Science of the Total Environment 866 (2023) 161339

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Science of the Total Environment

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Indirect potable water reuse to face drought events in Barcelona city. Setting
a monitoring procedure to protect aquatic ecosystems and to ensure a safe
drinking water supply
⁎
Antoni Munné a, , Carolina Solà a, Elisabet Ejarque a, Josep Sanchís a, Pere Serra b, Irene Corbella b, Mercè Aceves c,
Belen Galofré d, M. Rosa Boleda d, Miquel Paraira d, Jordi Molist a
a
Catalan Water Agency, c/ Provença 260, 08036 Barcelona, Spain
b
Public Health Agency of Catalonia, Roc Boronat 81, 08182 Barcelona, Spain
c
Barcelona Metropolitan Area, C/62, 18, Zona Franca, 08040 Barcelona, Spain
d
Aigües de Barcelona, General Batet 1-7, 08028 Barcelona, Spain

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• A real-scale indirect potable water reuse

trial under simulated drought conditions
was conducted.
• Three microbial indicators and 376 rele-
vant micropollutants were monitored.
• Trial showed ten micropollutants that
could pose a risk on aquatic life, and one
to human health.
• Chlorination of reclaimed water did not
improve microbiological quality but it
generated DBPs.
• Indirect potable water reuse becomes a
feasible alternative to cope with severe
droughts.

A R T I C L E I N F O A B S T R A C T

Editor: Damià Barceló The climate change and increasing anthropogenic pressures are expected to limit the availability of water resources.
Hence, active measures must be planned in vulnerable regions to ensure a sustainable water supply and minimize en-
Keywords: vironmental impacts. A pilot test was carried out in the Llobregat River (NE Spain) aiming to provide a useful proce-
Climate crisis
dure to cope with severe droughts through indirect water reuse. Reclaimed water was used to restore the minimum
Water scarcity
flow of the lower Llobregat River, ensuring a suitable water supply downstream for Barcelona. A monitoring was per-
Drought
Drinking water
formed to assess chemical and microbiological threats throughout the water treatment train, the river and the final
Indirect potable reuse drinking water, including 376 micropollutants and common microbiological indicators. The effects of water disinfec-
Microbiological load tion were studied by chlorinating reclaimed water prior to its discharge into the river.
Micropollutants Data showed that 10 micropollutants (bromodichloromethane, dibromochloromethane, chloroform, EDDP,
Llobregat River diclofenac, iopamidol, ioprimid, lamotrigine, ofloxacin and valsartan) posed a potential risk to aquatic life, whereas
one solvent (1,4-dioxane) could affect human health. The chlorination of reclaimed water mitigated the occurrence
of pharmaceuticals but, conversely, the concentration of halogenated disinfection by-products increased. From a mi-
crobiological perspective, the microbial load decreased along wastewater treatments and, later, along drinking
water treatment, ultimately reaching undetectable values in final potable water. Non-chlorinated reclaimed water

⁎ Corresponding author.
E-mail address: anmunne@gencat.cat (A. Munné).

