APPENDIX : Supplemental digital content

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APPENDIX : Supplemental digital content

eFigure 1. Flow diagram of the study sample. PELAGIE cohort, Brittany, France, 2002-2006.

PELAGIE cohort N=3421

Planned pregnancies N=2995

181 unplanned pregnancies 245 missing data

39 missing TTP

13 missing fish or shellfish consumption data

Available TTP and fish/shellfish consumption

N=2943

Liveborn singleton cohort N=3322

Available TTP N=2956

177 missing cord blood samples

Available TTP and POP measurements

N=349

N=333

Available TTP and mercury measurements

N=362

164 missing hair samples Sub-cohort randomly selected

N=601 19 unplanned pregnancies

50 missing data

Planned pregnancies N=532

6 missing TTP

Available TTP N=526

All available data for POP analyses

N=332

All available data for mercury analysis

N=353 N=318

9 missing data on covariates 17 missing data

on covariates 86 missing data on

covariates

All available data for fish/shellfish analysis

N=2870

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2 eAppendix : Chemical analyses of POP in cord blood serum samples

Plasma samples (2 mL) were extracted on a Strata-X 100 m (100 mg/ 3 mL; Phenomenex;

Torrance, California, USA) solid phase extraction (SPE) column. A mixture of

¹³

C-labeled

internal standards, plasma sample, formic acid and deionized water was slowly applied to the

column. After drying the column with pressurized nitrogen, the sample was extracted using 3

mL of dichloromethane. The extracts (taken up in 0.5 mL hexane) were cleaned up through a

column containing activated florisil (1 g) and the compounds were eluted using a mixture of

hexane/dichloromethane (3:1) and dichloromethane. The solvent was evaporated, taken up in

20 L of hexane and analyzed for PCBs and organochlorine pesticides on an Agilent 6890

Network gas chromatograph (GC) coupled with an Agilent 5973 Network mass spectrometer

(MS) (Agilent Technologies; Mississauga, Ontario, Canada). The GC was fitted with an

Agilent 60 m DB-XLB column (0.25 mm i.d., 0.25 µm film thickness). We added 125 L of

hexane to the fraction analyzed for the PCBs and organochlorine pesticides to measure all

PBDEs including PBDE209 on an Agilent 6890 Network GC coupled with an Agilent 5975

Inert XL MS. The GC was fitted with an Agilent 15 m DB-XLB column (0.25 mm i.d., 0.1

µm film thickness). For both analytical instruments, the carrier gas was helium, and all

injections were in splitless mode. The MS was operated in single ion monitoring (SIM),

using negative chemical ionization (NCI) with methane (99.97 %) as the reagent gas.

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3 eTable 1. Spearman’s rank correlation coefficient between each persistent organic pollutant (POP) among eligible women to time to pregnancy (N=349)

βHCH γHCH HCB HCE αendo-

sulfan Dieldrin DDE PCB118 PCB138 PCB153 PCB170 PCB180 PCB183 PCB187 PCB194 PCB203 PBDE209 Mercury^a

N > LOD 344 40 266 178 33 55 283 344 349 349 332 349 116 256 124 40 107 333

βHCH 1.00

γHCH 0.01 1.00

HCB 0.67** 0.22** 1.00

HCE 0.50** 0.12^* 0.62** 1.00

αendosulfan -0.03 0.05 -0.14* -0.06 1.00

Dieldrin 0.29** -0.04 0.37** 0.36** -0.03 1.00

DDE 0.54** 0.02 0.51** 0.28** -0.13* 0.18* 1.00

PCB118 0.66** 0.09 0.69** 0.49** -0.05 0.31** 0.51** 1.00

PCB138 0.69** 0.07 0.71** 0.47** -0.09 0.26** 0.64** 0.85** 1.00 PCB153 0.67** 0.07 0.69** 0.42** -0.07 0.27** 0.59** 0.82** 0.95** 1.00 PCB170 0.62** 0.07 0.63** 0.36** -0.08 0.25** 0.48** 0.71** 0.86** 0.95** 1.00 PCB180 0.61** 0.07 0.61** 0.35** -0.06 0.26** 0.49** 0.70** 0.84** 0.95** 0.99** 1.00 PCB183 0.58** 0.04 0.54** 0.40** -0.01 0.24** 0.53** 0.66** 0.81** 0.79** 0.76** 0.74** 1.00 PCB187 0.60** 0.03 0.55** 0.32** -0.05 0.25** 0.52** 0.71** 0.82** 0.90** 0.88** 0.89** 0.75** 1.00 PCB194 0.45** 0.08 0.47** 0.28** 0.01 0.23** 0.33** 0.51** 0.63** 0.74** 0.80** 0.82** 0.69** 0.75** 1.00 PCB203 0.35** -0.02 0.31** 0.17* 0.03 0.12* 0.27** 0.38** 0.45** 0.49** 0.49** 0.50** 0.55** 0.52** 0.63** 1.00 PBDE209 0.08 0.05 0.05 0.07 0.08 0.03 -0.01 0.08 0.11 0.13* 0.14* 0.14* 0.10 0.11 0.10 0.08 1.00 Mercury 0.17* -0.11* 0.17* 0.14* 0.002 0.24** 0.09 0.24** 0.25** 0.28** 0.25** 0.26** 0.19* 0.28** 0.22** 0.19* 0.13* 1.00 LOD, Limit Of Detection

Concentrations below the LOD were replaced by the LOD value divided by 2.

a From the 349 women who had POP measurements in cord blood, only 333 also had mercury measurement in their hairs, all with a detectable level.

