Establishment of analytical methods to assess endocrine disrupting compounds in a swedish wastewater treatment plant

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II

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24 𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 [%] = (

𝑃𝑟𝑒𝑠𝑝𝑖𝑘𝑒𝑑 𝑆𝑎𝑚𝑝𝑙𝑒𝑠

𝑃𝑜𝑠𝑡𝑠𝑝𝑖𝑘𝑒𝑑 𝑆𝑎𝑚𝑝𝑙𝑒𝑠

) ∙ 100

𝑀𝑎𝑡𝑟𝑖𝑥 𝐸𝑓𝑓𝑒𝑐𝑡 [%] = ((

𝑃𝑜𝑠𝑡𝑠𝑝𝑖𝑘𝑒𝑑 𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑀𝑎𝑡𝑟𝑖𝑥 𝐵𝑙𝑎𝑛𝑘

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25 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 (%) = (

𝑃𝑟𝑒𝑠𝑝𝑖𝑘𝑒𝑑 𝑆𝑎𝑚𝑝𝑙𝑒𝑠 [

𝑛𝑔

𝐿 ] − 𝑀𝑎𝑡𝑟𝑖𝑥 𝐵 [

𝑛𝑔

𝐿 ]

𝑁𝑜𝑚𝑖𝑛𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛

) ∙ 100

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26 .

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P G R -C 1 3 NOR -C 1 3 EE2 -C 1 3 E2 -C 1 3 C a rb o n L a b el ed H o rm o n es TTR An d ro g en s P G R NGT NOR G ES DIE ETO Pro g es tin s EE2 _E3 _E2 _E1 Est ro g en s C o m p o u n d 6 .0 3 5 .3 2 5 .9 3.7₅ 5.4₈ ₃.06 5.6₅ 5.3₂ ₆.35 4.7₅ 5.7₃ 325. 3.7₅ 5_.4 5.2₆ R et en tio n Ti me ( mi n ) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 R T W in d o w (mi n ) + + _- _- + + + + + + + _- _- _- _- P o la rit y 3 1 8 .2 5 3 0 1 .2 2 9 5 2 9 0 .2 2 8 9 3 1 5 .2 5 3 1 3 .2 2 9 9 .2 3 1 1 3 1 2 .2 3 2 5 2 9 5 2 8 7 .2 2 7 1 .1 2 2 6 9 P re cu rs o r (m/ z) 1 0 0 .1 1 1 1 0 9 .0 4 1 4 5 .0 4 1 4 6 .0 5 4 9 7 .0 4 9 7 .0 4 1 5 9 .0 4 1 0 9 .0 4 1 0 9 .0 4 1 3 5 .0 4 1 4 7 .1 1 1 1 4 5 .0 4 1 4 3 .0 4 1 4 5 .0 4 1 4 3 .0 4 P ro d u ct ( m/ z) 2 2 .3 4 2 6 .6 9 3 6 .0 4 55 22 .3 4 2 2 .0 8 2 5 .8 3 2 6 .2 3 2 5 .4 2 2 9 .2 1 79 40 .9 4 5 1 .5 6 4 0 .2 9 5 4 .7 C o lli si o n En er g y ( V ) 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 5 .1 0 7 24 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 5 .1 0 7 2 4 .0 9 1 2 4 .0 9 1 M in D w el l Ti me ( ms) 8 2 .4 8 6 .6 1 1 2 7 .8 9 1 7 6 .3 4 4 5 .0 8 47 56 .2 4 9 .5 3 4 8 .2 9 6 3 .3 7 4 9 .2 8 8 1 .1 7 8 6 .1 1 8 3 .1 5 7 8 .9 4 R F Le n s ( V )

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0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% SW EWIW SW EWIW SW EWIW SW EWIW SW EWIW SW EWIW SW EWIW SW EWIW SW EWIW SW EWIW E1 E2 EE2 E3 DIE E T O GE S NO R P GR T T R 4 mL 2 mL

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-250 -200 -150 -100 -50 0 50 100 150 200 M a tri x Eff e c t [% ] Hormones ACN MeOH 0 50 100 150 200 250 300 Ab s o lu te Rec o v e ry [% ] Hormones ACN MeOH

(A)

(B)

ND ND

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35 .

0 50 100 150 200 250

E1 E2 E3 EE2 ETO DIE GES NGT NOR PGR TTR

A b s o lut e R e v o c e ry [ % ] Hormones SW EW IW ND

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-120 -100 -80 -60 -40 -20 0 20 40 60 80

E1 E2 E3 EE2 ETO DIE GES NGT NOR PGR TTR

M a tri x Eff e c t [% ] SW EW IW

.

