3. CONTENTS
3.1. Introduction
Similarly to what happened last year, the current coronavirus pandemic precluded the collectionof the target samples, as the hospital services were mostly entirely dedicated to treat Covid-19
patients and non-urgent consultations were postponed and/or were reduced to a minimum. For
this reason, the collection of urine samples from early menopause women could not beperformed. As a contingency plan, previous urine samples already collected from volunteers
from Aveiro district were analyzed and data computed in order to understand any possiblechanges in the use of consumer products with endocrine disruptors. Particular attention wasdrawn to Bisphenol A (BPA) and substitutes. BPA is generally used in manufacture ofpolycarbonate (PC) plastics, epoxy resins and other polymeric materials, as well as paper
products (thermal receipts, e.g.) and other consumer products including dental sealants. Due totheir widespread use, humans are continuously exposed and given the endocrine disrupting andimmunotoxic potential of this compound and the public awareness of its toxicity, BPA regulations
have been implemented over the last decade and as a result, other bisphenols have been
increasingly used1. In January 2011, the European Commission prohibited the use of BPA in themanufacture of polycarbonate infant feeding bottles. In February 2018, the EU introduced
stricter limits on BPA in food contact materials. Currently (as of February 2022), in the EuropeanUnion (EU), BPA is still permitted for use in food contact materials under Regulation 10/2011/EU,
relating to plastic materials and articles intending to come into contact with foodstuff, except
some countries like France that have already completely banned the use of BPA in food packing,containers and utensils. In January 2020, EU-wide ban has entered into force on the use of BPA
in thermal paper (in a concentration equal to or greater than 0.02% by weight). Recently (21stDecember 2021), the Court of Justice in the EU confirmed that BPA must be listed as a “substanceof very high concern” for its hormone disrupting properties in humans2. Given the molting
evidences on BPA toxicity, the European Food Safety Agency (EFSA) proposed in December 2021
new safety standards for BPA, radically decreasing the recommended exposure limit by a factor2

of 100,000, to a recommended value of 0.04 ng per kg of body weight per day, this proposal was
until 22th February under public consultation. This new standard is so low that if approved, it willprobably ban the use of BPA in any products that come into contact with food. Therefore a drastic
decrease in BPA levels are expected in the future.
Hence, the aim of this report is to describe the levels of phenolic compounds, namely triclosan,
triclocarban, parabens, benzophenones and bisphenols in urine samples from volunteers living
is Aveiro district, to describe exposure levels and to understand any potential shift in theexposure to EDCs, particularly bisphenols.
3.2. Procedure
3.2.1. SamplingThis study was approved by the Ethics Committee of Centro Hospitalar do Baixo Vouga and by
the Portuguese National Data Protection Agency. Samples from volunteers inhabiting Aveiro
district were collected between 2017 and 2019. Three different groups were selected: men(n=24), women (n=20) and pregnant women (n=28). Urine samples were collected directly into
sterile containers and several aliquots of 1.5ml were prepared and immediately frozen. Twoaliquots of each sample were transported to CMES in cool conditions and preserved at -20oC
until analysis.
3.2.2. Chemical analysis
At CMES, levels of triclosan, triclocarban, parabens (methyl, ethyl, propyl, butyl), benzophenones
2-OH-4-MeO-BP (BP-3); 2,4-diOH-BP (BP-1); 2,2'-diOH-4-MeO-BP (BP-8); 2,2',4,4'-tetraOH-BP(BP-2); 4-OH-BP) and bisphenols (BPA, BPB, BPC, BPF, BPS, BPZ, BPAF, TBBPA) were quantified inurine samples. The target compounds were analyzed following the protocol described by Kunisue,et al. 3 after some modifications and optimization by the team members (see LaMer 2017/18
report). Briefly, 50 µL of Internal Standards (ISs) Mixture was added to each urine sample after
being hydrolised with β-glucuronidase/aryl-sulfatase for 16h at 370C. Afterwards, cold methanol,ultrapure water and ammonia solution (5% NH4OH) were added. The samples were then loaded
into a pre-conditioned OASIS MAX cartridge (MTBE, Methanol and ultrapure water). Prior toelution with formic acid:MTBE:methanol = 0.2: 3: 7 (v/v/v), the cartridges were washed with 5%
NH4OH, 5% NH4OH in methanol, Milli-Q-water: methanol = 0.2: 6: 4 (v/v/v) and afterwards
completely dried for 15 min. The eluted target compounds were evaporated to dryness under
nitrogen flux and re-dissolved with acetonitrile and 50 µL of mixture of naproxen-13C,d3 andketoprofen-13C,d3 (20 ng/mL each) was added. The samples were preserved in amber LC glassvials at 40C and before injection into the LC-MS/MS, Milli-Q water was added to a final volumeof 1mL. The internal standard (ISs) mixture contains 100 ng/mL of Triclosan-13C6, 20 ng/mL of3

