to top with 4 g of anhydrous sodium

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sulfate, 5 g of acid silica gel (w/w, 44% H2SO4), the prepared placenta sample-sorbent

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blend, and 3 g of anhydrous sodium sulfate. Then, the column was eluted with 100

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mL of hexane/dichloromethane (7:3, V/V). Prior to elution, approximately 40 mL of

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the mixed solvent was used to rinse the mortar and pestle a total of three times. The

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eluent was rotary-evaporated to approximately 2 mL and then blown to near-dryness 6


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with nitrogen. The solvent was changed to cyclohexane and to a final volume of 200

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µL.

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(є-hexachlorocyclohexane, purchased from Dr. Ehrenstorfer GmbH, Germany) was

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added in the vial and vortexed.

Prior

to

instrument

analysis,

10

ng

of

injection

standard

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Lipid content was determined gravimetrically after extraction with the mixture of

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hexane/dichloromethane (1:1, V/V), using accelerated solvent extraction (350, Dionex

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Canada Ltd., Oakville, ON, Canada) and solvent evaporation.

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Instrument Analysis and Quantification

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Instrumental analysis was performed using GC-QTOF-HRMS (Agilent 7200, Santa,

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Clara, USA). One injection enabled for the quick quantification of 24 SCCPs

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(C10-13Cl5-10) and 24 MCCPs (C14-17Cl5-10) formula congener groups. The

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identification of different CP congener groups was conducted by screening the

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extracting accurate masses (accurate to four decimal places) and by comparing the

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retention time and the signal shape with matching standards. The two most abundant

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isotope ions of [M-Cl]- were applied to quantification and confirmation, respectively.

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The accurate m/z values of the quantitative and qualitative [M-Cl]- ions and the

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corresponding retention time for the 24 SCCP and 24 MCCP formula congener groups

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were shown in Table S2. The detailed data analysis method for CPs by

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GC-QTOF-HRMS was reported in our previous work14 and is summarized in the SI.

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Statistical analysis was conducted with predictive analytics software (PASW)

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Statistics 18 and Origin pro 8.

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Quality Assurance/Quality Control

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To ensure the accurate identification and quantification of the target compounds,

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strict quality controls were employed. All glasswares were baked at a high

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temperature (450°C) and rinsed with dichloromethane and hexane three times in turn 7



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before use. Anhydrous sodium sulfate and silica gel were heated for 10 h to 650°C

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and 550°C, respectively, and florisil was heated to 140°C for 7 h before use. The

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MDL (method detection limit) for ΣSCCPs and ΣMCCPs were calculated using three

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times the standard deviation of the procedural blanks from all the sample batches

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(n=8). The MDLs for ΣSCCPs and ΣMCCPs were estimated to approximately 98.5

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ng/g lw (6.4 ng/g dw) and 72.3 ng/g lw (4.7 ng/g dw), respectively. One procedure

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blank was performed in each batch of 6-8 samples. The levels of all the SCCPs and

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MCCPs in blanks were below the MDL, therefore, the final concentrations of SCCPs

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and MCCPs in the human placentas were not blank-corrected. Ten nanogram of

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13

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samples as well as procedure blanks before extraction and 10 ng of injection standard

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(є-hexachlorocyclohexane) was added in the vial before instrument analysis. The

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internal recoveries of

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were in the range of 93.8% to 126.6% with a mean of 105.7%.

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Results and Discussion

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CPs Levels in Human Placentas

C10-1, 5, 5, 6, 6, 10-hexachlorodecane (internal standard) was added into all the

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C10-1,5,5,6,6,10-hexachlorodecane in all placenta samples

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The concentrations of ΣSCCPs, ΣMCCPs, C10-17 – CPs congener groups, and the

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lipid contents in the human placentas are summarized in Table S3. SCCPs could be

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detected in all 54 of the human placenta samples with concentrations in the range of

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98.5 to 3771 ng/g lw with an average value of 593 ng/g lw. MCCPs were detected in

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38 of the 54 placenta samples, and the concentrations were in the range of 80.8 to 954

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ng/g lw with a mean value of 316 ng/g lw. High levels of CPs indicated that these

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chemicals may cross the placenta barrier and reach human fetuses.

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To date, no other studies could be found on the level of CPs in the human placenta.