ND ND

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39 (a)

(b)

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_(d)

0 2 4 6 8 10 12

E1 E2 E3 EE2 ETO DIE NOR PGR TTR

Con c e n tra ti o n [n g /L ] 0 2 4 6 8 10 12

Con c e n tra ti o n [n g /L ] 0 2 4 6 8 10 12 14 16 18

Con c e n tra ti o n [n g /L ]

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_(b)

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0 20 40 60 80 100 120 140 160 W e e k 1 W e e k 2 W e e k 3 C o n c e n tr a tion [ n g /L ] ETO PGR DIE E2 E1 0 20 40 60 80 100 120 W e e k 1 W e e k 2 W e e k 3 C o n c e n tr a tion [ n g /L ] TTR

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0 2 4 6 8 10 12 W e e k 1 W e e k 2 W e e k 3 C o n c e n tr a tion [ n g /L ] PGR E3 E2 E1

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0 10 20 30 40 50 60 70 80 90 100

Con c e n tra ti o n [n g /L ] TIMFIE Composite Samples

0

10

20

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40

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60

70

80

90

100 Conc

en

tr

ati

on

[ng

/L]

TIMFIE Composite Samples ND ND ND ND

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0 1 2 3 4 5 6 7 8 9 10

Con c e n tra ti o n [n g /L ] TIMFIE Composite Samples ND ND ND ND

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P G R -C 1 3 NOR -C 1 3 EE2 -C 1 3 E2 -C 1 3 C a rb o n L a b el ed H o rm o n es TTR An d ro g en s P G R NGT NOR G ES DIE ETO Pro g es tin s EE2 _E3 _E2 _E1 Est ro g en s C o m p o u n d 6 .0 3 5 .3 2 5 .9 .7₅3 5.4₈ ₃6.0 5.6₅ 5.3₂ ₆.35 4.7₅ 5.7₃ ₂.35 3.7₅ 5.4 5.2₆ R et en tio n Ti me ( mi n ) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 R T W in d o w (mi n ) + + _- _- + + + + + + + _- _- _- _- P o la rit y 3 1 8 .2 5 3 0 1 .2 2 9 5 2 9 0 .2 2 8 9 3 1 5 .2 5 3 1 3 .2 2 9 9 .2 3 1 1 3 1 2 .2 3 2 5 2 9 5 2 8 7 .2 2 7 1 .1 2 2 6 9 P re cu rs o r (m/ z) 1 0 0 .1 1 1 1 0 9 .0 4 1 4 5 .0 4 1 4 6 .0 5 4 9 7 .0 4 9 7 .0 4 1 5 9 .0 4 1 0 9 .0 4 1 0 9 .0 4 1 3 5 .0 4 1 4 7 .1 1 1 1 4 5 .0 4 1 4 3 .0 4 1 4 5 .0 4 1 4 3 .0 4 P ro d u ct ( m/ z) 2 2 .3 4 2 6 .6 9 3 6 .0 4 55 22 .3 4 2 2 .0 8 2 5 .8 3 2 6 .2 3 2 5 .4 2 2 9 .2 1 79 40 .9 4 5 1 .5 6 4 0 .2 9 5 4 .7 C o lli si o n En er g y ( V ) 2 4 .0 9 1 2 4 .091 2 4 .0 9 1 2 5 .1 0 7 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 4 .0 9 1 2 5 .1 0 7 2 4 .0 9 1 2 4 .0 9 1 M in D w el l Ti me ( ms) 8 2 .4 8 6 .6 1 1 2 7 .8 9 1 7 6 .3 4 4 5 .0 8 47 56 .2 4 9 .5 3 4 8 .2 9 6 3 .3 7 4 9 .2 8 8 1 .1 7 8 6 .1 1 8 3 .1 5 7 8 .9 4 R F Le n s ( V )

