Persistent Organic Pollutants: Foods, Prenatal Exposure to Birth Outcomes

Prof. Zongwei Cai

State Key Laboratory of Environmental

and Biological Analysis

Hong Kong Baptist University

2019 Food and Water Submit, Singapore, Sept 12

Persistent Organic Pollutants

Persistent Organic Pollutants (POPs) are groups of

pollutants (including dioxins) that cannot be easily

degraded, are strongly lipophilic, can easily be enriched

in food chains, and can be transported for long distance.

DDT, 滴滴涕 Dioxins, 二噁英

(made by human) (produced unintentionally)

They are only a part of the ugly truth……

• POPs - persistence, long-range transportable, bio-

accumulative & chronic toxicity

• Stockholm Convention on POPs – 12 chemicals in

2001, 9 emerging POPs included in 2009 (more

have coming)

• >95% of dioxin & PCBs intake by dietary, mainly

animal-origin food (WHO)

1. Persistent

2. Long-distance transportation

North Pole

Detected!

Dioxin

in water

Dioxin in

Daphnia

Dioxin

in fish

Dioxin

in duck

Bioconcentration

bioaccumulation

3. Biomagnification

生物積累

生物濃縮

105

水

蚤

鱼

鸭

Air

(1) POPs - Dioxin and PCBs in foods

Food Maximum level (pg WHO-TEQ /g fat) Action level (pg WHO-TEQ /g fat)

Beef and mutton 4.5 2.5

Poultry 4 3

Pigs 1.5 1.1

Livers of terrestrial animal 12 8

Fish muscle (except eel)* 8 6

Eel muscle * 12 9

Raw milk and products 6 4

Hen egg products 6 4

Mixed animal fat 3 2.25

Vegetable oil 1.5 1

Marine oil 10 7.5

* Fresh weight basis

⚫ It occurs, incidents happened - 1999 Belgian

“Chicken gate”, latest 2011 German egg products

scandal

⚫ EU maximum and action level of dioxin like

compounds in food

Risk assessment

Authorities Time Tolerable intake

WHO 1998 TDI=1-4 pg TEQ/kg b.w. day

JECFA 2001 TMI=70 pg TEQ/kg b.w. month

SCF 2001 TWI=14 pg TEQ/kg b.w. week

⚫ Estimation of potential dietary exposure to specific

contaminants of various subgroups

⚫ Compare with built tolerable intake level based on

toxicology

Chemical analysis of pooled food samples

(a total of 174)

Weigh 5-10 g sample, freeze dried

Spike internal standard, Soxhlet extraction

Liquid-liquid extraction

Lipid content determined gravimetrically

Acidic silica, acidic alumina, florisil cleanup

Active carbon (PCDD/Fs) GPC (PCBs)

High resolution gas chromatography/

high resolution mass spectrometry

(HRGC/HRMS)

HRGC-HRMS (Waters Autospec)

•Double

focusing

magnetic-sector

Instrument

• High-

resolution (>

10,000) mass

spectrometry

• Ultra low-level

trace (ppq – sub

ppt) detection

for dioxins and

other organic

pollutants

Dioxin & PCB concentrations in foods

of Hong Kong

Pork

Ruminant meat

Concentrations in food subgroups (lipid weight basis)

Poultry

Concentrations in chicken and duck (lipid weight basis)

Dioxin & PCB concentrations in foods

of Hong Kong

Fish

Total TEQ of various fish species (lipid weight basis)

Fish subgroups

Concentrations in fish subgroups (lipid weight basis)

Summary of dioxins in food

Food Total TEQ of all composite samples

( pg WHO-TEQ 1998/g fat)

Maximum level (pg WHO-TEQ

/g fat)

Action level (pg

WHO-TEQ /g fat)

Mean Median Max

Beef and

mutton

1.4 1.17 4.03 4.5 2.5

Poultry 0.986 0.957 1.47 4 3

Pigs 0.563 0.548 1.07 1.5 1.1

Fish

muscle*

0.65 0.513 2.4 8 6

Raw milk

and

products

1.27 1.26 2.21 6 4

Hen egg

products

2.69 2.61 5.43 6 4

Mixed

animal fat

0.268 0.243 0.541 3 2.25

* Fresh weight basis

Risk assessment of dietary intake of

dioxin & PCBs of H.K. residents• By combining food consumption data of H.K. residents,

the dietary exposure to dioxins and PCBs was estimated

• Two scenarios : average and high risk group (95th

percentile)

