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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 ... - ACS Publications

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