Manuscript

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Wastewater based epidemiology (WBE), a tool to bridge
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biomarkers of exposure, contaminants and human
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5 health
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7 Dyana Vitalea, Maria Morales Suárez-Varelab,c and Yolanda Picó*a,c
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Food and Environmental Research Group (SAMA-UV), Research Desertification Centre (CIDE) (CSIC-
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10 University of Valencia-GV), Moncada-Naquera Road, Km 4.5, 46113 Moncada, Valencia, Spain
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Unit of Public Health and Environmental Care, Department of Preventive Medicine, University of
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Valencia, Valencia, Spain
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CIBER Epidemiologia y Salud Pública (CIBERESP), Institute of Health Carlos III, Madrid, Spain
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Highlights
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23  The concept of WBE as a tool to assess Human Health is outlined
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25  WBE early warning capacities for chemical exposure and disease outbreaks are listed
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27  State of art of applications to biomarkers and pathogens is critically discussed
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29  The development of the WBE in terms of human health assessment is reviewed
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31  Insights and future perspectives on WBE within human health are provided
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34 Abstract
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36 The concept of wastewater-based epidemiology (WBE) also known as sewage epidemiology was
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38 proposed by Daughton in 2001. Since then, WBE has become now a reality that makes it possible
39 to determine consumption or exposure to chemical substances or pathogens in a population by
40 measuring certain compounds (drugs of abuse, metabolites or biomarkers) or microorganisms
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(viruses, bacteria, parasites) in wastewater. The first and most developed application is the
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43 estimation of illicit drug consumption in communities or populations, but it can be used to
44 measure both consumption and exposure to a wide range of substances and pathogens. Its
45 recent application to measure the SARS-CoVID-2 loads in neighborhoods, towns and cities serves
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47 as an example of its usefulness As a result it can be helpful to determine lifestyle, exposure to
48 toxic agents and prevalence of disease in a given population. This review highlights how sewage
49 epidemiology has become a tool to bridge biomarkers of exposure, contaminants and human
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health.
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58 *
Corresponding author
59 E-mail: Yolanda.Pico@uv.es
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Phone: +34 963424216
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 Keywords: wastewater analysis, influents, chemicals, biomarkers, pathogens, community health
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 1. Introduction
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3 The definition of epidemiology has evolved greatly over time, nowadays the most accepted one
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5 defined it as “the study of distribution and determinants of health related states or events in
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8 specified populations, and the application of this study to control health problems” [1].
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10 Wastewater-Based Epidemiology (WBE) can provide information on the risk factors and health
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12 status of a population through analysis of their wastewater [2**,3*]. This tool has an
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15 unquestionable advantage over other classical epidemiological techniques such as data
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17 collection and analysis, surveys and/or direct monitoring of a large number of individuals, since
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it achieves a rapid (almost real time) data collection (which makes WBE an early warning tool)
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22 and in addition these data represent a pool of the global population or at least a very important
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24 percentage of it. The potential of wastewater to serve as a reflection of the substances
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27 consumed or to which the population is exposed was predicted by Daughton [4**] who already
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29 described wastewater as an instrument that through the measurement of chemical compounds
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31 and metabolites (biomarkers in a global way) would evaluate the population's health. This
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34 attractive theory was put into practice for the first time in the city of Milan, where the
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36 consumption of drugs of abuse was estimated from their presence in the wastewater that
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reached the treatment plants [5*]. Thanks to the efforts made by several research groups at
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41 European level, at present, this estimate is in common use and the European Monitoring Centre
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43 for Drug and Drug Addiction (EMCDDA) publishes annually the data obtained at European level
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46 on drug consumption estimated with this technique [6**]. WBE has become the method of
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48 reference to measure variations in the consumption of these substances over time in a given
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50 area [7-9], as well as to make comparisons between populations [10-13] as it provides a non-
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53 invasive, near-real-time analysis of drug use within the area served by the sampled sewer
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55 network or in special leisure events [13].
