Supplementary Information

Effect-based assessment of toxicity removal during wastewater treatment

Pia Välitaloa,b,*, Riccardo Masseic,d , Ilse Heiskanenb, Peter Behnische, Werner Brackc,d, Andrew J. Tindallf, David Du Pasquierf ,Eberhard Küsterc, Anna Mikolaa, Tobias Schulzec and Markus Sillanpääb

aAalto University, Department of Civil and Environmental Engineering, Tietotie 1E, 02150 Espoo, FinlandbFinnish Environment Institute, Laboratory Centre, Hakuninmaantie 6, 00430 Helsinki, FinlandcHelmholtz Centre for Environmental Research UFZ, Leipzig, GermanydInstitute for Environmental Research (Biology V), RWTH Aachen University, Aachen, GermanyeBioDetection Systems b.v., Amsterdam, The NetherlandsfWatchFrog S.A., 1 rue Pierre Fontaine, 91000 Evry, France

*Corresponding author: pia.valitalo@aalto.fi

SI, 1. Additional equations for REF calculation

The enrichment factor of the solid phase extraction was calculated for each samples using (Eq. 1).

enrichment factor SPE=V water

V extract (Eq. 1)

The dilution factor for each biotest was calculated according to (Eq. 2).

dilution factorbioassay=volume of extract added ¿bioassay ¿total volumeof bioassay

(Eq. 2)

SI, 2. Material and methods for biological analysis

SI, 2.1 Acute cytotoxic effects

The acute cytotoxic effects of the influent and effluent samples were investigated by using the neutral red

retention (NRR) assay with a permanent rainbow trout liver cell line (RTL-W1; (Lee et al., 1993). The

cells were cultured at 20 ºC in 75 cm2 plastic culture flasks in L15 medium with L-glutamine

supplemented with 9 % fetal calf serum. The cells were exposed to serial dilutions of the wastewater

extracts in 96-well microtiter plates with a concentration range of REF5, 7.5, 10, 12.5, 15, 17.5 and

REF20. 3,4-dichlorphenol (40 mg/L) was used as positive control and medium was used for negative

control. The cells were exposed for 48 h at 20 ºC, after which the cells in each well were stained with 100

µL of 0.004% neutral red solution (2-methyl-3-amino-7-dimethylamino-phenanzine) in medium and

incubated in darkness for 3 h at 20ºC. After incubation, the neutral red solution was discarded and wells

were washed twice with 100 µl of PBS and 100 µl of extraction solution (2.5 mL acetic acid (glacial) in

125 mL ethanol and 122.5 mL distilled water) was added to each well. Then the plates were shaken for 30

minutes. Subsequently, absorption by Neutral Red was measured at 540 nm with a reference wavelength

of 690 nm using a spectrophotometer (Victor3, Perkin Elmer, Singapore). Each sample was tested in three

replicates. The viability of the cells was calculated as a percentage of the negative controls (unexposed

cells).

SI, 2.2 Analysis of endocrine disrupting effects

SI, 2.2.1 CALUX® assays

The extracts were evaporated to dryness with EZ-Envi centrifugal evaporator (Genevac Ltd, Ipswich,

UK) and re-dissolved in dimethylsulfoxide (DMSO) with a concentration factor or 1000x. The samples

were stored at 4°C prior to analysis. The ER and AR CALUX® bioassays were then performed as

described previously (van der Linden et al., 2008). In short, cells were seeded into 96-well microtiter

plates with DF medium (without phenol red) that was supplemented with stripped (dextran-coated

charcoal treated) serum. After 24 h of incubation (37 °C, 7.5%CO2), the medium was replaced by

medium containing the water extracts (0.1% v/v DMSO) for agonistic activity testing (in triplicate). After

24 h of incubation, the medium was removed and the cells were lysed in 30 μL of Triton-lysis buffer. The

amount of luciferase activity was quantified using a 96 well plate luminometer reader (Mithras, Berthold

Technologies Gmbh, Germany). On all plates, a dose-response curve of the reference compound was

included for quantification of the response, which was estradiol (E2) or dihydrotestosterone (DHT) for the

ER- or AR- CALUX, respectively. All of the respective extracts were analyzed in triplicate. The dilution

series consisted of a non-diluted sample and 10, 30 and 100 times diluted sample.