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TTR P

G

R

NOR NGT G

ES DIE ETO EE2 E3 E2 E1

M at rix S tu d y S p ik in g Le v el 1 1 2 ± 3 107 ± 11 11₀ ± 4 – 101 ± 5 107 ± 5 – 114 ± 2 9 6 ± 7 1 1 3 ± 3 106 ± 5 M ill iQ Zh an g 2 0 1 7 N .S . 0 4 5 ± 4 2 3 ± 4 2 3 ± 5 4 5 ± 5 0 2 6 ± 17 50_± 5 2 6 0 ± 60 75_± 5 0 Th_is S tu d y 1 n g /L 8 4 ± 8 7 2 ± 8 6 0 ± 11 53 ± 23 84_± 4 0 6 5 ± 23 93_± 2 9 6 ± 14 73_± 9 7 2 ± 11 5 n_g /L 1 1 6 ± 5 105 ± 5 114 ± 4 – 111 ± 5 101 ± 5 – 112 ± 9 82 ± 11 11₉ ± 6 9 4 ± 6 SW Zh an g 2 0 1 7 N .S . – 10_1 ±   11 88_±  9 87  ±  1 3 90_±  1 6 – 82_±  1 3 – – – – 20₁ Sh_en 8 1 0 n g /L 1 1 0 1 1 0 1 1 0 1 1 0 2 1 0 0 1 1 0 1 2 0 1 1 0 1 1 0 1 1 0 1 2 0 Th is S tu d y 1 0 n g /L – – – – – – – 83_± 5 8 6 ± 6 8 8 ± 4 8 8 ± 3 EW K o h 2 0 0 7 1 5 n g /L 1 1 8 ± 7 108 ± 6 110 ± 7 – 97 ± 16 12₄ ± 7 – 9 6 ± 8 9 6 ± 5 9 9 ± 9 9 0 ± 7 Zh an g 2 0 1 7 N .S . – 10_2 ±   12 10 0 ±   12 91_±  1 5 96_±  1 4 – 94_±  1 7 – – – – Sh en 2 0 1 8 1 0 n g /L 9 5 .4 ± 1 .6 7 4 .2 ± 4 .8 1 2 1 ± 10 10₁ ± 4 .6 – – – 1 4 2 ± 0 .1 – 1 0 6 ± 1 .5 1 1 1 ± 3 .8 Liu 2 0 1 1 a 20 n g / L 1 1 0 ± 4 110 ± 2 100 ± 1 0 710 ± 47 14₀ ± 13 15₀ ± 9 36 ± 0 .2 1 1 0 ± 0 .9 1 1 0 ± 1 120 ± 3 Th is S tu d y 3 0 n g /L 98 ± 1 1 0 4 ± 1 94 ± 6 1 1 1 ± 5 – – – 89_± 7 – 1 2 3 ± 14 12₃ ± 15 IW Li u 2 0 1 1 a 50 n g / L 1 2 0 ± 3 108 ± 8 114 ± 9 – 103 ± 12 11₄ ± 5 – 119 ± 6 126 ± 9 106 ± 4 102 ± 8 Zh an g 2 0 1 7 N .S . 5 9 ± 8 5 4 ± 3 8 9 ± 2 0 6 5 0 ± 64 10₀ ± 5 68 ± 17 72 ± 14 12₀ ± 3 5 6 ± 5 6 7 ± 7 Th is st u d y 3 0 n g /L

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UPLC-MS/MS GC-MS/MS S/N LOQ [ng/L] IDL [ng/L] IQL on Column [pg] S/N IDL [ng/L] LOQ [ng/L] IQL on Column [pg] E1 6.3 1.6 0.48 2.4 3.3 4.6 15 9.1 E2 4.3 2.3 0.7 3.5 3.5 0.43 1.4 0.85 E3 13 0.8 0.24 1.2 3 10 33 20 EE2 3 3.3 1 5 4.5 3.3 11 6.7 ETO 9.1 1.1 0.33 1.6 ND ND ND ND DIE 7.3 1.4 0.41 2.1 ND ND ND ND GES 11 0.91 0.27 1.4 ND ND ND ND NGT 8 1.3 0.38 1.9 ND ND ND ND NOR 35 0.28 0.085 0.43 ND ND ND ND PGR 40 0.25 0.075 0.38 ND ND ND ND TTR 85 0.12 0.035 0.18 89 25 2.8 50

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Area Recovery [%] Mean SD cv [%] Matrix Effect [%] Prespike Prespike Postspike 1 2

E1 85000 96000 98000 89 98 93 6.7 7.2 -55 E2 20000 22000 24000 90 92 91 1.2 1.3 -65 E3 230000 230000 250000 99 92 96 4.8 5 -44 EE2 280000 310000 140000 89 220 160 96 61 -190 ETO 47000 45000 32000 100 140 120 26 21 -100 DIE 88000 97000 84000 90 120 100 19 18 -95 GES 6200000 5900000 7500000 110 79 92 19 20 160 NGT 0 0 0 0 0 0 0 0 0 NOR 42000 47000 53000 89 90 89 0.44 0.49 -95 PGR 38000 44000 66000 85 67 76 13 17 -99 TTR 170000 150000 320000 110 48 79 44 56 -90