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 The WBE concept proposed by Daughton is now well validated but is far away to reach its
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2 maturity yet. The measurement of the concentrations of chemicals or of microbiological
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5 indicators can provide information on the consumption and leisure habits of a population, as
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7 well as of the habits related to domestic waste management. Such is the importance that
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9 recently this journal has published a special issue on WBE covering different aspects [14**].
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12 These analyses are able to establish the personality of a city or a neighborhood. More recently,
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14 Daughton [3*] also defines the concept of “Sewage Chemical-Information Mining (SCIM)” that
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16 “involves the monitoring of sewage for the information that resides in the form of natural and
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19 anthropogenic chemicals that enter sewers as a result of the everyday actions, activities, and
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21 behaviors of humans”. This information will overpass the WBE as we now up to the momment
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and open an amazing horizon to improve our knowledge on the health status of the human
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26 being. Here is the moment to highlight the multidisciplinary an transdisciplinary character of
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28 SCIM and WBE, where the boundaries between the different scientific disciplines involved
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31 (anthropology, biology, chemistry, epidemiology, engineering, medicine, public health and
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33 sociology) are blurred to form a whole.
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36 This review will cover these aspects as a step forward of the traditional WBE to became a most
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ambitious proposal to evaluate biomarkers of exposure and contaminants in order to improve
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41 the health pattern of a city and closed the circle on the potential of sewage epidemiology to
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43 bridge biomarkers of exposure, contaminants and human health to evaluate community.
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46 2. Sate of the art of WBE
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49 The most important drawback that globally WBE still had is the uncertainty in the estimation of
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52 population. In order to get a deep knowledge on the population size changes, Hart and Halden
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54 [15] simulated repeated sampling at major U.S. WWTPs under constant biomarker loading
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conditions, to explore the potential sensitivity of WBE for generating skewed data using 2017
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59 nationwide data on geospatial population demographics as a test case. Alternatively, Baz-Lomba
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 et al. [16] used mobile phone data from 8-weeks in 2016 to train three linear models based on
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2 drinking water production, electricity consumption and online measurements of ammonium in
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5 wastewater. However, these sophisticated systems are far from being in widespread use either
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7 because they need extensive coordinated networks or data coming from for-profit interests.
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9 They usually seek to identify what physical-chemical parameter gives the best result. Currently,
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12 there is still a lot of research in finding a biomarker of human origin that can easily and accurately
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14 extrapolate the population. Hou et al. [17] developed a multi-parameter population model
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16 based on selected endogenous population biomarkers (cotinine, trans-3-hydroxy cotinine, 4-
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19 Pyridoxic acid and 1,4-methylimidazole acetic acid) and flow volume was established to reduce
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21 the population-related uncertainties in WBE and better estimate the population size. Instead,
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Pandopoulos et al. [18] evaluated the catecholamine metabolites – homovanillic acid and
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26 vanillylmandelic acid – as potential population biomarkers. There is not an appropriate
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28 agreement on this point yet.
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31 As reported in the introduction, nowadays, the estimation of drug of abuse through this
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34 technique has been generalized. However, research is still on-going by (i) revising excretion
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36 factors (e.g. ketamine [19]), (ii) determining chiral compounds [20] and (iii) improving analytical
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methods by applying passive samplers [21] or even better substituting liquid-chromatography
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41 by sensors and/or aptasensors [21,22]. Of special interest within the analytical methods is the
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43 development sensors and aptasensors to estimate the drug consumption since they can speed
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46 even more the process and be used for “in-situ” monitoring in the WWTPs [22]. Furthermore,
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48 similar studies have achieved the estimation of the consumption of alcohol [23-25], caffeine
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50 [26], nicotine [24,27-29], opiods [30], and psychoactive and other types pharmaceuticals [31].
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53 The biomarkers used to determine these compounds are reported in Table 1.