* p<0.05 ** p<0.0001

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4 eTable 2. Limit of detection (LOD) and geometric mean (GM) of persistent organic pollutants in cord blood and mercury in hair from the eligible sub-cohort (n=349) and the excluded women (n=45)

Eligible sub-cohort Excluded women

LOD μg/L

> LOD n (%)

GM (SE) μg/L

GM (SE) ng/g lipid

> LOD n (%)

GM (SE) μg/L

GM (SE) ng/g lipid Organochlorine pesticides

beta-hexachlorocyclohexane 0.01 344 (98.6) 0.044 (1.8) 11.9 (2.1) 45 (100.0) 0.047 (2.1) 14.0 (2.3)

p.p’-DDE 0.02 283 (81.1) 0.126 (3.9) 34.4 (4.0) 37 (82.2) 0.154 (4.5) 50.0 (4.4)

hexachlorobenzene 0.02 266 (76.2) 0.025 (2.6) 6.8 (2.9) 29 (64.4) 0.020 (3.0) 6.1 (3.6) heptachlor epoxide^a 0.01 178 (51.1) 0.009 (1.8) 2.3 (2.1) 16 (35.6) 0.007 (1.7)* 2.0 (2.1)

dieldrin^a 0.01 55 (15.8) 0.007 (1.9) 1.8 (2.1) 7 (15.6) 0.006 (1.9) 1.9 (2.3)

gamma-hexachlorocyclohexane^a 0.01 40 (11.5) 0.006 (1.6) 1.6 (2.0) 5 (11.1) 0.006 (1.4) 1.6 (2.1)

alpha-endosulfan^a 0.01 33 (9.5) 0.005 (1.3) 1.5 (1.8) 5 (11.1) 0.006 (1.4) 1.6 (1.9)

Polychlorinated biphenyls

PCB138 0.01 349 (100.0) 0.065 (1.7) 17.6 (2.0) 45 (100.0) 0.058 (1.8) 17.0 (2.1)

PCB153 0.01 349 (100.0) 0.111 (1.7) 30.2 (2.0) 45 (100.0) 0.098 (1.9) 28.5 (2.1)

PCB180 0.01 349 (100.0) 0.072 (1.7) 19.7 (2.0) 44 (97.8) 0.062 (2.0) 17.9 (2.3)

PCB118 0.01 344 (98.6) 0.027 (1.7) 7.4 (2.0) 43 (95.6) 0.025 (1.9) 7.2 (2.4)

PCB170 0.01 332 (95.1) 0.024 (1.8) 6.6 (2.1) 40 (88.9) 0.021 (2.1) 6.0 (2.3)

PCB187 0.01 256 (73.4) 0.012 (2.0) 3.3 (2.2) 31 (68.9) 0.012 (2.0) 3.4 (2.2)

PCB194 0.01 124 (35.5) 0.007 (1.7) 2.0 (2.0) 15 (33.3) 0.007 (1.7) 2.0 (2.0)

PCB183 0.01 116 (33.2) 0.007 (1.7) 1.9 (2.0) 13 (28.9) 0.007 (1.7) 2.0 (2.0)

PCB203^a 0.01 40 (11.5) 0.006 (1.4) 1.5 (1.8) 6 (13.3) 0.006 (1.4) 1.6 (1.8)

Total PCBs^{a ,b} NA NA 0.337 (1.7) 91.6 (2.0) NA 0.301 (1.8) 87.5 (2.1)

Polybrominated diphenyl ether

PBDE209^c 0.05 107 (32.3) 0.046 (2.8) 12.6 (3.1) 13 (28.9) 0.044 (2.5) 13.5 (2.7)

Metal

Mercury^d 0.02 362 (100.0) 0.623 (1.9) NA 45 (100.0) 0.570 (2.1) NA SE: Standard Error; NA: Not applicable

* : p-value of the Wilcoxon test <0.05 for comparison between concentrations of the eligible sub-cohort and the excluded women

a Among 348 samples of the eligible sub-cohort

b Total PCBs: sum of congeners 118, 138, 153, 170, 180, 183, 187, 194 and 203. Concentrations below the LOD were replaced by the LOD value divided by 2.

c Among 331 samples of the eligible sub-cohort

d Geometric mean (SE) expressed in microgram per hair gram, among the 362 women of the sub-cohort with hair-mercury measurement and TTP value

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5 Legend eFigure 2: (a) main population; (b) limited to primiparous women; (c) excluding women who conceive in the first month of trying; (d) adding women with contraceptive failure (TTP=0); (e) excluding women who used medical assistance to get pregnant; (f) censoring at 7 months; (g) censoring at 10 months; (h) censoring at 15 months.

eFigure 2: Adjusted fORs and 95% CIs for association of medium (solid line) and high (dotted

line) levels of shellfish intake, serum POP and hair-mercury with time-to-pregnancy (TTP)

compared with the lowest levels, according to various sensitivity analyses.