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Hence, these unique results showed a contrast between CPs and other POPs in the 8


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human placenta. Compared with other reports, the average concentration of ΣSCCPs

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and ΣMCCPs in placentas were all lower than the concentration of total polycyclic

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aromatic hydrocarbon (ΣPAHs) (890±330 ng/g lw) in the placentas of Beijing

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populations11, but considerably higher than the concentrations of ΣPBDEs (15.8±9.88

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ng/g lw)10, total dechlorane plus (ΣDPs) (0.92-197 ng/g lw)12, and dichlorodiphenyl

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trichloroethane (DDTs) (49.2±30.2 ng/g lw)8 in China. These findings may be related

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to the widespread applications and the high exposure levels of CPs to human beings in

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China.

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To date, studies on the human internal exposure to CPs are also scarce. Only a few

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reports of CP concentrations in breast milk from females in China and the UK can be

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found. As shown in Figure 1, comparison between CPs in breast milk and placenta

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samples indicated that the mean concentration of ΣSCCPs (593 ng/g lw) in placenta

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samples from present study was lower than the mean level (1861 ng/g lw) found in

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breast milk samples from Chinese women15 but significantly higher than the ΣSCCPs

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(mean: 180 ng/g lw) in the UK breast milk16. However, the mean concentration of

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ΣMCCPs in placenta samples were higher than both the ΣMCCPs in China with a

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mean of 176 ng/g lw and the UK breast milk samples with a mean of 21 ng/g lw15,16.

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This is due to the fact that the potential releasing capacity is higher than that of other

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countries in the world. In addition, the European Union banned the production and use

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of SCCPs in 200017, but there have been no restrictive regulations to be put into place

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in respect to this in China. This may be one of the main reasons that the

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concentrations of CPs in human samples in China were higher than in other counties.

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Overall, the concentrations of ΣSCCPs are higher than the ΣMCCPs in most human

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placentas. This finding may be closely related to the molecular features and the

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physicochemical properties of the substances. In general, small-molecular substances 9



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will more easily cross the placenta barrier than large-molecular substances18. However,

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in a few of placenta samples, ΣMCCPs are higher than the ΣSCCPs. The external

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exposure pathway of SCCPs and MCCPs should be further warrant since we didn’t

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divide sub-populations during the sampling campaign, which might be influenced by

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different factors such as residential environments, professions, and dietary habits of

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the donors, etc.

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Congener Group Abundance Profiles

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The distribution profiles of CPs in placentas (Figure 2, Figure S4) indicated that the

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congener group abundance of SCCPs and MCCPs were similar in most of the placenta

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samples with several exceptions, which might be due to different external exposure

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pathways and/or the metabolism mechanisms of mothers. C10-CPs and C11-CPs were

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found to be the most abundant SCCP congener groups with average contributions of

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58.7% and 35.6%, respectively (Figure 2). For the chlorine congener group profiles of

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SCCPs, Cl6-CPs and Cl7-CPs predominated in the human placenta samples and the

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average proportions were 39.0% and 52.2%, respectively. The congener group

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abundance profiles of SCCPs in the placenta samples were similar to the breast milk

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samples of the China population15. For MCCPs in the 38 detectable samples, C15 and

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Cl7 were the most abundant carbon and chlorine atom congeners, with C15H25Cl7

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accounting for approximately 80% of total MCCPs and the remaining 20% was

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almost all C14Cl7-8. These results were different from the MCCP congener group

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patterns found in the China breast milk samples, where MCCPs were predominated by

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C14Cl7-8 homologues15.

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In general, the primary pathways of human exposure to POPs include inhalation,

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skin contact and diet. The SCCP congener group patterns in the placenta samples in

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this work were similar to those reported in air19, 20 and completely consistent with the 10


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particle phase of air19, indoor dust21 and foodstuffs22 in China. In China, CP-42 and

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CP-52 are the major industrial CP mixtures, which account for approximately 80% of

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the total output, and their dominating congener groups are C10-11Cl6-8. The similarity

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of CP congener group patterns in human bodies, environmental matrices and

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industrial CP mixtures23 implied that air, dust and diet might be the important

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exposure pathways of CPs in humans.

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Relationships between CP Congeners and Demographic Characteristics

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Pearson analyses indicated that there was a significant linear relationship between

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ΣSCCPs and ΣMCCPs (r=0.684, p

Distribution and Pattern Profiles of Chlorinated Paraffins in Human

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