Overview of animal origin food intake of an average H.K. resident

H.K. population-based food consumption survey, final report

Are we safe?assumptions

• Both mean & median of dioxin & PCB

concentration were used for assessment

• Mean & 95th of food consumption used

• Food subgroup adjusted

We are safe, to some extent

Estimated daily intake of dioxin like compounds (A) TEQ 1998; (B) TEQ 2005

Estimated daily intake of NDL-PCBs

A B

High risk subgroup is in danger

Estimated weekly intake of dioxin like compounds (A) TEQ 1998; (B) TEQ 2005

Estimated monthly intake of dioxin like compounds (A) TEQ 1998; (B) TEQ 2005

A

A

B

B

Food group contribution of high risk

residents

Total TEQ

Mean concentration

21

(2) POPs - Organochlorine pesticides

➢ Organochlorine pesticides (OCPs) are chlorinated hydrocarbons usedextensively from the 1940s in agriculture and mosquito control.

Cl

Cl

Cl

Cl

Cl Cl

Hexachlorocyclohexane(HCH)

Cl

Cl

Cl

Cl

Cl Cl

Cl

Cl

Cl

Cl

Cl Cl

Cl Cl

Cl Cl

Cl

Cl Cl

Cl Cl

Dichlorodiphenyltrichloroethane(DDT) and metabolites

(DDE and DDD)

Cl Cl

Cl Cl

Cl Cl

Cl

Cl

Cl

Cl Cl

ClCl

Cl Cl

ClCl

Prenatal exposure

Birth outcomes

Hypothesis

OCP levels

22

➢ In fast-growing stages oflife, developing fetuses aremore vulnerable to thepotentially adverse effects.

23

Objectives

Exposure Levels Effects of Exposure

Method development:

Establish an accurate, sensitive and robust method for analysis of OCPs.

Effects of prenatal exposure:

Evaluate the associations of prenatal exposure and birth outcomes.

24

Challenges in the analysis of OCPs and other POPs

➢Various polarity of analytes➢Complicated matrix for biomonitoring studies

Plasma/Serum Breast milk Adipose tissue

➢ Low concentrations and limited sample volumes

Trace levels of POPs in human bodies

▪ ng/g lipid, pg/mL

Solution

➢ Sensitive➢ Accurate➢ Robust➢ High-throughput

➢ Large sample number

25

Typical instrumental analysis of OCPs

➢ Gas chromatograph-electron capture detector (GC-ECD)

The electronegative analytes capture electrons and reduce the current.

Require standards or referencefor identification

➢ Gas chromatograph-mass spectrometry (GC-MS)

Electron ionization (EI) Extent fragmentations in the source

Negative chemical ionization (NCI) Restricted to specific chemical

Triple quadrupole

➢ High sensitivity in complicated matrix

➢ More accurate & confident quantification

➢ Soft ionization provides high abundance of molecular ions

➢ High levels of matrix tolerance

➢ Ability to inject less sample matrix, reducing effect of contamination

➢ Capabilities for various MS detector

➢ Traditionally for LC-MS

➢ Now for GC-MS (APGC)

26

Atmospheric pressure chemical ionization

APCI

High intensity

EI

Extensive fragmentation

27

Soft ionization under APCI

➢ Under APCI, the mass spectrum only showed the molecular ion of p,p’-DDE.

➢ Under EI, the mass spectrum showed the fragment ion [M-Cl2]+ with high intensity.

• p,p’-DDE

Anal. Bioanal. Chem. 411(2019):4185-4191.

28

Method validations

• Linearity, recovery and repeatability

➢ The recoveries were acceptable, ranging from 74.0 to 126.7%.

➢ The relative standard deviations were lower than 20%.

➢ The calibration linearity ranged from 0.01 ng/mL to 10 ng/mL (R2>0.99)

• Limits of detection

J. Chromatogr. B 1040(2017) : 180-185

29

Method validations

• Sensitivity

APCI EI

➢ BDE-99 and BDE-100 was not detected in GC-EI-MS/MS analysis, but could be successfully quantified by GC-APCI-MS/MS.

➢ The sensitivity of GC-APCI-MS/MS was better than those of the GC-EI-MS/MS method.

Chemosphere 225(2019) : 288-294

30

Trouble and shooting

31

Accuracy of GC-APCI-MS/MS (APGC)

• Relative errors

32

Prenatal exposure

Maternal

Placental barrier

Fetus

➢ OCPs have been reported to transfer from mothersto infants by the placental transfer.

