In future study, extra treatment groups involving plastics additives alone such as DEHP and BPA should be included to compare with the effects of PVC MPs.
The sample size used in the study should be increased to generate more meaningful data and avoid the loss of sample due to oral gavage complications.
Duration of the MPs treatment should be lengthened to study the chronic effects of MPs in murine models. Moreover, different types, sizes, density, and
61 concentrations of MPs can be used to study the potential adverse health effects of caused different MPs and the plasticizers added. Furthermore, histopathological studies and detection of proinflammatory cytokines in liver tissues should be carried out to determine the infiltration of MPs and inflammatory cells. Another treatment group of mice without oral gavage administration can be included to compare the effects of stress and oral gavage complications in mice. In addition, proteomic study involving Western Blot analysis should be caried out to determine the abundance of corresponding protein. Last but not least, molecular analysis can be included in the study to determine the epigenetic effects of MPs in the targeted genes.
62 CHAPTER 6
CONCLUSION
In this study, no significant difference was observed in the body weight changes of all mice among five treatment groups throughout the 28-day repeated single dose oral gavage administration of PVC MPs. Liver index of the mice in PVC MPs treated groups showed increment as compared to control. Liver transcriptome results showed a dose-dependent relationship in the gene expression profile in which the effect was greater in the high dosage group followed by medium and low dosage group. Moreover, the mRNA expression of BCL-2 was down-regulated, and the mRNA expression of caspase-3 was up- regulated in mice liver after exposure to PVC MPs via ingestion. This implies that PVC MPs could induce mitochondrial disruption and influence the mitochondrial apoptotic pathway in mice liver. PVC MPs exposure led to alterations of both BCL-2 and caspase-3 gene expression. These results showed the potential risk of PVC MPs in promoting cancer formation. The results obtained in this study could be served as an indicator of potential adverse health effects caused by PVC MPs exposure to human due to the similarity in genes of mice and human. However, further studies are required due to the limited sample size and human errors in this study. The results of this study are useful to serve as preliminary data which can enhance the understanding on the impacts of PVC MPs exposure through ingestion in murine models, specifically the liver transcriptome results of BCL-2 and caspase-3.
63 REFERENCES
Agency for Toxic Substances and Disease Registry, 2016. Toxicological profile for vinyl chloride [Online]. Available at:
https://wwwn.cdc.gov/TSP/ToxProfiles/ToxProfiles.aspx?id=282&tid=51 [Accessed: 5 January 2022].
Alfaro-Núñez, A. et al., 2021. Microplastic pollution in seawater and marine organisms across the Tropical Eastern Pacific and Galápagos. Scientific Reports, 11(1), pp.1–8.
Andrady, A.L. and Neal, M.A., 2009. Applications and societal benefits of plastics. Philosophical Transactions of the Royal Society B: Biological Sciences, 364(1526), pp.1977–1984.
Ašmonaitė, G. et al., 2020. Microplastics as a Vector for Exposure to Hydrophobic Organic Chemicals in Fish: A Comparison of Two Polymers and Silica Particles Spiked With Three Model Compounds. Frontiers in
Environmental Science, [e-journal] 8.
https://doi.org/10.3389/fenvs.2020.00087
Barboza, L.G.A. et al., 2018. Marine microplastic debris: An emerging issue for food security, food safety and human health. Marine Pollution Bulletin, 133, pp.336–348.
Bes-Rastrollo, M. et al., 2005. Olive oil consumption and weight change: The SUN prospective cohort study. Lipids, 41, pp. 249-256.
Business Wire, 2018. Global Polyvinyl Chloride (PVC) Market Size, Demand Forecasts, Industry Trends and Updates (2018-2025) [Online]. Available at:
https://www.businesswire.com/news/home/20181024005331/en/Global- Polyvinyl-Chloride-PVC-Market-Size-Demand-Forecasts-Industry-Trends- and-Updates-2018-2025---ResearchAndMarkets.com [Accessed: 5 January 2022].
Campanale, C. et al., 2020. A detailed review study on potential effects of microplastics and additives of concern on human health. International Journal of Environmental Research and Public Health, 17(4), p. 1212.