The results are presented as bioanalytical equivalent concentrations (ng E2 eq./L for ER-CALUX, and ng

DHT eq./L for AR-CALUX). The results were calculated based on a calibration concentration series,

which was added to each microtiter plate. After measuring luminescence, the calibration concentrations

were used for constructing a calibration curve. The equation used was a curve fit with the dose response

sigmoidal formula (Eq. 3):

(Eq. 3)

Where: y= The response in relative light units RLU (corrected for the RLU result of DMSO)

                x= The concentration in pM for reference substance in the well.

                a0=The maximum response

                a1=The EC50 of the curve

                a2=The slope of the curve

Data was corrected for DMSO RLU of the assay (determined by cells that are only exposed to DMSO).

By interpolation of the DMSO corrected sample in the calibration curve, the sample result is expressed as

a CALUX EQ-value.   Finally, the CALUX EQ-value of the sample is expressed as ng reference per unit

of processed material.

SI, 2.2.2 Measurement of endocrine activity using transgenic larval models

Extracts of the water samples were reconstituted in DMSO and frozen as stock aliquots. Immediately

prior to testing, the stock aliquots were diluted in glass-bottled Evian water to give a final concentration

of 0.2% DMSO for the estrogen assay and 0.3% DMSO for the thyroid assay.

The estrogen axis activity of the samples was determined using eleuthero-embryos of a stable transgenic

line of medaka harbouring the chgh-gfp transgene (Spirhanzlova et al., 2016) . Exposures were carried out

as previously described in the presence (spiked mode) or absence (unspiked mode) of testosterone

(Spirhanzlova et al., 2016). Key steps and modifications to the previously published protocol are noted

here briefly. A 24 h embryo-lethality test was performed and a ten-fold dilution was applied to the lowest

concentration inducing lethality. Estrogen axis activity was then determined by exposing day post hatch

zero eleuthero-embryos to the diluted extracts in the presence or absence of testosterone. Eight embryos

per treatment were exposed for 24 h at 26°C in six-well plates. All controls were carried out in glass

bottled Evian water. In unspiked mode the controls consisted of a nine-point ethinyl estradiol (EE2)

standard curve with the 0 ng/L EE2 group acting as the negative control. In spiked mode the negative

control was a group exposed to testosterone alone at the concentration used to spike the samples (30

µg/L). The positive controls were testosterone plus 64 ng/L EE2 for activation of the estrogen axis and

testosterone plus fadrozole (10 µg/L) for inhibition of the estrogen axis.

Following 24 h exposure to the test solutions, embryos were anaesthetised to allow image capture of their

livers at 8x magnification using a TXD 14C camera (Baumer) fitted to a MZ10F stereomicroscope

(Leica). Embryos were illuminated with a Lumen 200W fluorescence source (Prior Scientific) and ET-

GFP long-pass filters (excitation 480/40, emission 510LP, Leica). Image treatment and data analysis was

carried out as previously described (Spirhanzlova et al., 2016). Two replicate experiments were

performed, the results were normalised to the testosterone control group and pooled. GraphPad Prism

version 5.04 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. In cases where the

data in all groups followed a Gaussian distribution, a Students T-Test (pairwise comparison) or one-way

ANOVA and Dunnett’s post-test (comparing multiple groups) were carried out. In cases where the data of

one or more groups did not follow a Gaussian distribution, a Mann-Whitney test (pairwise comparison) or

Kruskal-Wallis and Dunn’s post-test (comparing multiple groups) were carried out. In unspiked mode, all

groups were compared to the solvent control group, in spiked mode all groups were compared to the

testosterone control. A nine point ethinyl estradiol (EE2) dose response curve was modelled and used to

determine the hormonal equivalence of each sample (SI, Fig. 1). This curve was modelled in Graphpad

Prism using the asymmetric Weibull model (ref. PMID:11351447) and fluorescence induction for the

environmental samples were read from the curve.

SI, Figure 1. a) The full concentration response curve for the EE2 standard curve and b) a partial curve showing the lower concentrations. The partial curve was used for the curve fit for calculating the hormone equivalence.