Prenatal Exposure to OCPs

33

Health risk of prenatal exposure

OCP exposure Birth outcomesAssociation

34

Study population and sample collection

• Collected during 2014 and 2015

• N = 1046

Selection criteria:

• <16 gestational weeks• singleton pregnancy• plan to give birth at the study

hospital• residents of Wuhan City• volunteer to take part in this

birth cohort

This study:

35

Study design

Instrumental analysis

Demographic information:• Maternal age• Prepregnancy BMI• Parity• Education & income• etc.

Anthropology parameters:• Birth weight & length• Sex• Gestational age• etc.

Statistical analyses:• Multiple linear regression models• Stratified analyses

36

Detection rates

HCHs DDTs

> Limits of detection (LODs)

➢ The presence of OCPs in cord serum indicated the prenatal exposure.

➢ Detection rates of HCHs and p,p’-DDT and its metabolites were larger than 50%.

➢ The β-HCH and p,p’-DDE were detectable in almost all samples.

37

Predictors of OCPs

➢ Maternal age ➢ Prepregnancy BMI

➢ Maternal education➢ Passive smoking

Ecotox. Environ. Safe. 174(2019): 263-269

38

Associations with birth weight

β : regression coefficientβ>0 : positive associationβ<0 : negative association

➢Birth weight were negativelyassociated with exposure to β-HCHand total HCH for male infants. (p< 0.05)

Sci. Total Environ. 654(2019): 678-683

39

Associations with birth length

➢Associations between the OCP levels and birth length were not significant.(p > 0.05)

40

Conclusion

A GC-APCI-MS/MS method was established with highersensitivity for the analysis of OCPs in serum samples.

A total of 1046 cord serum samples were analyzed and thepredominant OCPs were β-HCH and p,p′-DDE.

The OCP levels were associated with maternal age,prepregnancy BMI, education levels, and passive smoking.

Negative relationships of OCP levels with birth weight andponderal index were observed in boys but not in girls.

41

Publications

1) Comparison of different mass spectrometric approaches coupled to gas chromatography for the analysis of organochlorine pesticides in serum samples. Jing Fang, et al. J. Chromatogr. B 2017, 1040, 180-185.

2) Simultaneous determination of eighteen nitro-polyaromatic hydrocarbons in PM2.5 by atmospheric pressure gas chromatography tandem mass spectrometry, YanhaoZhang, et al, Chemosphere, 198 (2018) 303–310.

3) Determination of PM2.5-bound polyaromatic hydrocarbons and their hydroxylatedderivatives by atmospheric pressure gas chromatography-tandem mass spectrometry, Yanhao Zhang, et al, Talanta, 195 (2019) 757-763.

4) Evaluation of gas chromatography-atmospheric pressure chemical ionization tandem mass spectrometry as an alternative to gas chromatography tandem mass spectrometry for the determination of polychlorinated biphenyls and polybrominated diphenyl ethers. Jing Fang, et al. Chemosphere 2019, 225, 288-294.

5) Performance of atmospheric pressure gas chromatography-tandem mass spectrometry for the analysis of organochlorine pesticides in human serum. Jing Fang, et al. Anal. Bioanal. Chem. 2019, 411, 4185-4191.

6) Applications of atmospheric pressure chemical ionization coupled to gas chromatography mass spectrometry for the analysis of persistent organic pollutants. Jing Fang, et al. Trends Environ. Anal. 2019, (Accepted)

Acknowledgement

Collaborator Huazhong Uni of Sci and TechProf. Shunqing XuDr. Wei XiaDr. Yuanyuan Li

SKLEBA membersMiss Jing FangDr. Yanjun HongDr. Zhu YangDr. Hongzhi ZhaoMiss Jiufeng LiMiss Yanqiu ZhouOther lab members

Resaech Grants

NSFC 21437002GRF 12319716C2014-14E(Research Grant Council Hong Kong, SAR)

42

Department of Chemistry, HKBU

Dioxin Analyss laboratory, BKBU

Fuzhou UniversityDr. Yu HE

43

Concentrations of OCPs

➢ The predominant OCPs were β-HCH and p,p’-DDE .

➢ p,p‘-DDE accounted for 84.4% of ΣDDTs.

< LOD LOD/2

Co

nce

ntr

atio

ns

(ng/

g lip

id)