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56 Table 1. Biomarkers to determine alcohol, caffeine, nicotine and some pharmaceuticals.
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Compound Biomarkers Characteristics Ref.
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Alcohol Ethyl sufate Determined by LC-MS [23-25]
60 Stable in WWTPs (several days)
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 Nicotine Cotinine Determined by LC-MS [24,26-29]
Hydroxycotinine Anabasine and anatabine specific biomarkers
1 Nicotine of tobacco.
2 Anabasine Stable in WWTPs but anabasine and anatabine
3 Anatabine were less stable than cotinine and
4 hydroxycotinine
5 Caffeine Caffeine Determined by LC-MS [26,32]
Dimethyluric acid Stable in WWTPs
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Psychoactive [30,31]
7 pharmaceuticals
8 Efedrine Efedrine Determined by LC-MS
9 Methamphetamine Methamphetamine Not data about stability “in-sewer”
10 Mepedirine Mepedirine
11 Phendimetrazine Phendimetrazine
12 Phentermine Phentermine
Zolpidem Zolpidem 4-phenyl
13 carboxylic acid
14 Cis-Tramadol Cis-Tramadol
15 Codeine Codeine
16 Morphine Morphine
17 Oxycodone Oxycodone
18 Hydrocodone Hydrocodone
Oxymorphone Oxymorphone
19 Hydomorphone Hydomorphone
20 Pharmaceuticals Determined by LC-MS [15]
21 Citalopram N-desmethylcitalopram Sufficiently stable in WW for an average
22 Enalapril Enalaprilat hydraulic retention time in sewers of 8 h and
23 Losartan EXP-3174 24 h of sampling performed at 4 .
24 Ramipril Ramiprilat
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28 Within this topic, recently, the possibility to assess the adherence of the patients to some
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30 pharmacological treatments in a defined area through WBE has been tested [15]. Low adherence
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33 to pharmacological therapy is one of the main reasons for poor success, reducing the patient's
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35 wellbeing, affecting health-care effectiveness and involving higher cost. The results show
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37 agreement between metabolite and parent compound concentrations in several cases, thus
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40 providing approximation to treatment compliance (e.g. for enalapril), but in other cases the
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42 approach is not correct and as a consequence the interpretation is complicated.
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45 The knowledge about potential biomarkers to establish exposure, consumption, lifestyle habits,
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48 health and wellbeing have been reviewed recurrently [33*,34**,35*,36**,37*,38**]. Up to the
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50 moment, information obtained from the analysis of specific biomarkers in urban wastewater
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52 can also be used to estimate the exposure of the human being to several contaminants or
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55 natural toxins, such as mycotoxins [39], pesticides [35*], phthalates [34***,40],
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57 organophosphorus flame retardants and bisphenol A [41]. The most relevant biomarkers applied
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59 in those studies are reported in Table 2.
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 Table 2. Most relevant biomarkers of human exposure to contaminants
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3 Compound Biomarkers Characteristics Ref.
Deoxynivalenol (DON) DON Determination by LC-MS [39]
4 Fumonisins (FB1, FB2, FB3) FB1, FB2, FB3 Differences in the concentrations after 24 h
5 at room temperature (-3, -37%) were larger
6 than at 4 °C (-1, -26%).