Triclocarban-13C6, 20 ng/mL of Methyl
paraben-13C6, 20 ng/mL of Butyl paraben13

C6, 20 ng/mL of BP-3-d5, 100 ng/mL ofBisphenol A-d16, 100 ng/mL of Bisphenol F13

C6, 20 ng/mL of Bisphenol S-13C12, 20ng/mL of Bisphenol AF-d4 and 20 ng/mL of
Tetrabromobisphenol A-13C12. The activityof β-glucuronidase/aryl-sulfatase was 290
units per mL of urine. The solution wasprepared twice a week by adding 4.7 ml of
1.0 mol/L ammonium acetate; 5.3 mL of 1.0mol/L acetic acid and 50 µL of βglucuronidase/aryl-sulfatasesolution(116,000 units/mL). The crude mixture of βglucuronidase/sulfatase from Helix pomatia(Type HP-2, aqueous solution, 116,000units/mL glucuronidase and 1020 units/mLsulfatase) was purchased from SigmaAldrich (St. Louis, MO, USA); (G7017). The
method detection limit was 0.147 ng/mL for
Triclosan, 0.073 ng/mL for Triclocarban,
1.101 ng/mL for Methyl paraben, 0.036
ng/mL for Ethyl paraben, 0.64 ng/mL for
Propyl paraben, 0.015 ng/mL for Butylparaben, 0.015 ng/mL for BP-1, 0.025

Figure 1 - Detection frequency (%) of each EDC in the urine samples frommen (n=24, blue bars), women (n=20, green bars) and pregnant women(n=28, red bars).

ng/mL for BP-2, 0.471 ng/mL for BP-3, 0.046 ng/mL for BP-8, 0.223 ng/mL for 4-OH-BP, 2.2
ng/mL for Bisphenol A, 0.076 ng/mL for Bisphenol AF, 0.325ng/mL for Bisphenol B, 2.175 ng/mLfor Bisphenol C, 0.910 ng/mL for Bisphenol F, 0.199 ng/mL for Bisphenol S, 0.438 ng/mL forBisphenol S, and 0.386 ng/mL for TTBBPA.
3.3. Results
Of the 19 compounds analyzed, only triclocarban, namely BPAF, BPB, BPC, BPZ and TBBPA were
not detected in all groups analyzed. BP-2 was not detected in men and pregnant women, and all
the remaining EDCs were detected in at least one individual of each group (Figure 1). Ethyl
paraben was the only EDC detected in all the all samples analyzed for all groups. The EDCs withthe highest detection frequencies in men were Ethyl paraben (100%), Benzophenone-1 (96%),4

Bisphenol F (92%) and Methyl paraben (83%). In women, the EDCs with the highest detection
frequencies were Ethyl paraben (100%), BPF (100%), Methyl paraben (100%) andBenzophenone-1 (95%). In pregnant women, the EDCs with the highest detection frequencies
were Ethyl paraben (100%), Benzophenone-1 (100%), BPF (96%), Methyl paraben (93%),
Triclosan (82%), and Benzophenone-3 (82%).
The profiles for each phenolic compounds detected in urine and dust samples are described inFigures 2-4. Overall, in urine samples the highest average concentration detected was for Methyl
paraben in women and pregnant women (Figure 2), followed by benzophenone-3 in pregnantwomen (Figure 3).

[Parabens] ng/g
4000
3500
3000
2500
2000
1500
1000
100
80
604020reg)

(♀
))

(PPBu

BuP(♂
PBuPrP(Preg)

(♀
)

)

rP(♂
PP)
reg(PPEtPr(♀
))

tPEg)

(♂
PEt

(PreePePMMePM(♂

(♀
))

0

Figure 2 - Scatter plot summarizing the values of parabens in the analyzed urine samples in men (n=24,blue bars), women (n=20, green bars) and pregnant women (n=28, red bars).

Regarding bisphenols (Figure 4), generally men displayed the lowest levels. Interestingly, in
pregnant women the levels of BPA were lower than the levels of BPF and BPS, two widely usedalternatives for BPA. This tendency is probably associated with the concern of women to avoid
BPA products containing BPA.

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[UV Filters] ng/g400035003000
2500
2000
1500
1000
100
80
60
40
20
04-O

HP-B)
(♂
4-O

HP-B)
(♀

HO4--BPre(Pg)
BP-1)
(♂
BP-1)
(♀
-1BPg)
re(PBP-2)
(♂
BP-2)
(♀
-2BPg)
re(PBP-3)
(♂
BP-3)
(♀
-3BPg)
re(PBP-8)
(♂
BP-8)
(♀
-8BPg)
re(PFigure 3 - Scatter plot summarizing the values of UV-filters in the analyzed urine samples in men(n=24, blue bars), women (n=20, green bars) and pregnant women (n=28, red bars).
[Bisphenols] ng/mL
4035302520151050BPA)))))))))))))))))))))))(♂ (♀ reg F (♂ F (♀ reg( ♀ reg F(♂ F(♀ reg( ♂ ( ♀ reg( ♂ ( ♀ reg Z (♂ Z (♀ reg A(♂ A(♀ reg
B
B P
S
S P
CC PP
PPPP
A
A
PPP
PP
P
P
PPP
P
P
((((((((PP
B
B
B
B
B
B
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B
B
B
B
B
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F
Z
A
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C
B
B
AF
T B T B BPBP
BP
BP
BPBP
BPBPTB)(♂

PAFigure 4 - Scatter plot summarizing the values of bisphenols in the analyzed urine samples inmen (n=24, blue bars), women (n=20, green bars) and pregnant women (n=28, red bars).