64 Carpinetti, P. et al., 2021. Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues. PLoS ONE, [e-journal] 16(7).
https://doi.org/10.1371/journal.pone.0255245
Chemical Safety Facts, 2022. Polyvinyl Chloride [Online]. Available at:
https://www.chemicalsafetyfacts.org/polyvinyl-chloride/ [Accessed: 6 January 2022].
Cole, M., Lindeque, P., Halsband, C. and Galloway, T.S., 2011. Microplastics as contaminants in the marine environment: A review. Marine Pollution Bulletin, 62(12), pp.2588–2597.
Conserved Domain Database, 2018a. Apoptosis regulator. [Online] Available at:https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=
10&seltype=2&uid=TIGR00865&&aln=1,0,149,39 [Accessed: 20 October 2021].
Conserved Domain Database, 2018b. Caspase. [Online] Available at:
https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=10
&seltype=2&uid=smart00115&&aln=1,0,151,51 [Accessed: 20 October 2021].
Cox, K.D. et al., 2019. Human Consumption of Microplastics. Environmental Science and Technology, 53(12), pp.7068–7074.
de Sá, L.C. et al., 2018. Studies of the effects of microplastics on aquatic organisms: What do we know and where should we focus our efforts in the future? Science of the Total Environment, 645, pp.1029–1039
Deng, Y., Zhang, Y., Lemos, B. and Ren, H., 2017. Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Scientific Reports, [e-journal] 7. https://doi.org/10.1038/srep46687
DeNovix, 2019. Purity ratios explained [online]. Available at:
https://www.denovix.com/tn-130-purity-ratios-explained/ [Accessed 6 January 2022].
65 Ding, T. et al., 2021. Microplastics altered contaminant behavior and toxicity in natural waters. Journal of Hazardous Materials, 425, p.127908.
Dong, C. et al., 2020. Polystyrene microplastic particles: In vitro pulmonary toxicity assessment. Journal of Hazardous Materials, 385, p.121575.
Ejaredar, M., Nyanza, E.C., ten Eycke, K. and Dewey, D., 2015. Phthalate exposure and childrens neurodevelopment: A systematic review.
Environmental Research, 142, pp.51–60.
El-Mihi, K.A. et al., 2017. Naringin attenuates thioacetamide-induced liver fibrosis in rats through modulation of the PI3K/Akt pathway. Life Sciences, 187, pp.50–57.
Elmore, S., 2007. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol, 35(4), pp.495-516.
Enyoh, C.E. et al., 2020. Microplastics exposure routes and toxicity studies to ecosystems: An overview. Environmental Health and Toxicology, [e-journal]
35(1). https://doi.org/10.5620/eaht.e2020004
European Chemicals Agency, 2021. Chloroethylene [Online]. Available at:
https://echa.europa.eu/brief-profile/-/briefprofile/100.000.756 [Accessed: 5 January 2022].
Eweda, S., Newairy, A., Abdou, H. and Gaber, A., 2019. Bisphenol A-induced oxidative damage in the hepatic and cardiac tissues of rats: The modulatory role of sesame lignans. Experimental and Therapeutic Medicine, 19(1), pp.33-44.
Fu, G. et al., 2020. The role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat.
Toxicology and Applied Pharmacology, 404, p.115151.
Gao, H.T. et al., 2017. Effects of six priority controlled phthalate esters with long-term low-dose integrated exposure on male reproductive toxicity in rats.
Food and Chemical Toxicology, 101, pp.94–104.
66 Geiser, M. et al., 2005. Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environmental Health Perspectives, 113(11), pp.1555–1560.
GeneCards Database, 2016a. BCL-2 gene. [Online] Available at:
https://www.genecards.org/cgi-bin/carddisp.pl?gene=BCL2 [Accessed: 20 October 2021].
GeneCards Database, 2016b. CASP3 gene. [Online] Available at:
https://www.genecards.org/cgi-bin/carddisp.pl?gene=CASP3 [Accessed: 20 October 2021].
Ghatei, N. et al., 2017. Evaluation of bax, bcl-2, p21 and p53 genes expression variations on cerebellum of BALB/c mice before and after birth under mobile phone radiation exposure. Iranian Journal of Basic Medical Sciences, 20(9), pp.
1037–1043.
Hahladakis, J.N. et al., 2018. An overview of chemical additives present in plastics: Migration, release, fate and environmental impact during their use, disposal and recycling. Journal of Hazardous Materials, 344, pp.179–199.
Hasan Anik, A. et al., 2021. Microplastics pollution: A comprehensive review on the sources, fates, effects, and potential remediation. Environmental Nanotechnology, Monitoring and Management, [e-journal] 16.
https://doi.org/10.1016/j.enmm.2021.100530
Hasegawa, A. et al., 2012. Efficient production of offspring from japanese wild- derived strains of mice (Mus musculus molossinus) by improved assisted reproductive technologies. Biology of Reproduction, 86(5), pp. 1-7.
Hauser, R. and Calafat, A.M., 2005. Phthalates and human health. Occupational and Environmental Medicine, 62(11), pp.806–818.
Horton, A.A. and Barnes, D.K.A., 2020. Microplastic pollution in a rapidly changing world: Implications for remote and vulnerable marine ecosystems.
Science of the Total Environment, 738, p.140349.
67 Howdeshell, K.L., Rider, C. v., Wilson, V.S. and Gray, L.E., 2008. Mechanisms of action of phthalate esters, individually and in combination, to induce abnormal reproductive development in male laboratory rats. Environmental Research, 108(2), pp.168–176.
Huang, F.M. et al., 2018. Bisphenol A exhibits cytotoxic or genotoxic potential via oxidative stress-associated mitochondrial apoptotic pathway in murine macrophages. Food and Chemical Toxicology, 122, pp.215–224.
Ijaz, M.U. et al., 2021. Dose-Dependent Effect of Polystyrene Microplastics on the Testicular Tissues of the Male Sprague Dawley Rats. Dose-Response, [e- journal] 19(2). https://doi.org/10.1177/15593258211019882
Johnson, K.J., Heger, N.E. and Boekelheide, K., 2012. Of mice and men (and rats): Phthalate-induced fetal testis endocrine disruption is species-dependent.
Toxicological Sciences, 129(2), pp.235–248.
Jones, C.P., Boyd, K.L., and Wallace, J.M., 2016. Evaluation of mice undergoing serial oral gavage while awake or anesthetized. Journal of the American Association for Laboratory Animal Science, 55(6), pp.805-810.
Kannan, K. and Vimalkumar, K., 2021. A Review of Human Exposure to Microplastics and Insights Into Microplastics as Obesogens. Frontiers in Endocrinology, [e-journal] 12. https://doi.org/10.3389/fendo.2021.724989
Kelly, F.J. and Fussell, J.C., 2012. Size, source and chemical composition as determinants of toxicity attributable to ambient particulate matter. Atmospheric Environment, 60, pp.504–526.
Kerr, L.E. et al., 2004. Mice overexpressing human caspase 3 appear phenotypically normal but exhibit increased apoptosis and larger lesion volumes in response to transient focal cerebral ischaemia. Cell Death and Differentiation, 11(10), pp. 1102–1111.
Kobroob, A., Peerapanyasut, W., Chattipakorn, N. and Wongmekiat, O., 2018.
Damaging effects of bisphenol a on the kidney and the protection by melatonin:
Emerging evidences from in vivo and in vitro studies. Oxidative Medicine and Cellular Longevity, [e-journal] 2018. https://doi.org/10.1155/2018/3082438
68 Lee, Sijoon et al., 2022. Toxicity Study and Quantitative Evaluation of Polyethylene Microplastics in ICR Mice. Polymers, 2022(14), p.402.
Leibowitz, B. and Yu, J., 2010. Mitochondrial signaling in cell death via the Bcl-2 family. Cancer Biol Ther. 9 (6), pp. 417-422.
Leslie, H.A. et al., 2022. Discovery and quantification of plastic particle pollution in human blood. Environment International, p.107199. Available at:
https://linkinghub.elsevier.com/retrieve/pii/S0160412022001258 [Accessed: 3 April 2022].
Li, L. et al., 2021. Typical phthalic acid esters induce apoptosis by regulating the PI3K/Akt/Bcl-2 signaling pathway in rat insulinoma cells. Ecotoxicology and Environmental Safety, 208, p.111461.
Li, Y. et al., 2021. Research on the influence of microplastics on marine life.
IOP Conference Series: Earth and Environmental Science. IOP Publishing Ltd.
Limonta, G. et al., 2019. Microplastics induce transcriptional changes, immune response and behavioral alterations in adult zebrafish. Scientific Reports, [e- journal] 9. https://doi.org/10.1038/s41598-019-52292-5
Lu, L. et al., 2018. Polystyrene microplastics induce gut microbiota dysbiosis and hepatic lipid metabolism disorder in mice. Science of The Total Environment, 631–632, pp.449–458.
Lu, Y. et al., 2016. Uptake and Accumulation of Polystyrene Microplastics in Zebrafish (Danio rerio) and Toxic Effects in Liver. Environmental Science and Technology, 50(7), pp.4054–4060.
Malaguarnera, G. et al., 2012. Toxic hepatitis in occupational exposure to solvents. World J Gastroenterol, 18(22), pp. 2756-2766.
Meruvu, S., Zhang, J., Bedi, Y.S. and Choudhury, M., 2016. Mono-(2- ethylhexyl) phthalate induces apoptosis through miR-16 in human first trimester placental cell line HTR-8/SVneo. Toxicology in Vitro, 31, pp.35–42.
69 News European Parliament, 2018. Microplastic: sources, effects and solutions
[Online]. Available at:
https://www.europarl.europa.eu/news/en/headlines/society/20181116STO1921 7/microplastics-sources-effects-and-solutions [Accessed: 4 January 2022].
Omnexus, 2022. Comprehensive guide on polyvinyl chloride (PVC) [Online].
Available at: https://omnexus.specialchem.com/selection-guide/polyvinyl- chloride-pvc-plastic [Accessed: 4 January 2022].
Persad, R. et al., 2004. Overexpression of caspase-3 hepatocellular carcinomas.
Modern Pathology, 17(7), pp.861–867.
Plastics Europe, 2021. Plastics – the facts 2021. [online]. Available at:
<https://plasticseurope.org/wp-content/uploads/2021/12/Plastics-the-Facts- 2021-web-final.pdf> [Accessed: 31 December 2021].
Prata, J.C. et al., 2020. Environmental exposure to microplastics: An overview on possible human health effects. Science of the Total Environment, 702, p.134455.
Rahman, A. et al., 2021. Potential human health risks due to environmental exposure to nano- and microplastics and knowledge gaps: A scoping review.
Science of the Total Environment, 757, p.143872.
Redza-Dutordoir, M. and Averill-Bates, D.A., 2016. Activation of apoptosis signalling pathways by reactive oxygen species. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1863(12), pp.2977–2992.
Rezg, R., El-Fazaa, S., Gharbi, N. and Mornagui, B., 2014. Bisphenol A and human chronic diseases: Current evidences, possible mechanisms, and future perspectives. Environment International, 64, pp.83–90.
Santangeli, S. et al., 2017. Effects of BPA on female reproductive function: The involvement of epigenetic mechanism. General and Comparative Endocrinology, 245, pp.122–126.
70 Saravia, J. et al., 2014. Early-life exposure to combustion-derived particulate matter causes pulmonary immunosuppression. Mucosal Immunology, 7(3), pp.694–704.
Schirinzi, G.F. et al., 2017. Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells. Environmental Research, 159, pp.579-587.
Singh, S. and Li, S.S.L., 2012. Epigenetic effects of environmental chemicals bisphenol A and phthalates. International Journal of Molecular Sciences, 13(8), pp.10143–10153.
Somade, O.T., Ajayi, B.O., Olunaike, O.E. and Jimoh, L.A., 2020. Hepatic oxidative stress, up-regulation of pro-inflammatory cytokines, apoptotic and oncogenic markers following 2-methoxyethanol administrations in rats.
Biochemistry and Biophysics Reports, 24, p.100806.
Tagorti, G. and Kaya, B., 2021. Genotoxic effect of microplastics and COVID- 19: The hidden threat. Chemosphere, 286, p.131898.
Trejo-Solís, C. et al., 2018. Autophagic and apoptotic pathways as targets for chemotherapy in glioblastoma. International Journal of Molecular Sciences, 19(12), p.3773.
Tripathi, A. et al., 2019. Di-(2-ethylhexyl) phthalate (DEHP) inhibits steroidogenesis and induces mitochondria-ROS mediated apoptosis in rat ovarian granulosa cells. Toxicology Research, 8(3), pp.381–394.
Valavanidis, A., Vlachogianni, T., Fiotakis, K. and Loridas, S., 2013.
Pulmonary oxidative stress, inflammation and cancer: Respirable particulate matter, fibrous dusts and ozone as major causes of lung carcinogenesis through reactive oxygen species mechanisms. International Journal of Environmental Research and Public Health, 10(9), pp.3886–3907.
Watts, A.J.R. et al., 2015. Ingestion of Plastic Microfibers by the Crab Carcinus maenas and Its Effect on Food Consumption and Energy Balance.
Environmental Science and Technology, 49(24), pp.14597–14604.
71 Wen, B. et al., 2018. Microplastics have a more profound impact than elevated temperatures on the predatory performance, digestion and energy metabolism of an Amazonian cichlid. Aquatic Toxicology, 195, pp.67–76.
World Health Organization, 2022. Dioxins and their effects on human health [Online] Available at: https://www.who.int/news-room/fact- sheets/detail/dioxins-and-their-effects-on-human-health [Accessed: 5 January 2022].
Wright, S.L., Rowe, D., Thompson, R.C. and Galloway, T.S., 2013.
Microplastic ingestion decreases energy reserves in marine worms. Current Biology, 23(23), pp.1031-1033.
Xia, B. et al., 2022. Secondary PVC microplastics are more toxic than primary PVC microplastics to Oryzias melastigma embryos. Journal of Hazardous Materials, 424, p.127421.
Xiao, M. et al., 2018. Deoxypodophyllotoxin induces cell cycle arrest and apoptosis in human cholangiocarcinoma cells. Oncology Letters, 16(3), pp.3177–3182.
Xin, F. et al., 2014. Bisphenol A induces oxidative stress-associated DNA damage in INS-1 cells. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 769, pp.29–33.
Xu, X.Y. et al., 2017. Microplastic ingestion reduces energy intake in the clam Atactodea striata. Marine Pollution Bulletin, 124(2), pp.798–802.
Yacobi, N.R. et al., 2008. Polystyrene nanoparticle trafficking across alveolar epithelium. Nanomedicine: Nanotechnology, Biology, and Medicine, 4(2), pp.139–145.
Yip, K.W. and Reed, J.C., 2008. Bcl-2 family proteins and cancer. Oncogene, 27(50), pp.6398–6406.
72 Yoshida, K. and Miki, Y., 2010. The cell death machinery governed by the p53 tumor suppressor in response to DNA damage. Cancer Science, 101(4), pp.831–
835.
Yu, J. and Zhang, L., 2005. The transcriptional targets of p53 in apoptosis control. Biochemical and Biophysical Research Communications, 331(3), pp.851–858.
Zhang, J.N. et al., 2019. Di (2-ethylhexyl) Phthalate Exposure Impairs the microRNAs Expression Profile During Primordial Follicle Assembly. Frontiers in Endocrinology, [e-journal] 10. https://doi.org/10.3389/fendo.2019.00877
Zhang, P. et al., 2018. Roles and potential mechanisms of selenium in countering thyrotoxicity of DEHP. Science of The Total Environment, 619–620, pp.732–739.
Zhao, L. et al., 2021. Prolonged oral ingestion of microplastics induced inflammation in the liver tissues of C57BL/6J mice through polarization of macrophages and increased infiltration of natural killer cells. Ecotoxicology and Environmental Safety, 227, p.112882.
Zhao, Z. B. et al., 2019. Di(2-ethylhexyl) phthalate promotes hepatic fibrosis by regulation of oxidative stress and inflammation responses in rats.
Environmental Toxicology and Pharmacology, 68, pp.109–119.
Zimmermann, K.C. and Green, D.R., 2001. How cells die: Apoptosis pathways.
Journal of Allergy and Clinical Immunology. 2001 Mosby Inc.
73 APPENDICES
Appendix A
PVC particle size measurement report
74 Appendix B
Ethical approval from SERC, UTAR
75
76 Appendix C
List of materials, chemicals and instruments used
List of chemicals used in the study
Chemicals Manufacturer, Country
Tris 1st BASE, Singapore
Ethylenediaminetetraacetic Acid (EDTA)
R&M Chemistry, Malaysia
Boric Acid MERCK, Germany
Phosphate buffer saline MERCK, Germany Absolute ethanol (99.5 %) Chemsol, Malaysia
Agarose powder 1st BASE, Singapore
Diethylpyrocarbonate (DEPC) Himedia, India
GeneRuler 1kb DNA Ladder Fermentas, United States Novel Juice (DNA staining
reagent)
BIO-HELIX, China
Pairwise primers Integrated DNA Technologies, United States
GENEzol™ TriRNA Pure Kit Geneaid, Taiwan - GENEzol™ Reagent
- Pre-Wash Buffer - DNase I
- DNase I Reaction Buffer - Wash Buffer
- RNase-free Water
HiScript® II 1st Strand cDNA Synthesis Kit
Vazyme, China - RNase-free ddH2O
- RT Mix (2X)
- HiScript II Enzyme Mix - Oligo (dT)23VN (50 µM) - Random hexamers (50 µM) ChamQ Universal SYBR qPCR Master Mix
Vazyme, China - ChamQ Universal SYBR qPCR
Master Mix (2X)
77 List of instruments and consumables used
Consumables/Instruments Manufacturer, Country Conical flask (50 mL) Favorit, Malaysia
Measuring cylinder (50 mL, 100 mL) Favorit, Malaysia Schott bottle (250 mL, 500 mL, 1000
mL)
Favorit, Malaysia Beaker (100 mL, 500 mL, 1000 mL) Favorit, Malaysia
Centrifuge tubes (15 mL) Axygen Scientific, USA 1.5 mL microcentrifuge tubes Greiner Bio-one, Germany
PCR tubes Axygen Scientific, USA
qPCR strip tubes Axygen Scientific, USA Micropipette (10A, 100A, 1000A)
Micropipette tips Quality Scientific Plastic, China
Weighing machine Adventurer, USA
Electrophoresis Set (power supply, gel tank, gel cast tray, comb)
Major Science, Taiwan
Vortex mixer Gemmy Industrial Corporation,
Taiwan
Microwave Sharp, Malaysia
Chiller (4 °C) Panasonic, Malaysia
Freezer (-20 °C) Panasonic, Malaysia
Microcentrifuge machine (1.5 mL) Thermo Scientific, United States NanoDrop 2000 Spectrophotometer Thermo Scientific, United States
PCR machine Bio-rad, United States
Gel Documentation System Bio-rad, United States Real-time PCR machine Bio-rad, United States 1.0 mL disposable syringe Terumo, Philippines Extra virgin olive oil Naturel, Spain
Wood Shave Delikate, Malaysia
Food Pellets Gold Coin, Malaysia
GENEzol™ TriRNA Pure Kit Geneaid, Taiwan - RB Columns
- Collection Tubes (2 mL)
78 Gold coin food pellets nutritional facts
Ingredients Percentage (%)
Crude Protein, Minimum 21.0 Crude Fibre, Maximum 5.0
Crude Fat, Minimum 3.0
Moisture, Minimum 13.0
Ash, Maximum 8.0
Calcium, Minimum 0.8
Phosphorus, Minimum 0.4
79 Appendix D
Dilution method of dosage preparation
Stock solution:
20 mg PVC MPs + 8 mL extra virgin olive oil
= 2.5 mg/mL
Low dosage:
C1V1 = C2V2
(2.5) (V1) = (0.05) (6000) V2 = 120 µL
Medium dosage:
C1V1 = C2V2
(2.5) (V1) = (0.5) (6000) V2 = 1200 µL
80 Appendix E
OECD guideline for mice feeding volume
(Adapted from Oghenesuvwe et al., 2014)
81