7 Pesticides [42-44]
8 Triazines (Atrazine, Terbutylazine desethyl (DES) DES specific of terbutylazine and AM of the
9 terbutylazine, simazine, Atrazine desisopropyl (DIA) atrazine
propazine) Atrazine desethyl (DEA)
10 Atrazine mercapturate (AM)
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12 Pyrethroids (trans- 3-phenoxybenzoic acid (PBA) BPA is metabolite of more than 20
13 permethrin, cypermethrin, 3-(2,2-dichlorovinyl)-2,2-dimethyl- pyrethroids
14 cyfluthrin and others) (1-cyclopropane) carboxylic acid
15 (trans-DCCA and cis-DCCA)
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Organophosphorus 3,5,6-trichloro-2-pyridinol (TCPY)
17 (Chlorpyrifos, chlorpyrifos- malathion monocarboxylic acid
18 methyl, malathion, diazinon (MMA)
19 and several other 2-isopropyl-6-methyl-4-
20 organophosphorus pyrimidinol (IMPY)
21 pesticides) Diethylphosphate (DEP)
Diethyl thiophosphate (DETP)
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Dimethyl phosphate (DMP)
23 Dimethyl thiophosphate (DMTP)
24 Phthalates [i-(2-ethylhexyl) monomethyl phthalate (MMP) Determination by LC-MS [1]
25 phthalate (DEHP) and other monoethyl phthalate (MEP) MMP, MEP, MnBP, MiBP and MBzP for low
26 Phthalate esters] mono-n-butyl phthalate (MnBP) molecular weight phthalates
27 mono-i-butyl phthalate (MiBP) MEHHP and MEOHP for DEHP
28 monobenzyl phthalate (MBzP)
mono-(2-ethyl-5-hydroxyhexyl)
29 phthalate (MEHHP)
30 mono-(2-ethyl-5-oxohexyl)
31 phthalate (MEOHP)
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Organophosphorus flame [45]
33 retardants (OFRs)
34 2-ethylhexyldiphenyl 2-ethyl-5-hydroxyhexyldiphenyl Determined by LC-MS
35 phosphate (EHDPHP) phosphate (OH-EHDPHP) DPP is a minor metabolite of EHDPHP can
36 2-ethylhexyl phenyl phosphate also be a plasticizer and can be metabolite
37 (EHPHP) of other OFRs
38 Diphenyl phosphate (DPP)
39 Tris-(2- Bis-(2-butoxyethyl)phosphate
40 butoxyethyl)phosphate (BBOEP)
41 (TBOEP) Bis-(2-butoxyethyl)-3’-hydroxy-2-
42 butoxyethyl-phosphate (HQ-
43 TBOEP)
44 2-hydroxyethyl bis-(2-butoxyethyl
phosphate (BBOEHEP)
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46 Bis-(2-chloroisopropyl)phosphate
47 Tris-(2-chloroisopropyl) (BClPP)
48 phosphate (TClPP) 1-hydroxy-2-propyl bis(1-chloro-2-
49 propyl) phosphate (BClPHIPP)
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51 Diphenylphosphate (DPHP)
Triphenylphosphate (TPP) Hidroxyphenyl-phenylphosphate
52 (OH-DPHP)
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54 Bis(1,3-dichloroisopropyl)
55 Tris(1,3-dichloroisopropyl) phosphate (BDClPP)
56 phosphate (TDClPP)
57 Tris(chloroethyl)phosphate (TCEP)
Tris(chloroethyl)phosphate
58 (TCEP)
59 Bisphenol BPA, BPB, BPF, BPP, BPS, BPZ, Important nonhuman excretion sources of [41]
60 BPAF, and BPAP BPA in municipal wastewater
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 [bisphenol A (BPA), bisphenol
F (BPF), bisphenol S (BPS),
1 bisphenol P (BPP), bisphenol
2 AP (BPAP), bisphenol B (BPB),
3 bisphenol Z (BPZ), and
4 bisphenol AF (BPAF),}
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8 This approach has already been used to assess the population exposure to three classes of
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pesticides: triazines, organophosphates, and pyrethroids. Recently, Devault and Karolkav [35*]
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13 review the state-of-art to obtain the data (excretion rates) and characteristics (pesticide and
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15 metabolites stability, including in-sewer one) for other pesticides (2,4-D, aldrin, carbaryl,
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chlorobenzilate, dieldrin, diquat, ethion, glufosinate, glyphosate, folpet, malathion, parathion,
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20 penconazole, and tebuconazole) to their future application to WBEs.