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6

eFigure 3. Estimation of PCB 153 concentration at pregnancy attempt by modeling

bioaccumulation with age and parity

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7

eTable 3. Median level (or 90

^e

percentile) of persistent organic pollutant measured in cord blood expressed in microgram per liter (N=349) and mercury measured in hair expressed in microgram per gram (N=362) for women eligible to time to pregnancy, according to seafood consumption.

Median level for compounds detected in at least 50% samples 90^e percentile for compounds detected in less than 50% samples Median level

N β

HCB HCE DDE PCB PCB PCB PCB PCB PCB γ

HCH

αendo-

dieldrin PCB PCB PCB PBDE

N Mercury

HCH 118 138 153 170 180 187 sulfan 183 194 203 209

N ≥ LOD ^a 344 266 178 283 344 349 349 332 349 256 40 33 55 116 124 40 107 362

Fish consumption ** * ****

Less than monthly 66 0.042 0.032 0.008 0.160 0.024 0.056 0.094 0.023 0.068 0.010 0.013 0.010 0.020 0.015 0.014 0.005 0.170 66 0.481 1-4 times a month 192 0.042 0.032 0.010 0.190 0.028 0.064 0.110 0.024 0.070 0.013 0.005 0.005 0.021 0.015 0.018 0.010 0.250 201 0.602 At least twice a week 89 0.039 0.036 0.010 0.170 0.027 0.065 0.110 0.026 0.073 0.014 0.012 0.005 0.025 0.017 0.019 0.011 0.320 94 0.863

Shellfish consumption * * *

Less than twice a week 228 0.044 0.032 0.010 0.175 0.026 0.060 0.110 0.023 0.068 0.010 0.011 0.005 0.024 0.015 0.016 0.005 0.300 240 0.602 2-8 times a month 85 0.040 0.034 0.005 0.190 0.029 0.069 0.120 0.026 0.077 0.014 0.011 0.010 0.022 0.017 0.018 0.012 0.180 85 0.642 At least twice a week 35 0.039 0.037 0.005 0.170 0.025 0.061 0.120 0.027 0.080 0.015 0.005 0.005 0.021 0.016 0.019 0.011 0.100 37 0.702

Mollusk consumption * ** **

Less than monthly 278 0.043 0.033 0.010 0.175 0.027 0.061 0.110 0.023 0.068 0.013 0.011 0.005 0.023 0.016 0.016 0.005 0.250 291 0.602 Once a week or less 68 0.040 0.033 0.005 0.180 0.026 0.071 0.120 0.027 0.083 0.016 0.013 0.010 0.024 0.016 0.021 0.012 0.270 70 0.702 At least twice a week 1 0.035 0.048 0.005 0.010 0.029 0.052 0.110 0.027 0.073 0.017 0.005 0.005 0.005 0.005 0.005 0.005 0.090 1 3.410 Small crustacean

consumption

Less than monthly 250 0.043 0.032 0.010 0.180 0.027 0.060 0.110 0.024 0.069 0.013 0.011 0.005 0.024 0.016 0.016 0.010 0.300 262 0.602 Once a week or less 95 0.039 0.035 0.005 0.170 0.029 0.069 0.110 0.026 0.076 0.014 0.012 0.005 0.022 0.016 0.018 0.010 0.160 97 0.642 At least twice a week 2 0.032 0.041 0.008 0.170 0.033 0.084 0.138 0.034 0.091 0.015 0.005 0.005 0.005 0.016 0.016 0.012 0.025 2 0.702 Large crustacean

consumption * * * * * * *

Less than monthly 309 0.043 0.033 0.010 0.180 0.027 0.062 0.110 0.024 0.069 0.013 0.011 0.010 0.023 0.016 0.016 0.010 0.250 321 0.602 Once a week or less 37 0.039 0.034 0.005 0.160 0.032 0.070 0.120 0.027 0.080 0.014 0.013 0.005 0.021 0.015 0.019 0.010 0.300 39 0.702 At least twice a week 2 0.067 0.068 0.010 0.260 0.058 0.140 0.200 0.043 0.115 0.026 0.005 0.005 0.005 0.016 0.016 0.012 0.025 2 0.853 LOD: Limit of detection

a Concentrations below the LOD were replaced by the LOD value divided by 2.

* p<0.10 ** p<0.05 *** p<0.001 **** p<0.0001 (Kruskal-Wallis test)