Thyroid axis activity was determined by exposing stage 45 eleuthero-embryos of a stable transgenic

Xenopus line harbouring the THbZIP-GFP transgene to the diluted extracts in the presence (spiked mode)

or absence (unspiked mode) of T3 hormone (Fini et al., 2007). Ten embryos per treatment were exposed

for 48 h at 26°C in six-well plates. All controls were carried out in glass bottled Evian water. In unspiked

mode the controls consisted of a four point T3 standard curve with the 0 µg/L T3 group acting as the

solvent control. In spiked mode the negative control was a group exposed to T3 alone at the concentration

used to spike the samples (3.25 µg/L). The positive controls were 6.5 µg/L and 16.5 µg/L T3. Following

exposure to the test solutions for 48 h, embryos were anaesthetised to allow fluorescence quantification

by imaging using a macrofluo stereomicroscope (Leica), a robotized platform (Märzhäuser Wetzlar,

Wetzlar, Germany) and a high-resolution ORCA-AG camera (Hamamatsu Photonics, Hamamatsu City,

Japan). Using SimplePCI software (Hamamatsu Photonics), mean pixel intensity of each well was

quantified and mean pixel intensity of empty wells was subtracted from the wells on a plate-by-plate basis

for normalization of background fluorescence. GraphPad Prism version 5.04 (GraphPad Software, La

Jolla, CA, USA) was used for statistical analysis. In cases where the data in all groups followed a

b)a)

Gaussian distribution, a Students T-Test (pairwise comparison) or one-way ANOVA and Dunnett’s post-

test (comparing multiple groups) were carried out. In cases where the data of one or more groups did not

follow a Gaussian distribution, a Mann-Whitney test (pairwise comparison) or Kruskal-Wallis and

Dunn’s post-test (comparing multiple groups) were carried out. In unspiked mode, all groups were

compared to the solvent control group, in spiked mode all groups were compared to the T3 3.25 µg/L

control. A four point T3 dose response curve was modelled using the results obtained with the T3

concentrations and used to determine the hormonal equivalence of each sample. The concentration data

was log transformed, giving a linear concentration-response profile. The equation of the line describing

the concentration-response was determined using Microsoft Excel. This equation was then used to

calculate the T3 equivalents for the environmental samples.

SI, 2.3 Genotoxicity assays

The genotoxic activity of the wastewater samples were analyzed with two assays, the umuC test and the

p53-CALUX assay.

The umuC test was performed according to ISO standard 13829 (2000) with minor modifications. The

principle of the test is to determine the induction of the umuC-operon by analyzing the activity of ß-

galactosidase. In short, the genetically modified bacterium Salmonella typhimurium (strain

TA1535/pSK1002) were exposed to the sample extracts at four different concentrations (REF40, 20, 10

and 5) with and without metabolic activation (S9) using 96-well microtiter plates. Positive control

(aminoantracene (2-AA)) samples were prepared according to the guideline, except in the test with

metabolic activation, where aminoantracene (2-AA) was diluted 200-fold instead of 500-fold.

Furthermore, NADPH (tetrasodium salt) 16.88 mg in 5 mL 10 x TGA was used as a cofactor solution.

The plates were incubated for 4 hours at 37 ºC after which the bacterial growth was measured in plate

with iEMS Reader MF 1401 microplate reader (Labsystems, Finland). A sample was evaluated as

genotoxic is the induction factor was >1.5 while the growth factor was >0.5. If the growth factor is <0.5

the sample is considered cytotoxic and no evaluation of genototoxicity can be made.

The p53 and p53+S9 CALUX® bioassays were performed as described previously (van der Linden et al.,

2014). In short, cells were seeded into 96-well plates with DF medium that was supplemented with serum.

After 24 h of incubation for the p53 CALUX® and 48 h of incubation for the p53+S9 CALUX® (37 °C,

7.5%CO2), the medium was replaced by medium containing the water extracts (1 % v/v DMSO) for

agonistic activity testing (in triplicate). In case of the p53+S9 CALUX®, a S9 enzyme mixture was added

for 3 h to the cells and then the medium was removed with new medium. After 24 h of incubation, the

medium was then finally removed and the cells were lysed in 30 μL of Triton-lysis buffer. The amount of

luciferase activity was quantified using a 96 well plate luminometer reader (Mithras, Berthold

Technologies Gmbh, Germany). On all plates, a dose-response curve of the reference compound was

included for quantification of the response, which was actinomycin D or cyclophosphamide for the p53 or

p53+S9 CALUX, respectively. An induction factor was calculated for every point (Relative light unit

(RLU) concentration divided by RLU in wells with only DMSO) for the reference substance and the

samples. If the sample had an induction factor higher than 1.5, a dose-response fit was used to calculate at

which sample concentration the induction factor 1.5 is reached. The calculated 1.5 induction factor (IF) of

the sample together with the concentration of the reference substance (at IF 1.5) were applied to produce

the equivalence values. The dilution series consisted of a non-diluted sample and 10, 30 and 100 times

diluted sample.