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23 The recent quantification of SARS-CoVID-2 in wastewater to track the spread of the virus in a
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25 particular community has been very mediatic and popular. There are a high number of reviews
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28 [46**,47,48,49,50,51,52,53,54,55*] and in this moment the scientific literature is saturated of
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30 examples of the determination of SARS-CoVID-2 in wastewater treatment plants around the
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world. The detection of this virus is mostly based on nucleic acid–based polymerase chain
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35 reaction (PCR) approaches (nested real time (RT)-PCR, RT qPCR, etc.). Most of these reviews
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37 highlight the need to fully validate and homogenized the methodology [46**,47,50,53].
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40 The emerging field of microfluidics also known as the lab-on-a-chip (LOC) has also found
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43 application in the detection SARS-CoVID-2 with so called point of care (POC). Interestingly, Mao
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45 et al. [56] discuss the feasibility of an integrated point-of-care (POC) biosensor system with
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47 mobile health for WBE (iBMW) that is suitable early warning of COVID-19, screening and
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50 diagnosis of potential infectors, and improving health care and public health. In any case, during
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52 this pandemic WBE has been able to demonstrate a great capacity as an early warning system
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to know the population's viral load.
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 3. Future prospects within WBE
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3 The emergence of novel pathogenic organisms and the re-emergence of infections that were
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5 once controlled are a reality. The previous reported CoVID pandemic is a good and recent
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8 example. The main reasons reported are the climate change and unprecedented population
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10 growth with accelerated rates of antimicrobial resistance [57**].
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13 Monitoring temporal changes in virus concentration and diversity excreted in community
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wastewater, in combination with monitoring metabolites and biomarkers for population
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18 adjustments, allows early detection of outbreaks (critical moments for the onset of an outbreak)
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20 [58*]. Future monitoring efforts are the enlargement of the scope to consider also parasitic
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23 protozoa (Cryptosporidium spp. and Giardia lamblia) and enteric bacteria (Campylobacter
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25 jejuni).
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28 Table 3. Summary of human viruses detected in wastewater
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30 Virus Biomarker Characteristics Ref.
31 Determination by droplet digital (dd)PCR
Adenoviruses Nucleic acid [59-61]
qPCR (both DNA and RNA)
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Astroviruses Nucleic acid qPCR (both DNA and RNA) [61,62]
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Enteroviruses Nucleic acid qPCR (both DNA and RNA) [61,62]}
34 Hepatitis A virus Nucleic acid qPCR (both DNA and RNA) [62]
35 Determination by LC-MS
36 Hepatitis B Lamiduvine Lamivudine was found to be stable after 4 months' [63]
37 storage at −80 °C
38 Hepatitis E virus Nucleic acid qPCR (both DNA and RNA) [64]
39 Noroviruses Nucleic acid Determination by ddPCR [59,61]
40 Rotaviruses Nucleic acid qPCR (both DNA and RNA) [60]
41 Aichi virus (AiV) RNA Conventional PCR [65]
42 Bocavirus (HBoV) RNA Conventional PCR [65]
Human Polyomaviruses (HPyVs) DNA Conventional PCR [66]
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Salivirus Nucleic acid qPCR [67]
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Sapovirus Nucleic acid qPCR [61]
45 Herpesvirus Nucleic acid qPCR [61]
46 Coronaviruses Nucleic acid qPCR (both DNA and RNA [64]
47 RNA occurred after 21 days at 25 °C and after 8.5
48 Zika virus RNA [68]
days at 35 °C
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52 As can be observed along this discussion, key challenge in WBE is the selection of biomarkers
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55 which are specific to human metabolism, excreted in sufficient amounts, and stable in sewage
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57 [34**]. Nowadays, there is interest in fully develop the potentiality of WBE to establish the
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health, wellbeing and lifestyle biomarkers to be able to determine the health fingerprinting of a
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 community [37*]. The most investigated biomarkers up to the moment have been those related
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2 to dietary characteristics and oxidative stress are closely linked to the wellbeing of individuals.