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TTR P GR NOR GE S ETO DI E E3 EE2 E2 E1 350000 73000 190000 65 * 10 5 36000 200000 380000 33000 14000 82000 _Inf luen t 4 mL 8100 5800 0 11* 10 5 3800 3800 13000 0 4600 4000 4100 2 mL 430000 65000 150000 72 * 10 5 44000 150000 330000 24000 16000 92000 4 mL 7000 4800 19000 12* 10 5 3500 4700 160000 4900 4800 4300 2 mL 390000 69000 170000 68* 10 5 40000 170000 360000 28000 15000 87000 Av. 4 mL 7600 5300 9600 12* 10 5 3700 4200 150000 4800 4400 4200 Av. 2 mL 56000 5400 28000 490000 5500 33000 36000 6500 1000 7300 _Inf luen t S T DV 14 7. 8 16 7.2 14 19 10 23 6.9 8.3 CV

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TTR P GR NOR GE S ETO DI E E3 EE2 E2 E1 900000 590000 380000 210000 100000 370000 14000 12000 24000 64000 S ur fa ce W ater 4 mL 3500 9200 2600 14000 2000 3100 8900 1100 1800 1800 2 mL 900000 940000 360000 15 * 10 6 370000 520000 47000 39000 67000 190000 E ff luent 4 mL 7800 11000 3500 3* 10 6 8100 5200 22000 8400 4100 3000 2 mL 870000 980000 360000 14 * 10 6 350000 610000 49000 39000 66000 180000 4 mL 7400 11000 0 26* 10 5 8000 4200 19000 6500 4200 3200 2 mL 890000 960000 360000 15* 10 6 360000 560000 48000 39000 67000 190000 Av. 4 mL 7600 11000 1800 28* 10 5 8100 4700 20000 7400 4200 3100 Av. 2 mL 26000 30000 810 980000 18000 61000 1700 260 750 6000 S T DV 3 3. 1 0.22 6.7 4.9 11 3.5 0.66 1.1 3.2 CV

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In flu en t Blan k Da y 1 In flu en t Blan k Da y 2 In flu en t sp ik ed 1 n g /L In flu en t S p ik ed 5 n g /L In flu en t S p ik ed 5 n g /L Da y 2 57 48 55 55 32 61 53 57 73 44 64 38 22 22 E1 21 16 34 28 41 36 35 36 45 35 42 48 27 37 E2 > 100 >100 >100 >100 >100 >100 >100 100> >100 >100 >100 >100 >100 >100 E3 9. 7 7.7 16 16 23 15 17 20 13 6.8 4.3 6.6 5.9 6 EE2 29 27 15 12 27 61 36 23 67 48 60 22 13 19 ETO < 0. 37 < 0. 21 < 0. 52 < 0. 55 < 0. 41 2.9 2.6 3.7 11 46. 7.7 8.4 3.9 4.9 DI E > 100 >100 >100 >100 >100 >100 100> >100 >100 >100 >100 >100 >100 > 1 0 0 GE S < 8. 6 < 4. 9 < 12 <13 <9. 5 < 12 <9. 6 < 12 >100 >100 >100 >100 >100 >100 NGT < 2. 2 < 1. 2 13 18 39 24 16 18 16 26 23 15 19 18 NOR 51 33 14 15 33 19 14 17 22 14 16 23 13 17 PGR 24 18 4. 2 4.7 10 12 9.1 11 26 18 24 5 3.1 4.1 TTR

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Ef flu en t B la n k Ef flu en t B la n k D ay 2 Ef flu en t S p ik ed 1 n g /L Ef flu en t S p ik ed 5 n g /L Ef flu en t S p ik ed 5 n g /L d ay 2 1 .4 0.8₈ 0.7₆ 3.5 3.6 .64 9.8 10 11 7.5 6.7 9.3 E1 2 .9 1.9 1.8 2.4 3 3_.1 _.28 9_.7 10 8_.6 6_.3 9_.3 E2 3 .5 1.4 1.8 2.7 3.9 7.3 9.9 9.4 10 8.3 6 8_.2 E3 < 2 .9 < 2 .3 < 2 .1 < 2 .1 <2 3.5 6 5.2 9.5 6.8 3.5 5 EE2 37 28 25 25 20 33 17 17 17 12 10 12 ETO < 0 .1 3 < 0 .1 2 < 0 .1 2 1 .2 1.2 1.5 4 4_.7 5_.4 4_.9 3_.4 5_.2 DIE > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 G ES < 3 .1 < 2 .7 < 2 .7 < 3 .5 <3 < 4 .4 < 3 .8 < 3 .8 < 4 .1 < 4 .2 < 3 .1 < 3 .3 NGT < 0 .7 9 < 0 .7 < 0 .6 9 < 0 .8 8 < 0 .7 7 < 1 .1 6.5 4 3 6.5 3.5 3.1 NOR < 0 .2 7 < 0 .2 3 < 0 .2 3 2 .2 1.9 2.5 7.6 8.3 .68 7.9 5.7 8.9 PGR < 0 .2 3 < 0 .2 0 .8 9 1 .1 1 1_.5 6_.4 6_.1 3_.6 5_.2 7_.2 4_.4 TTR