SI, 2.4 Fish embryo toxicity assay

The FET test was conducted in the laboratory at UFZ (Leipzig, Germany). Wild type adult zebrafish

(Danio rerio, strain UFZ-OBI) were kept for several generations at the UFZ. The fish were bred in

several 14 L aquaria with 25 — 30 fish in each with a sex distribution of 1:2 (female to male). The light-

dark rhythm was 14:10 h and the water temperature was 26 ± 1 °C. Water parameters were measured

frequently (pH 7—8; water hardness 2—3 mmol/L, conductivity 540—560 µS/cm, Nitrat < 2,5 mg/L,

Nitrit <0,025 mg/L, Ammonia < 0.6 mg/L, oxygen saturation 87—91 %). The fish were fed twice a day

with Artemia salina. Within 30 min after spawning, eggs were collected using fertilization trays as

described in Nagel et al. (2002). Developmental stages were identified according to (Kimmel et al.,

1995).

Briefly, eggs were collected and washed twice with the exposure medium. Unfertilized eggs were

separated from fertilized eggs at the four- to eight- cell stage by microscopical observations. Only

fertilized eggs with no apparent disorders were used for testing. Exposure was started no later than 90

min post fertilization.

Embryos were exposed to effluent extracts from the selected WWTPs (except WWTP6) in three

concentrations (REF2.5, REF5, REF10,) and experiments were performed in 3 independent biological

replicates. For each replicate, 10 embryos were exposed to 10 mL of test medium (1 embryo / 1 mL

exposure medium). Test medium was prepared an hour before exposure with a maximum amount of 0.1

% (=10 µL) of extract. Negative control (ISO-Water with 0.1 % methanol) and positive control (3,4

dichloroaniline, 3.2 mg/L) were performed in parallel with each replicate. The exposure medium was not

renewed during exposure.

The exposure was conducted for 96 hours at a temperature of 26 +/-1 °C and a photoperiod of 12:12

light/dark. Mortality and malformations were checked every 24 hours. Toxicity and sub lethal effects in

embryos were detected according to the OECD TG 236 (SI, Table 1). The pH and conductivity in the

controls and in the highest test extract concentration were measured in the beginning of the test and at the

end of the test to make sure that there are no changes.

SI, Table 1. List of all the observations on toxicity that are recorded during the exposure in the FET test. The embryos are checked for these lethal, sub-lethal and teratogenic endpoints at 24h, 48, 72h and 96h.

Endpoint Test duration

24 h 48 h 72 and 96 hLethal coagulated embryos + + +

lack of somite formation + + +non-detachment of tail + + +lack of heartbeat + +

Sub-lethal no formation of eyes + + +no spontaneous movements +no blood circulation + +heartbeat frequency + +no pigmentation + +edema (yolk/pericardial) + +hatching rate + +hatching without movements + +

Teratogenic retardation (age) + + +malformation of head + + +malformation of saccule/otoliths

+ + +

malformation of tail + + +malformation of heart + + +malformation of tip of tail + + +modified structure of chorda + + +scoliosis + + +rachischisis + + +yolk deformation + + +length of tail + + +behavior (shivering, tremor) + + +

SI, Table 2. The cytotoxicity of influent and effluent samples from 7 WWTPs analyzed with NRR-assays. Results are given as viability (%) of the RTL-W1 cells at different sample concentrations. Standard deviation is given in parenthesis to show bioassay variability. The samples were analyzed as three replicates, except WWTP 6 MBR effluent sample which was only analyzed once due to limited sample volume (n.d. =no data).

Influent EffluentViability%

5x 7.5x 10x12.5

x 15x17.5

x 20x 5x 7.5x 10x12.5

x 15x17.5

x 20x >95%

WWTP1

116.0

(6.6)

109.2

(6.1)

105.2

(4.9)94.1(5.7)

86.2

(2.0)

68.9(3.4

)

50.3(14.

5)

99.3(1.1

)

94.0(1.4

)

95.1(1.9

)

95.6(2.9

)

89.4(2.0

)

89.9(2.6

)

91.4(4.0

)80%-95%

WWTP2

83.9(6.3

)73.6(4.7)

59.3(4.5)

43.4(1.7)

4.4(1.4)

2.4(1.4

)1.9

(0.7)

107.2

(2.5)

105.0

(0.0)

104.3

(3.8)

101.4

(4.7)

102.7

(6.2)

90.8(1.9

)

91.8(4.9

) 60-80%

WWTP3

110.9

(3.3)

102.7

(7.8)

96.4(14.