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5 In recent years, various urinary biomarkers of food and oxidative stress have been proposed for
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7 use in wastewater-based epidemiology (WBE), in efforts to objectively monitor the food
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9 consumed and the oxidative stress experienced by individuals in a wastewater catchment. Table
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12 4 summarizes most of this biomarkers. However, it is not clear whether such biomarkers are
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14 suitable for wastewater-based epidemiology. Choi et al. [69**,70] in the most comprehensive
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16 presented a suite of 30 urinary food and oxidative stress biomarkers and evaluates their
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19 applicability for WBE studies. However, the human metabolism is very complex and there are a
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21 large number of substances that can be included. So far, most studies looking for new
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biomarkers, begin by conducting a study of the prior knowledge that exists on metabolites of
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26 different compounds or different health states created by urine and feces. They then study
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28 whether these compounds are stable and whether they can be detected in wastewater. This
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31 process, although it has given its fruits is improvable, and in this aspect there are very interesting
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33 examples. Ferrando-Climent et al [71] applied a suspect screening methodology to detect
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35 chemotherapy and radiotherapy drugs and their related compounds such metabolites and/or
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38 biomarkers in wastewater. Successful identification of cancer-related suspects included two
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40 antineoplastic hormones, two X-ray contrast agents and a pyrrolizidine alkaloid related to an
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herbal medicine. This system could be much more straight forward to identify suitable
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45 biomarkers. Another obstacle to the development of this technique is the general lack of
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47 knowledge of the biomarkers that appear in each stress situation. In this sense, Etteieb et al.
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50 [72] proposed a model assess the overexpression of some genes produced exposing to toxic
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52 substances human intestinal epithelial Caco‐2 cells and the biomarkers synthesized as results.
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54 That can enlarge our knowledge on the topic. Although there is still much to be done in this field
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57 and is still fully open horizon, all these studies open a range of unsuspected possibilities to
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59 achieve a better future that guarantees a better health status of populations.
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 Table 4. Most relevant biomarkers of Exposure, Nutrition and Heath