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S u rf ac e W at er B la n k S u rf ac e W at er B la n k D ay 2 S u rf ac e W at er sp ik ed w ith 1 n g /L S u rf ac e W at er sp ik ed w ith 5 n g /L S u rf ac e W at er sp ik ed w ith 5 n g /L D ay 2 < 0 .4 6 < 0 .4 4 < 0 .4 8 0 .8 2 0 .8 4 1 .5 1.7 2.6 10 9.8 11 9.2 10 10 E1 2 .7 2.7 3.4 2.1 2 1_.4 1_.9 1_.4 9_.7 10 11 9_.7 10 7_.9 E2 < 1 .1 <1 < 1 .1 < 0 .6 3 < 0 .8 8 < 0 .7 5 < 0 .7 7 < 0 .9 8 8 .3 8 9 7 8_.1 7_.5 E3 <3 <2 .9 < 3 .2 < 1 .8 < 2 .5 < 2 .1 < 2 .1 < 2 .7 10 7.5 7.9 7.8 9.6 10 EE2 < 0 .9 4 < 0 .9 2 < 0 .9 9 < 0 .5 5 < 0 .7 8 < 0 .6 6 < 0 .6 7 < 0 .8 6 4 2 .9 4.5 3.7 3.7 4.1 ETO < 0 .0 8 2 < 0 .0 7 2 < 0 .0 7 7 < 0 .0 5 4 < 0 .0 7 7 0 .4 8 0 .4 8 0 .7 4 4 .8 5.3 5.8 5 5 5 DIE 46 42 37 27 30 26 25 22 5 .1 2.4 2 3_.7 6_.3 4_.3 GES < 1 .9 < 1 .7 < 1 .8 < 1 .3 < 1 .8 < 1 .2 < 1 .3 < 2 .3 <2 < 2 .3 < 2 .4 <2 <2 < 2 .1 NGT < 0 .4 9 < 0 .4 3 < 0 .4 5 < 0 .3 2 < 0 .4 5 < 0 .2 9 < 0 .3 3 < 0 .5 8 6 .3 7.3 7.5 4.2 4.6 6.9 NOR < 0 .1 6 0 .3 0.2₈ < 0 .1 1 < 0 .1 5 1 .5 1.4 1.6 7.2 7.8 7.8 7 7_.7 7_.5 PG R < 0 .1 4 < 0 .1 3 < 0 .1 3 < 0 .0 9 3 < 0 .1 3 0 .6 8 0 .5 8 0 .6 6 4 .3 7.5 7.5 4.5 4.7 5.6 TTR

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M il li Q Blan k Mi lli Q S p ik ed with 1 n g /L M il liQ S p ik ed with 5 n g /L < 0 .3 6 < 0 .3 6 < 0 .4 1.1 0 .8 9 1 9 .4 9.1 9.6 E1 4 .7 1.7 2.3 0.87 0 .8 6 1 .2 8.5 9.4 10 E2 < 0 .8 4 < 0 .8 4 < 0 .9 4 1 .4 1.6 1.6 6.7 6.9 8.5 E3 2 .3 2_.3< <2_.6 <1_.8 _.8<1 2 6_.5 6_.4 10 EE2 < 0 .7 4 9 .7 9.7 1.2 1.8 1.4 5.8 6.5 6.2 ETO < 0 .0 8 5 < 0 .0 8 5 < 0 .0 7 2 0 .9 1 0 .9 9 0 .7 8 8 .3 7.9 8.8 DIE < 0 .4 7 < 0 .4 7 < 0 .4 0 .3 7 0 .5 8 0 .4 2 5 .1 3 7_.6 GES <2 <2 <₁ .7 < 1 .3 < 1 .2 <1 7.8 8.4 9.1 NGT < 0 .5 < 0 .5 < 0 .4 3 0 .3 5 0 .5 1 0 .4 7 7 .4 5.8 5 NOR < 0 .1 7 < 0 .1 7 < 0 .1 4 1 .1 1.2 1 6_.1 7_.7 7_.6 PGR < 0 .1 5 < 0 .1 5 < 0 .1 3 0 .9 5 0 .9 0.8 8.2 7.4 6.5 TTR