8)

82.1(21.

1)

60.8

(2.3)

56.3(0.4

)51.5(0.3)

105.7

(6.2)

105.9

(2.0)

104.7

(3.9)

106.2

(4.4)

98.7(0.6

)

97.2(2.3

)

91.2(5.4

) 30-60%

WWTP4

97.0(4.3

)

91.4(10.

2)

81.2(15.

8)

67.8(21.

1)

36.0

(4.8)

29.9(1.5

)25.3(1.1)

102.0

(2.6)

100.3

(2.5)

99.9(3.7

)

94.5(0.2

)

90.6(0.7

)

85.2(1.7

)

81.4(2.7

) <30%

WWTP5

97.8(2.9

)80.4(8.1)

59.2(6.8)

17.3(17.

5)

1.9(0.4)

1.9(0.2

)2.0

(0.2)

98.3(5.3

)

104.1

(1.0)

102.9

(4.6)

98.2(7.3

)

99.0(4.9

)

99.3(5.2

)

95.9(4.7

)

WWTP6 MBR

65.4(1.1

)7.4

(0.3)0.8

(0.1)0.8

(0.0)

0.8(0.1)

0.8(0.1

)0.8

(0.1)

52.8(n.d.

)

32.1(n.d.

)

0.7(n.d.

)

0.8(n.d.

)

0.8(n.d.

)

0.9(n.d.

)

0.8(n.d

.)

WWTP6

65.4(1.1

)7.4

(0.3)0.8

(0.1)

0.8(0.0

0)

0.8(0.1)

0.8(0.1

)0.8

(0.1)

104.5

(2.2)

104.1

(3.3)

104.4

(2.3)

104.0

(6.3)

100.9

(5.5)

99.8(5.6

)

97.6(3.2

)

WWTP7

109.3

(8.2)

96.0(3.3)

80.2(3.5)

69.5(5.3)

69.9

(2.3)

67.4(4.6

)

57.0(10.

1)

104.3

(5.9)

96.5(7.9

)

87.3(2.3

)

76.1(3.7

)

72.9(3.3

)

69.9(3.3

)

62.0(6.5

)

WWTP1 WWTP2 WWTP3 WWTP4 WWTP5 WWTP6 WWTP70

10

20

30

40

50

60

70

80

ng D

HT

eq.

/L

SI, Figure 2. Androgenic activity of influent samples from seven WWTPs analyzed with AR-CALUX. Results are calculated as dihydrotestosterone (DHT) equivalents (ng/L). Results for WWTP3 and 7 were below the limit of detection (0.62 ng DHT eq./L). The error bars represent standard deviation of bioassay replicates.

WW

TP1

WW

TP2

WW

TP3

WW

TP4

WW

TP5

WW

TP6

WW

TP702468

1012141618

EE2

equi

vale

nce

(ng/

L)

SI, Figure 3. Estrogenic activity of effluent samples (REF1) analyzed with the transgenic line of medaka embryos tested in the unspiked mode. Hormonal (EE2) equivalents were calculated based on the EE2 standard curve performed in the assay (0 – 488 ng/L). The estrogenic activity in WWTP1 was below the limit of detection. The error bars represent 95 % confidence interval.

WWTP1

WWTP2

WWTP3

WWTP4

WWTP5

WWTP6

WWTP70

1000

2000

3000

4000

5000

6000

7000

InfluentEffluent MBR

ug c

yclo

phos

pham

ide

eq./L

SI, Figure 4. The genotoxic potency of influent samples collected from seven WWTPs and one MBR effluent sample analyzed with the p53-CALUX® + S9 assay. The genotoxic activity was below the LOD (53 µg cyclophosphamide eq./L) in WWTP3 influent sample and all of the effluent samples. The error bars represent standard deviation of bioassay replicates.

SI, Table 3. Genotoxicity of influent and effluent samples analyzed with the umuC-assay with and without metabolic activation by S9. The results are given as induction ratios (I), if I > 1.5 the sample is considered genotoxic. The sample is cytotoxic if the growth factor of the bacteria is < 0.5. The standard deviation is given in parenthesis to show bioassay variability.