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3 Compound Biomarkers Characteristics Ref.
Vitamin
4 Vitamin B2 Rivoflavin Determined by LC-MS [69**]
5 Vitamin B3 Nicotinic acid [69**]
6 Nicotinamide
7 MNA (1-methylnicotinamide)
8 2PY (1-methyl-2-pyridone-5-carboxamide)
9 4PY (1-methyl-4-pyridone-5-carboxamide)
Vitamin B5 Panthothenic acid [69**]
10 Vitamin B6 4-Pyridoxic acid [69**]
11 Vitamin E αCEHC (alpha-carboxyethyl [69**]
12 hydrochroman)
13 γCEHC (gamma-carboxyethyl
14 hydrochroman)
15 Plant derived foods [69**]
Lignan Enterodiol [69**]
16
Enterolactone
17 Fruit Hippuric acid [69**]
18 Proline betaine
19 Phloretin
20 Wholegrain DHPPA (3-(3,5-dihydroxyphenyl)-1- [69**]
21 propionoic acid)
Animal food [69**]
22
Meat CMPF (3-Carboxy-4-methyl-5-propyl-2- [69**]
23 furanpropionic acid)
24 3MH (3-methylhistidine)
25 Carnosine
26 AC (acetylcarnitine)
27 PC (propionylcarnitine)
28 Meat & Fish TMAO (trimethylamine N-oxide) [69**]
Anserin
29 Artificial sweteners
30
Ciclamate Cyclamate Determined by LC-MS [73-75]
31 Acesulfame Acesulfame Already tested to WBE
32 Sucralose Sucralose Stability of sucralose during WWT
33 Sacharine Sacharine at most plants
34 Household products
35 Parabens Methyl 3,4-dihydroxybenzoate (Methyl Only detected in urine (not applied [34**]
36 Methylparaben (MeP) protocatechuate; 3-OH-MeP) yet to WBE
37
38 Ethyl 3,4-dihydroxybenzoate (Ethyl
39 Ethylparaben (EtP) protocatechuate; 3-OH-EtP)
40 3-Hydroxy-n-butylparaben (3-OH-BuP)
41 n-Butylparaben (BuP)
42 2-Hydroxy-iso-butylparaben (2-OH-iBuP)
43 iso-Butylparaben (iBuP)
44 UV filters Determined by LC-MS [34**]
45 Benzophenone-3 Benzophenone-3 (Oxybenzone; BP-3) BP-3, 5-OH-BP-3, Hydroxy
(Oxybenzone; BP-3) 2,5-Dihydroxy-4-methoxybenzophenone avobenzone, Desmethylhydroxy
46 (5-OH-BP-3) avobenzone,
47 Desmethylavobenzone carboxylic
48 Octisalate Hydroxybenzoyl)oxy)methyl)heptanoic acid, Dehydrated dihydrohydroxy
49 (2-ethylhexyl salicylate; acid (5-cx-EPS) avobenzone and 3,3,5-
50 EHS) 2-Ethyl-5-hydroxyhexyl 2-hydroxybenzoate Trimethylcyclohexanol not
51 (5-OH-EHS) previously reported in water
2-Ethyl-5-oxohexyl 2-hydroxybenzoate (5-
52
oxo-EHS)
53
54 Enzacamene (3-(4- 3-(4-Carboxybenzylidene)-6-
55 methylbenzylidene)camphor; hydroxycamphor
56 4-MBC) 3-(4-Carboxybenzylidene)camphor
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Octocrylene (2- 2-Cyano-3,3-diphenylacrylic acid (CPAA)
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ethylhexyl 2-cyano-3,3- 2-(Carboxymethyl)butyl 2-cyano-3,3-
59 diphenyl-2-acrylate; OC) diphenyl acrylate (DOCCA)
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 2-Ethyl-5-hydroxyhexyl 2-cyano-3,3-
diphenyl acrylate (5-OH-OC)
1 Avobenzone
2 (butyl Hydroxy avobenzone
3 methoxydibenzoylmethane) Desmethylhydroxy avobenzone
4 Desmethylavobenzone carboxylic acid
5 Dehydrated dihydrohydroxy avobenzone
Homosalate (3,3,5-
6
trimethylcyclohexyl 3,3,5-Trimethylcyclohexanol
7 salicylate)
8 Plasticizers Already reported in raw [34**]
9 Di-2-ethylhexyl adipate Mono-2-ethylhexyl adipate (MEHA) wastewater, with the exception of
10 (DEHA) Mono-5-carboxy-2-ethylpentyl adipate MEHA
11 (5cx-MEPA)
12 Mono-2-ethylhydroxyhexyl adipate (5OH-
MEHA)
13 Mono-(2-ethyl-5-oxohexyl) adipate (5oxo-
14 MEHA)
15
16 Di(2-ethylhexyl) 1-Mono-(2-ethyl-5-carboxyl-pentyl)
17 terephthalate (DEHTP) terephtahalate (5cx-MEPTP)
18 1-Mono-(2-ethyl-5-hydroxy-hexyl)
terephtahalate (5OH-MEHTP)
19 1-Mono-(2-ethyl-5-oxo-hexyl)
20 terephtahalate (5oxo-MEHTP)
21 1-Mono-(2-carboxyl-methyl-hexyl)
22 terephtahalate (2cx-MEHTP)
23
24 Cyclohexane-1,2-dicarboxylate-mono-(7-
Di(isononyl)cyclohexane- hydroxy-4-methyl) octyl ester (OH-MINCH)
25
1,2-dicarboxylate (DINCH) Cyclohexane-1,2-dicarboxylic mono