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3.5. Future perspectives
Due to the Covid pandemic the computation of clinical data (that was performed by a Medicaldoctor) was delayed and for this reason the multivariate statistical analysis that was scheduled
to be finish in 2021 are still ongoing. The research papers projected in the previous report to bepublished in 2021 will only be published in 2022. Nevertheless, we have published a review paper,an encyclopedia entry and submitted another review paper (see achievements section).
Furthermore, the manuscript entitled “The RESPIRA Project: Unraveling the environmental
determinants of high burden respiratory diseases in Estarreja Region” is going to be submitted
in the beginning of March (see Achievements section) and another paper entitled “Riskperception and attitudes of pregnant women regarding exposure to endocrine disrupting
chemicals: a narrative review” is currently under preparation. All these manuscripts willacknowledge LaMer funding.
We expect to continue sampling and to obtain a representative number of samples so that incollaboration with CMES we can conclude the study on the effects of exposure to phenolic
endocrine disruptors and human reproductive health. Besides female reproductive health, a new
study will be implemented focusing on male reproductive health.
Furthermore, based on the results here described, and given the new regulation on BPA that will
likely enter into force in 2022 or 2023, we also aim to perform a new study to evaluate thetemporal trends in bisphenols levels in the same individuals.

3.6. Achievements
Oral Communications by Invitation
 Sousa ACA, Pastorinho MR, Coelho SD, Isobe T, Tanoue R, Kunisue T, Tanabe S (2021)
Hormones messed up - a tale of endocrine disruptors in the XXI century (Invited lecture).
XXI SPB - National Congress of Biochemistry: Tuning Biochemistry with Life Sciences andSociety. 14-16 November 2021, Évora, Portugal, p. 36
Publications
 Sousa ACA, Souto-Miranda S, Valente C, Tanoue R, Kunisue T, Tanabe S, Taborda-BarataL, Pastorinho MR and the RESPIRA team (under preparation) The RESPIRA Project:
Unraveling the environmental determinants of high burden respiratory diseases inEstarreja Region. To be submitted by March 10th
 Rato L, Sousa ACA (2021) The impact of endocrine-disrupting chemicals in malefertility: focus on the action of obesogens. Journal of Xenobiotics 11, 163–196; Feature
Paper https://doi.org/10.3390/jox11040012
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 Sousa ACA and Rato L (2022) The Impact of Endocrine-Disrupting Chemicals in MaleFertility. Scholarly Community Encyclopedia. https://encyclopedia.pub/20789
Editorial activities
 Special Issue: “Monitoring of Organic Contaminants”, Processes, Guest Associate Editor
 Special issue: “Obesogens in the XXI century: emerging health challenges”, Frontiers inEndocrinology, Guest Associate EditorSpecial Issue: “Environmental Exposures and Epidemiological Studies on Maternal andChild Health”, International Journal of Environmental Research and Public Health, Guest
Associate Editor

Outreach activities
Sousa ACA (2021) Imposex and Obesity: the story of an environmental contaminant.
Lecture for the general public under the framework of the 6th Coffee with Scienceprogram. Centro de Ciência Viva de Lagos. 2 July 2021. Online, available at:https://www.facebook.com/watch/?extid=WA-UNK-UNK-UNK-IOS_GK0TGK1C&v=262701795803074 (LaMer ackowlegment min 53:40)

Sousa ACA (2021) Environmental Contaminants and endocrine disruptors in the global
obesity epidemic. Ciclo de Palestras do CEBE, 22th May 2021, online

References:
1. Chen D, Kannan K, Tan H, et al. Bisphenol Analogues Other Than BPA: Environmental
Occurrence, Human Exposure, and Toxicity—A Review. Environ Sci Technol 2016;50(11):543853.
2. General Court of the European Union. Confirmation of the inclusion of Bisphenol A as asubstance of very high concern on account of its properties as a substance toxic forreproduction. In: General Court of the European Union, ed. T-185/17. Luxembourg, 2021.
3. Kunisue T, Wu Q, Tanabe S, et al. Analysis of five benzophenone-type UV filters in human urine
by liquid chromatography-tandem mass spectrometry. Analytical Methods 2010;2(6):707-13.
doi: 10.1039/B9AY00324J

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