SI, Figure 5. Sublethal effects observed during the exposure to wastewater extracts in % of the survived embryos in samples from different WWTPs.

SI, Table 4. Score bands for the bioassays used in figure 6 a and b. For the umuC assay a score of 6 or 7 depending on the induction factor (higher induction factor gets a 7) was given if effects were observed in REF40 and REF20. Following the same criteria regarding the induction factor, a score of 3 or 4 was given if effect were observed only in REF40. A score of 1 was given if in all cases no effects were observed.

Score

Cytotoxicity Genotoxicity (p53-CALUX)

Estrogenicity (ER-CALUX)

Androgenicity (AR-CALUX)

Estrogenicity (medaka)

% viability at REF12.5

µg cyclophosphamide eq./L ng E2 eq./L ng DHT eq./L ng EE2 eq./L

1 > 90 % <LOD <LOD <LOD <LOD2 80-90% <50 >LOD-1 >LOD-5 >LOD-53 60-80% 50-300 1-5 5-10 5-104 40-60% 300-500 5-15 10-15 10-155 20-40% 500-600 15-30 15-20 15-206 10-20% 600-1000 30-40 20-30 20-307 <10% >1000 >40 >30 >30

  Influent EffluentInduction ratio, I Test with S9 Test without S9 Test with S9 Test without S9Sample concentration (REF) 40 20 10 5 40 20 10 5 40 20 10 5 40 20 10

WWTP11.59

(0.07)1.32

(0.01)1.21

(0.01)1.11

(0.02)1.56

(0.07)1.33

(0.03)1.22

(0.03)1.14

(0.03)1.47

(0.06)1.29

(0.07)1.17

(0.02)1.08

(0.01)1.97

(0.06)1.60

(0.02)1.42

(0.07)1.29

(0.04)

WWTP21.52

(0.17)1.29

(0.03)1.20

(0.05)1.17

(0.05)1.97

(0.06)1.52

(0.04)1.23

(0.02)1.13

(0.01)1.43

(0.05)1.33

(0.02)1.19

(0.02)1.09

(0.02)1.98

(0.06)1.44

(0.03)1.28

(0.12)1.16

(0.12)

WWTP31.49

(0.06)1.28

(0.05)1.17

(0.02)1.09

(0.09)1.58

(0.04)1.35

(0.03)1.22

(0.03)1.10

(0.03)1.34

(0.03)1.23

(0.08)1.17

(0.01)1.06

(0.00)1.82

(0.04)1.45

(0.04)1.28

(0.13)1.11

(0.02)

WWTP41.78

(0.39)1.39

(0.06)1.24

(0.03)1.14

(0.05)2.98

(0.12)1.82

(0.07)1.37

(0.02)1.16

(0.04)1.56

(0.07)1.22

(0.18)1.17

(0.05)1.06

(0.06)1.99

(0.08)1.60

(0.05)1.35

(0.04)1.23

(0.05)

WWTP51.45

(0.19)1.19

(0.03)1.16

(0.07)1.09

(0.14)2.14

(0.31)1.47

(0.03)1.18

(0.06)1.24

(0.02)1.32

(0.02)1.16

(0.17)0.97

(0.01)0.99

(0.04)1.94

(0.11)1.48

(0.02)1.27

(0.09)1.24

(0.00)

WWTP61.22

(0.03)1.16

(0.02)1.05

(0.03)1.01

(0.04)1.09

(0.01)1.09

(0.01)1.12

(0.01)1.08

(0.02)1.15

(0.03)1.09

(0.03)1.01

(0.01)1.02

(0.05)1.05

(0.05)0.99

(0.05)1.02

(0.03)0.98

(0.09)

WWTP6 MBR1.22

(0.03)1.16

(0.02)1.05

(0.03)1.01

(0.04)1.09

(0.01)1.09

(0.01)1.12

(0.01)1.08

(0.02)1.20

(0.03)1.11

(0.03)1.03

(0.03)1.00

(0.05)1.29

(0.07)1.17

(0.10)0.95

(0.07)0.99

(0.11)

WWTP71.63

(0.10)1.38

(0.05)1.06

(0.02)1.10

(0.05)1.89

(0.18)1.40

(0.06)1.31

(0.09)1.09

(0.01)1.31

(0.06)1.08

(0.12)0.97

(0.04)0.97

(0.03)1.68

(0.11)1.45

(0.01)1.17

(0.06)1.06

(0.08)

SI, 3. References

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