26 oxoisononyl ester (oxo-MINCH)
27 Cyclohexane-1,2-diarboxylic mono
28 carboxyisononyl ester (cx-MINCH)
29 Monoisononyl-cyclohexane-1,2-
30 dicarboxylate (MINCH)
Cyclohexane-1,2-dicarboxylic acid mono
31
carboxyhexyl ester (MCHxCH)
32 Cyclohexane-1,2-dicarboxylic acid mono
33 carboxybutylester (MCBCH)
34 Cyclohexanol-1,2-dicarboxylic acid mono
35 carboxyoctyl ester (MCHeCH)
36
37
38
39 Stress [69]
40 Oxidative DiY (L,L-dityrosine) [69]
41 ClY (chlorotyrosine)
42 BrY (bromotyrosine)
43 NY (3-nitrotyrosine)
HPMA (3-hydroxypropyl mercapturic acid)
44 8OHdG (8-hydroxy-2'deoxyguanosine)
45 8OHG (8-hydroxyguanosine)
46 8-iso-prostaglandin F2α (PGF2α) Tested to WBE [76-79]
47 dinor-11β-Prostaglandin F2α (dnPGF2α) PGF2α, dnPGF2α and PGE2 are
48 Prostaglandin E2 (PGE2) sufficiently stable under typical
49 sewer conditions
Hydroxynonenal–mercapturic acid (HNE- Tested to WBE [77]
50
MA) Successful determination along a
51 8-nitroguanine (8-NO2Gua) week
52 8-hydroxy-2-deoxyguanosine (8-OHdG)
53 Diabetis Metformin (pharmaceutical) Tested to WBE [32,80]
54 Cancer “sample-to-answer” platform that can be Tested to WBE [81]
55 (including breast, renal and used for the quantitative monitoring of enrichment into a low cost lateral
gastric cancers) genetic biomarkers (human-specific flow-based test
56
mitochondrial DNA (mtDNA) LOD < 40 copies of DNA
57
58
59
60
61
62 12
63
64
65
 1
2
3
4
5
6 4. Conclusions
7
8
9 Since its proposal in 2001, WBE has evolved. Nowadays, the estimation of consumption of illicit
10
11 drugs and other substances of abuse as alcohol, nicotine, caffeine is already a well established
12
13
14 reality. However, within these analysis there are still some important gaps that needs to be
15
16 solve. A global problem of WBE is the uncertainty on the determination of the population. Many
17
18
19
research is still on-going to reduce it. However, it is clear that highly sophisticated simulations
20
21 and inclusion of more specific human biomarkers promise to be the solution. The research on
22
23 biomarker of human exposure to several chemicals (phthalates, pesticides, bisphenol A,
24
25
26 organophosphorus flame retardants) is one of the fields currently under development and can
27
28 offer very useful data on human exposure. Highlighted by the current outbreak of SARS-CoVID-
29
30 2, the future trends on this approach are much more ambitious aiming at establish biomarkers
31
32
33 of human health and of the lifestyle and habits of communities that become a fingerprint of the
34
35 different communities. The main advances in this field are currently in the monitoring and
36
37
growth of infectious diseases and some indicators of stress, nutritional status and cancer.
38
39
40 However, a search for bioindicators of the chronic diseases that most affect our society is still
41
42 lacking. It is expected that in the near future these markers will be included.
43
44
45 Acknowledgments
46
47
48 This work has been supported by the Spanish Ministry of Science, Innovation and Universities
49
50
51 and the ERDF (European Regional Development Fund) through the project CICLIC -subproject
52
53 WETANPACK (RTI2018-097158-B-C31), by the Generalitat Valenciana through the project
54
55
56
ANTROPOCEN@ (PROMETEO/ 2018/ 155).
57
58
59 References
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62 13
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Graphical Abstract (for review)
Consumption
Exposure

METABOLISM
Health state

EXCRETION

Wastewater based epidemiology (WBE)

Analysis
Population Raw wastewater
Declaration of Interest Statement

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: