4. Research report
Due to delay in the approval of the study “ARTEMIS: Assessing Risk from exposure
To Environmental contaminants in Portuguese Mother-Infant pairS”, it was notpossible to collect the urine samples that were supposed to be analyzed during thisperiod at CMES. As a contingency plan, urine samples from pets, that are consideredgood sentinels for human exposure to indoor contaminants were studied.
4.1. Title of research project
Levels of organohalogen and per- and polyfluoroalkyl contaminants in cats’ and dogs’
serum from Portugal
4.2. Project members
Ana Catarina Sousa, Department of Biology and Comprehensive Health ResearchCentre (CHRC), University of Évora, Évora, Portugal
M. Ramiro Pastorinho, Department of Medical Sciences and Comprehensive HealthResearch Centre (CHRC), University of Évora, Évora, Portugal
Hugo Vilhena, Department of Veterinary Medicine, Vasco da Gama Universitary
School, Coimbra, Portugal
Aika Sato, Center for Marine Environmental Studies (CMES), Ehime University,Matsuyama, Japan
Hazuki Mizukawa, Department of Science and Technology for Biological Resourcesand Environment, Graduate School of Agriculture, Ehime University, Matsuyama,Japan
Rumi Tanoue, Center for Marine Environmental Studies (CMES), Ehime University,Matsuyama, JapanKey Nomiyama, Center for Marine Environmental Studies (CMES), Ehime University,Matsuyama, Japan
Tatsuya Kunisue, Center for Marine Environmental Studies (CMES), Ehime University,Matsuyama, Japan
4.3. Purposes
To access, for the first time in Portugal, the exposure of pet cats and dogs toorganohalogen compounds and per- and polyfluoroalkyl substances (PFAS).
4.4. Methods
Sampling
This study was developed under the umbrella of the PETTO Project, approved by thePortuguese Veterinary Authority (Direção Geral de Agricultura e Veterinária-DGAV
0421/000/2013). Blood samples were collected from the jugular or cephalic veins ofpets’ cats (n=12) and dogs (n=8) by a certified veterinarian at the veterinary hospital
after the owner signed an informed consent form. Samples were immediately
centrifugated and serum was collected. Whenever possible two aliquots of 1.5ml wereprepared and immediately frozen. Serum samples were transported to CMES in cool
conditions and preserved at -20oC until analysis.
3/6Chemical analysis
Levels of organohalogen compounds including PCBs and hydroxylated metabolites(OH-PCBs); PBDEs and metabolites (OH-PBDEs, MeO-PBDEs), and BPhs wereanalyzed in 12 pet cats’ samples and 8 pet dogs’ samples following the protocol fully
described in Nomiyama, Yamamoto et al. (2022).
Levels of per- and polyfluoroalkyl substances (PFAS), including PFOS, PFHxS,PFDoA, PFUdA, PFDA, PFNA, PFOA, PFHpA and PFBA were analyzed following theprotocol recently implemented at CMES and not yet published. Briefly, for PFAS
analysis, 100 µL of serum was treated with acetonitrile containing 5% formic acid toinduce protein precipitation. The resulting supernatant was subjected to solid phaseextraction using an Oasis WAX cartridge, followed by concentration under a nitrogenstream. Qualitative and quantitative analysis of the 34 PFASs was then performed
using a liquid chromatograph-quadrupole time-of-flight mass spectrometer (LCQToF/MS; SCIEX X500R) (Sato et al., 2023, Joint Conference on EnvironmentalChemicals).
4.5. Results
Organohalogen compounds
Levels of PCBs, OH-PCBs, PBDEs, OH-PBDEs, MeO-PBDEs, and BPhs in both cat
and dog serum samples are described in Figure 1. Significantly higher concentrationsof BPhs (mean: 1700 pg.g−1) were found in cat serum, followed by PBDEs (430 pg.g−1),
PCBs (median: 380 pg.g−1), OH-PCBs (median: 280 pg.g−1), and OH-PBDEs (median:99 pg.g−1). The lowest levels were registered for MeO-PBDEs (median: 2.4 pg.g−1).
A different profile was observed for dogs’ serum samples, with significantly higher
concentrations of OH-PCBs (median: 530 pg.g−1), followed by PCBs (130 pg.g−1),
PBDEs (median: 87 pg.g−1), BPhs (41 pg.g−1) and MeO-PBDEs (<1.0 pg.g−1). Suchresults underline that dogs have a stronger capacity to metabolize PCBs than cats andsuggests that the low exposure to naturally occurring halogenated compounds might
be a consequence of lower exposures from pet food.

Figure 1. Median (min-max) concentrations (pg g−1 wet wt.) of organohalogen
contaminants in cat (n=12) and dog (n=8) serum samples.

Per- and polyfluoroalkyl substances (PFAS)
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For PFAS only cat serum samples were analyzed (n= 11). The main contaminants are
PFOS and PFOA (Figure 2), yet long-chain PFCAs (C9~C12) have also been detectedwhich suggests that cats may be less likely to excrete long-chain PFCAs. Overall, thePFAS levels are relatively low (Figure 3) with ΣPFAS varying between 1.4 and 4.5
ng.mL-1 (median: 2.6 ng.mL-1).

Figure 2. Profiles of per- and polyfluoroalkyl substances analyzed in the 11 cat serum
samples.

Figure 3. Concentrations (ng.mL−1) of per- and polyfluoroalkyl substances in cat
serum samples (n=11).
4.6. Future challenges
Due to time constraints, it was not possible to analyze PFAS in dog serum samples,
however we intend to analyze these compounds in the future so that a manuscript
comparing the levels of organohalogen compounds and per- and polyfluoroalkyl
substances in both species can be prepared and submitted to an international journal(IF>3).
4.7. Achievements
The following papers were submitted and are currently under review (both with LaMer
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acknowledgments)
Coelho SD, Maricoto t, Taborda-Barata L, Annesi-Maesano I, Isobe T, Sousa ACA(under review, 2nd round) Relationship between flame retardants and respiratory
health– A Systematic review and meta-analysis of observational studies.
Environmental Pollution, Journal IF 2023: 8.9
Mascarenhas C, Sousa ACA, Rato L (under review, 2nd round) Effects ofpharmaceutical substances with obesogenic activity on male reproductive health.
International Journal of Molecular Sciences, Special Issue Endocrine Disruptors
Exposure and Human Health. Preprints
https://doi.org/10.20944/preprints202401.0324.v1, Journal IF 2023: 6.208

References
Nomiyama, K., Y. Yamamoto, A. Eguchi, H. Nishikawa, H. Mizukawa, N. Yokoyama, O.
Ichii, M. Takiguchi, S. M. M. Nakayama, Y. Ikenaka and M. Ishizuka (2022). "Healthimpact assessment of pet cats caused by organohalogen contaminants by serum
metabolomics and thyroid hormone analysis." Science of The Total Environment:
156490.
佐藤愛佳, 佐藤楓夏, 水川葉月, 池中良徳, 田上瑠美, 横山望, 滝口満喜, 中津賞, 野見山
桂 (2023) コンパニオンアニマルの PFAS 汚染：イヌ・ネコの種差要因と汚染の地域
差. 環境化学物質 3 学会合同大会（徳島；2023 年）

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