http://dx.doi.org/10.1016/j.scitotenv.2022.161339
Received 25 September 2022; Received in revised form 28 December 2022; Accepted 29 December 2022
Available online 2 January 2023
0048-9697/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A. Munné et al.
showed a lower log reduction of E. coli and coliphages than chlorinated water. However, the effect of disinfection
vanished once reclaimed water was discharged into the river, as the basal concentration of microorganisms in
the Llobregat River was comparable to that of non-chlorinated reclaimed water. Overall, our study indicates that
indirect water reuse can be a valid alternative source of drinking water in densely populated areas such as Barcelona
(Catalonia - NE Spain). A suitable monitoring procedure is presented to assess the related risks to human health and the
aquatic ecosystem.
1. Introduction
Droughts, which are common worldwide, can have severe effects on
water uses and the environment when they are persistent or repeated
over time (Wilhite and Glantz, 1985; Hisdal et al., 2001; European Commis-
sion, 2006; Mendicino et al., 2008). They cause water shortages that can
affect human water-dependent activities such as industrial and agricultural
production, and jeopardize the drinking water supply to urban areas
(Wilhite and Glantz, 1985; Folke et al., 2002; European Commission,
2007). On the other hand, droughts can be considered an important driving
force for the structure of the biological community and aquatic ecosystem
dynamics (Larned et al., 2010), especially in rivers located in Mediterra-
nean climate areas, where droughts are naturally common (Lake, 2003;
Acuña et al., 2005; Prat and Rieradevall, 2006; Bonada et al., 2007). It is
well known that Mediterranean ecosystems are well adapted to their intrin-
sic high flow regime variability, combining dry and wet periods, and
occasionally droughts (Williams, 1996, 2006; Gasith and Resh, 1999).
This makes Mediterranean areas resilient since they present efficient strat-
egies to cope with dry periods and even drought events which result in
scarce or zero flows for a long time (Gasith and Resh, 1999; Lake, 2003;
Williams, 1996). However, an excessive water demand and significant
human alterations over the natural river flow can generate a chronic situa-
tion of water scarcity, which together with drought periods can trigger or
exacerbate negative effects on aquatic ecosystems (Davey and Kelly,
2007; Magalhães et al., 2007; Boix et al., 2008).
Therefore, the occurrence of dry periods, and especially those that last
for a long time, can result in many economic, social, and environmental
impacts (Folke et al., 2002; Vörösmarty et al., 2010). The current society re-
quires a consistent and continuous input of water for domestic, agricultural,
and industrial uses, in the same manner that aquatic ecosystems need
suitable water flows, and water supply failure can result in major impacts.
As a result, urban and industrial areas need additional non-conventional
water resources to face temporary water shortages, and aquatic ecosystems
need to be protected ensuring minimum environmental flows.
On the other hand, climate change has been identified as a driving force
which is worsening droughts through its influence on both rainfall and
evaporation. Rising temperatures are increasing evapotranspiration
through vegetation and therefore drying out soils and diminishing water
runoff and infiltration into groundwater (Gallart et al., 2002; Yan et al.,
2021). With regard to climate impacts, the analysis of global and regional
climate models concludes that under all representative concentration path-
ways (RCPs), potential evapotranspiration (PET) will increase worldwide
as a consequence of increasing surface temperatures and surface water va-
pour deficit (Sherwood and Fu, 2014). According to the IPCC AR5 (IPCC,
2013), a decrease in soil moisture can be detected in Mediterranean
regions. Changes in climate and in land use have resulted in higher aridity
and more droughts in some drylands, with the rising role of precipitation,
wind, and evaporation on desertification (Fischlin et al., 2007). The IPCC
Special Report on Global Warming of 1.5 °C (SR15) (Chapter 3; Hoegh-
Guldberg et al., 2018) concluded with ‘medium confidence’ that global
warming by >1.5 °C increases considerably the risk of aridity in Southern
Europe, and extreme events (i.e. droughts and floods) will increase in the
Mediterranean region (Giannakopoulos et al., 2009). In this context, the
coming recurrent droughts require proactively planned policy instruments
in order to both be well-prepared to respond when they occur, and also
undertake ex-ante actions to mitigate their impacts by strengthening
resilience against them (Gerber and Mirzabaev, 2017).
2
Science of the Total Environment 866 (2023) 161339
Because of this, the European Union (EU) agreed to take an initiative on
water scarcity issues (European Commission, 2007). Droughts must be
managed with prevention measures, and emergency actions should be
planned in advance. This is a new approach to traditional water planning
and society as a whole must make changes to the existing water allocations
(management of water demand) and planning must incorporate extraordi-
nary water resources not used in normal conditions (mitigation measures)
and suitable ecosystem protection requirements (adaptation measures).
The work described herein aims to provide useful information and a
procedure to cope with severe drought scenarios setting a suitable monitor-
ing program through a pilot exercise undertaken in a Mediterranean urban
area (Barcelona city). Reclaimed water from Barcelona's urban wastewater
treatment plant (WWTP) was used to restore minimum flows into a lower
part of a Mediterranean river (the Llobregat River) with a low dilution,
which allowed to ensure water supply for urban uses downstream. The
trial analysed the main chemical and microbiological threats that reclaimed
water could pose to both aquatic life in the river and the drinking water
supply. This pilot test allowed to set a suitable monitoring procedure to
properly protect aquatic life and to ensure a healthy drinking water supply
to Barcelona city when indirect potable water reuse must be undertaken to
face severe drought events.
2. Study area and sampling sites
The pilot test to analyse indirect potable water reuse was carried out in
the lower Llobregat River, close to Barcelona, in Catalonia (NE Spain)
(Fig. 1). Barcelona is located in a Mediterranean climate area that suffers
periodic water scarcity and occasional droughts. The Llobregat basin has
a total drainage area of 4948.3 km2 and the average of accumulative annual
rainfall is 737 mm. However, a high variability is recorded over time which
can range from close to 450 mm up to 1200 mm per year (i.e. 1170 mm in
1971; and 470 mm in 1972).
The Llobregat basin is one of the most industrialized and populated
areas in Europe with over 400 inhabitants per km2, which results in
elevated nutrient loads, high salt concentration and water conductivity
(mainly from mining activity), and the presence of some priority and
hazardous chemical substances due to the scarce environmental dilution
and high concentration of urban and industrial spots, mainly located in
the lower basin (Prat and Rieradevall, 2006; Muñoz et al., 2010). The
Llobregat water discharge is 22 m3/s on average (ACA, 2004), around
15 % of which comes from urban wastewater treatment plants located up-
stream. In drought periods the percentage of water flow that comes from
treated wastewater can account for over 70 % at the river mouth (Munné
et al., 2012). The water discharge has a high variability, with sudden floods
reaching over 500 m3/s, and low flow regimes during the dry season
(summer) of about 5 m3/s, or even close to 2 m3/s in severe drought periods
occasionally (ACA, 2004; Munné et al., 2012). The river flow is regulated
by three dams located at the Llobregat and Cardener River headwaters
(Fig. 1) with a total water storage capacity of 213.3 Hm3, which ensure
minimum flows required for water uses and environmental quality down-
stream. A drinking water treatment plant (DWTP) is located at the lower
Llobregat River, close to the river mouth, which provides water to over
2 million inhabitants of Barcelona and its surrounding area. The water for
drinking supply is mainly obtained from the Llobregat River through to
the DWTP, although groundwater can be also used, especially when severe
droughts may result in a lack of surface water availability or due to an even-
tual episode of poor water quality in the Llobregat River. On the other hand,
A. Munné et al.
Fig. 1. Location of the Llobregat River basin and Barcelona. Location of the monitoring sampling sites of the pilot test carried out to increase water flow in the lower Llobregat
River through indirect water reuse. Barcelona's urban wastewater treatment plant (WWTP), the water reuse treatment plant (WRTP), and the drinking water treatment plant
(DWTP) are also shown. Site W0 is located at the WWTP influent; W1 is at the WWTP effluent; W2 site is located at the WRTP effluent just before the chlorination facility; W3
is located at the water reuse discharge to the river; R0 and R1 are sites located in the river, R0 is upstream and R1 is downstream from the water reuse discharge; and D1 site is
located at the DWTP output to Barcelona's potable water supply network.
a significant amount of wastewater from the Barcelona is treated by a large
urban wastewater treatment plant (WWTP) also located close to the river
mouth. An average of 3.23 m3/s treated urban wastewater from Barcelona
is discharged into the Mediterranean Sea through a 6 km-long marine out-
fall. However, a total of 62 % of this treated wastewater (up to 2 m3/s) can
be reused via a water reuse treatment plant (WRTP) located next to the
Barcelona's WWTP and then be pumped and released 16.6 km upstream
to increase the water flow in the Llobregat River (Fig. 1). This procedure
can provide an additional water flow in the lower Llobregat River aiming
to ensure both the aquatic ecosystem's quality and the water supply to
Barcelona.
Barcelona's urban wastewater currently undergoes a primary process
which consists of an initial roughing and flocculation/settlement followed
by a conventional secondary biological treatment by activated sludge
with nutrient removal. Up to 70 % of the total nitrogen and phosphorus is
removed in the secondary treatment using nitrification and denitrification
processes. Additionally, water from the secondary treatment can be
partially treated in a WRTP with a flocculation and sedimentation process
ballasted with microsand (ActifloDisc®) followed by lamellar decanters
and a 10 μm-mesh sieve. Disinfection is fulfilled by using low pressure/
high intensity UV light in open channel (50 mJ/cm2), and optional chlorine
addition. The chlorine dose for the chlorinated campaigns of this pilot test
was 13 mg/L.
The drinking water treatment plant (DWTP) takes raw water from the
lower Llobregat River. Surface water is collected from the river, chlorine
dioxide and carbon dioxide are added as initial disinfection, followed by
a coagulation/flotation step. Afterwards, the water is driven through sand
filters. At this point, underground water from the Llobregat aquifer can be
added, and then, the water is split into two parallel treatment lines. The
upgraded conventional line consists in ozonation followed by granular acti-
vated carbon filtering (GAC). The advanced treatment involves membrane
technology (ultrafiltration followed by reverse osmosis). Water from both
lines is mixed and chlorine is added to ensure the total disinfection before
the water is pumped into the supply network.
The pilot test consisted of an investigative monitoring which was car-
ried out in order to carefully analyse possible effects on the aquatic ecosys-
tem as well as on the quality of drinking water in terms of human health
3
Science of the Total Environment 866 (2023) 161339
resulting from the reused water discharged upstream of the water intake
of Barcelona (Fig. 1). Sampling sites were set in raw wastewater just before
the WWTP (W0), after the biological wastewater treatment (W1), after the
water reuse treatment (W2), and just before the water discharge to the
Llobregat River 16.6 km upstream from the WWTP (W3). Two sampling
sites were located in the Llobregat River, one just before the reused water
discharge to the river (R0), and another 8.5 km downstream after the
water discharge (R2) just before the water withdrawal towards the drinking
water treatment plant (DWTP). An additional sampling site was located at
the DWTP output, which supplies drinking water to Barcelona, to ensure
quality standards for drinking water distribution (D1). Samples were
collected sequentially over time according to the hydrological retention in
the water treatments and flow velocity in the pipes and in the river to
allow comparing results among sampling sites (Table 1). Chemical samples
collected in W1 and W2 were integrated samples for a period of 4 h in order
to be representative enough, whereas chemical samples in W3, R0, R2 and
D1 and all microbiological samples were single samples. Only microbiolog-
ical samples were taken in W0 in order to assess microbiological health-
based targets (in terms of log reductions of pathogens) along the whole
water treatment process. Chemical and microbiological samples were
taken at W1, W2, W3, R0, R1 and D1 in order to analyse the WRTP's perfor-
mance, the effect of chlorination of reclaimed water, the chemical and
microbiological abatement throughout the treatment train, pipe transport
and river abatement, and finally the DWTP's performance when water
was supplied to Barcelona. The quality elements analysed in each sampling
site can be found in Table 1.
The pilot test was planned for six weeks during June and July of 2019
(summer) in order to find a dry period with scarce water dilution, aiming
for the worst scenario (like a severe drought). The first samples were col-
lected on June 4, and the last ones on July 24 (Table 2). We also analysed
the effect of adding chlorine after the WRTP on microbiological elements
(disinfection) and chemical compounds (chemical abatement/removal
and appearance of chlorination by-products). Chlorine was not added
after the WRTP for campaigns 1 to 4, whereas it was added just after the
W2 sampling site in campaigns 5 and 6 (Table 2). The chlorine dose
added after the W2 site was 13 mg Cl2/L. On the other hand, chlorine
was always added at the drinking water treatment plant (DWTP) effluent,
A. Munné et al. Science of the Total Environment 866 (2023) 161339

Table 1
Sampling sites of the pilot test carried out to in the lower Llobregat River. The pilot test lasted six weeks during two months (June and July). Samples were collected sequen-
tially at different times according to the treatment process time and river flow. Site W0 is located at the WWTP influent; W1 at the WWTP effluent; W2 site is located at the
WRTP effluent but just before the chlorination facility; W3 is located at the reused water discharge to the river; R0 and R1 are sites located in the river, R0 is upstream and R1
is downstream of the reused water discharge; and D1 site is located at the DWTP output to the Barcelona potable water supply network (see Fig. 1).
Sampling site code Time of sample collection Quality elements Type of sample collection

W0 20:00 on the previous day Microbiological Grab sample

Grab sample (microbiological)
W1 7:00 Microbiological and chemical
Integrated samples 4 h. (chemical)
Grab sample (microbiological)
W2 9:00 Microbiological and chemical
Integrated samples 4 h. (chemical)
W3 12:00 Microbiological and chemical Grab sample
R0 12:00 Microbiological and chemical Grab sample
R1 16:00 Microbiological and chemical Grab sample
D1 22:00 Microbiological and chemical Grab sample

just before the D1 sampling site, to assure quality standards for drinking microbiological elements were selected and prioritised considering envi-
water supply. ronmental and health criteria.
Microbiological elements were analysed in all sampling sites (W0, W1, A preliminary list of micropollutants that could potentially be present in
W2, W3, R0, R1 and D1) in campaigns 1, 2, 3, 5 and 6, whereas wastewater and reclaimed water was drawn up. This list of candidates was
micropollutants were analysed in W1, W2, W3, R0, R1 and D1 sampling set by using several sources (Fig. 2). Daily doses of drugs prescribed at the
sites for all campaigns. Basic Health Areas (ABS) in Barcelona were obtained from the Public
The river flow was set as low as possible (around 1-2 m3/s) during the Health Agency of Catalonia (ASPCAT). The list was enlarged by drugs
pilot test by regulating the flows from the reservoirs located upstream (Baells used in hospitals located in the Barcelona urban area (i.e., antivirals,
and St. Ponç reservoirs), while reclaimed water was set at about 1.6 m3/s in cytostatic compounds, radiological contrast agents, hormones, etc.). There-
order to achieve a dilution rate close to 1:1 or 1:3 (as low as possible) simu- fore, a total of 570 pharmaceuticals and their metabolites were included in
lating the worst-case scenario of an extreme drought. Most samples were the preliminary list (Table S1 - Supplementary material). Additionally,
taken at the expected dilution rates or slightly above (Table 2). The pilot chemicals from industrial activity were also included. Thus, discharge
test was temporarily suspended between campaigns 1 and 2 (in the second water licenses for industries located in Barcelona and surrounding areas
week of July), after 1:1 rate dilution, due to a sudden increase of primary connected to the Barcelona WWTP were screened to enlarge the prelimi-
production (eutrophication) and high concentration of organic matter nary list of micropollutants (e.g., PBDEs, PFOS, organic solvents, PAHs,
(DOM) detected in the river, which put the drinking water potabilization etc.). Pesticides used in Barcelona by local authorities in parks, streets
at risk and required increase river flow. The trial was resumed after the and gardens, and biocides used in sewer network control, mainly insecti-
quality of the river water was restored and the DOM decreased. cides and rodenticides (e.g. brodifacoum, difenacoum, difetialona,
bromaliodone, etc.), were also included. Moreover, chemicals found in pre-
vious analyses carried out in the lower Llobregat River or Barcelona
3. Methods WWTP's water discharge (e.g. SCARCE project, 2010) were also added in
the preliminary list, including mainly household products such as food
3.1. Selection of micropollutants and microbiological elements to be analysed additives and preservatives, biocides, body care products, etc. We also
checked and included chemicals previously found at relevant concentra-
The pilot test aimed to identify the main chemical and microbiological tions in monitoring programs of the Catalan Water Agency (public water
threats that reclaimed water could pose on both the river ecosystem authority in charge of water quality in Catalonia) in the Llobregat River
and the drinking water supply. Therefore, chemical compounds and close to the river mouth (data can be found at the Catalan Water Agency
webpage), as well as those detected in monitoring surveillance of drinking
Table 2 water by Aigües de Barcelona (a company in charge of drinking water
The sampling date (in bold) and the day before are shown for non-chlorination and potabilization and supply to Barcelona). Possible chlorination by-products
chlorination campaigns. Dates in bold indicate the day that the sample was taken at were also considered and included in the preliminary list taking into
W1, W2, W3, R0, R1 and D1 (see Table 1), with the exception of microbiological account that reclaimed water can be chlorinated (e.g., NDMA, THMs,
samples at W0, which were taken at 20:00 h the day before. River flow at R0 and HAAs). Finally, regulated chemicals in Europe with current quality stan-
R1, and dilution of WRTP discharge vs. river flow (at R0) is shown. dards for drinking water (EU Directive 2020/218/EU) and for water quality
Option Campaigns Date River flow River flow Dilution (WRTP in rivers (EU Directive 2013/39/EU) were added if they had not already
at R1 at R0 flow vs. river been included in the preliminary list (e.g., some VOCs, pesticides, heavy
(m3/s) (m3/s) flow at R0)
metals, etc.). Therefore, a total of 835 micropollutants were added to the
Without 1 03/06/19 4.14 1.4 1:2 preliminary list as candidates to be detected at the Barcelona WWTP afflu-
chlorination 04/06/19 2.78 1.6 1:1
ent (pharmaceuticals listed in Table S1 - Supplementary material, plus non-
after W2 site 2 18/06/19 4.93 1.7 1:2
19/06/19 4.78 1.7 1:2 pharmaceuticals listed in Table S2 - Supplementary material).
3 25/06/19 7.08 1.7 1:3 Because of the high number of pharmaceuticals and their metabolites
26/06/19 7.19 1.6 1:3 (n = 570) obtained from Basic Health Areas and hospitals, they were re-
4 02/07/19 6.78 1.6 1:3 viewed and some of them were rejected according to their low possibility
03/07/19 6.01 1.6 1:3
to be found at concentrations of concern. Thus, pharmaceuticals that can
With chlorination 5 16/07/19 6.54 1.7 1:3
after W2 site 17/07/19 11.13a 1.6 1:6 be found at the wastewater effluent were assessed using the defined daily
6 23/07/19 6.29 1.6 1:3 dose (DDD) obtained for all pharmaceuticals supplied by Basic Health
24/07/19 5.71 1.7 1:2 Areas and hospitals. The DDD is the assumed average maintenance dose
a
A higher river flow was discharged from upstream reservoirs with the aim to per day for a drug used for its main indication in adults, and it is recom-
increase dilution from the WRTP as a preventive measure in the first chlorination mended by the WHO as a measuring unit for drug monitoring (WHO -
campaign. Collaborating Centre for Drug Statistics Methodology, 2021). The estimated

4
A. Munné et al.
Fig. 2. Flowchart of the selection of chemical compounds to analyse chemical hazards for the aquatic ecosystem and human health from water reuse. Guide values are based
on guidelines used in Australia (NRMMC-EPHC-NHMRC, 2008).
concentration of each drug in the wastewater influent (ug/L) was calculated
by assuming the indication dose used in adults for DDD (from WHO) with
the total DDD (DDDt) obtained from Basic Health Areas and hospitals in
Barcelona, and considering the wastewater inflow per day that comes to
the Barcelona WWTP:
μg
Estimated concentration in waste water influent
L hormones) which due to their potential carcinogenicity must be considered
mg 1000 μg
DDDt  ðWHOÞ 1 mg
¼ DDD  3
m 1000 L
waste water inflow
day 1 m3
On the other hand, a guide value (GV) was calculated for all 570 pharmaceu-
ticals and X-ray contrast agents. The WHO Drinking Water Guidelines include
guide values for a wide range of potential chemical contaminants in drinking
water, including naturally-occurring chemicals, pesticides, and industrial
micropollutants (WHO, 2017). However, for many pharmaceuticals there
are no established guide values for drinking waters nor to assess chemical sta-
tus in rivers, so other approaches must be used to set guide values such as the
guidelines used in Australia (NRMMC-EPHC-NHMRC, 2008) or PNEC values
for aquatic life. Regarding human health, we applied the Australian guide-
lines for pharmaceutical guide values derivation on human health, which
follow an approach based on two different pharmaceuticals groups: those
used only for humans and those used for veterinary purposes (some of
which can also be used for humans) (Fig. 2).
Thus, regarding drugs used for veterinary purposes, experts such as the
Joint FAO-WHO Expert Committee Report on Food Additives (JECFA) and
the European Medicines Agency (EMEA) established an acceptable daily
intake (ADI) that can be used to set the guide values:
ADI ðmg=kg bw=day Þ  bw ðkg Þ  P
Guide Value ðGV Þ ¼
V ðL=day Þ
where: V: water consumption per person (2 L/day); ADI: acceptable daily
intake; bw: body weight; P: proportion of ADI attributable to water
consumption (100 %).
When ADI values were not available, such as those used only for
humans, guide values were set by using the minimum therapeutically
effective dose (MTED) following a similar calculation approach. Since the
5
Science of the Total Environment 866 (2023) 161339
minimum therapeutically effective doses are well established, and given
that they generally have a high therapeutic index (ratio beneficial dose/
toxic dose), the use of the minimum therapeutically effective dose for deri-
vation of guide values gives a sufficient safety margin for human health.
When establishing guide values, the minimum dose with therapeutic effect
was divided by safety factors of 1000 (for most drugs) or 10,000 in the case
of hazardous compounds (e.g., cytotoxic compounds or synthetic or natural
with a higher safety factor. Pharmaceutical metabolites may also be present
in water. However, their activity is usually considered lower than the active
ingredient. The application of safety factors of 1000 and 10,000 provides a
sufficient safety buffer to consider the possible effects of metabolites. There-
fore, a guide value can be assessed by using MTED for those drugs without
ADI data available for human health analysis:
 
mg
MTED  P  103
day
Guide Value ðGV Þ ¼
SF  V ðL=dayÞ
where: V: water consumption per person (2 L/day); MTED: minimum
therapeutically effective dose; P: proportion of MTED attributable to
water consumption (usually 100 %); SF: safety factor (1000 for most
drugs; 10,000 for hormones, cytotoxic and hazardous compounds).
Once GVs were obtained for all 570 pharmaceuticals, we then assessed a
risk coefficient (RC) as a ratio between the estimated concentration for each
drug at the wastewater effluent divided by the guide value (GV) for each
compound. Thus, a RC < 1 indicates that the estimated concentration is
under the guide value. We selected pharmaceuticals with a RC < 10 to be
removed from the initial list. A coefficient risk (RC) below 10 was consid-
ered safe enough regarding their possible occurrence in the reclaimed
water did not pose a significant potential risk.
Estimated concentration in wase water effluent
RC ðRisk Coefficient Þ ¼
Guide Value ðGV Þ
Therefore, a total of 111 out of the 570 pharmaceuticals and X-ray contrast
agents prescribed in Basic Health Areas and used in hospitals located in
Barcelona were selected. Therefore, the total amount of chemicals,
considering selected pharmaceuticals and the potential remaining
A. Munné et al.
micropollutants that could be found at the Barcelona WWTP influent, were
376 (Table S2 - Supplementary material), which were analysed in sampling
sites W1, W2, W3, R0, R1 and D1 (Table 1 and Fig. 1).
Microbiological elements were also selected to be analysed according to
both the main international recommendations for safety wastewater reuse
(WHO, 2017) as well as the new European Union regulation on water
reuse (EU, 2020b). Microbial indicators associated with major groups of
pathogens (bacteria, viruses, and protozoa) were selected: E. coli as an indi-
cator of pathogenic bacteria, somatic coliphages as indicators of pathogenic
viruses, and Clostridium perfringens spores as an indicator of protozoa. These
microbial indicators were analysed in all the sampling sites (W0, W1, W2,
W3, R0, R1 and D1).
Regarding water for human consumption, the WHO Guidelines for
Drinking Water Quality (WHO, 2017) establish the tolerable burden of dis-
ease (caused by either a chemical or an infectious agent) as an upper limit of
10–6 Disability Adjusted Life Years (DALYs) per person and per year (pppy).
They are mainly used for microbiological risks in drinking water. The toler-
able burden of disease of 10-6 DALYs, or level of acceptable risk, must be
translated into a system performance target (log reductions) required
with the treatment steps to achieve the health goal. Therefore, microbiolog-
ical safety is usually defined in terms of achieving performance targets
expressed as logarithmic reduction value (LRV) of reference pathogens
potentially present in the source waters (wastewater).
No quantitative microbial risk assessment was performed because of the
absence of system-specific pathogen data. The performance targets were
established considering the WHO guidelines (WHO, 2017), the Australian
guidelines for water reuse practices (NRMMC–EPHC–AHMC, 2008), and
EPA Guidelines for Water Reuse (US Environmental Protection Agency,
2012). Therefore, the logarithmic reduction values of microbiological
indicators were used to assess microbiological risk.
3.2. Analytical methods
Because of the wide variety and heterogeneity of micropollutants
included in the present study, their chemical analysis was conducted
following a multi-methodological approach. A comprehensive list of the
analytes and their respective methods can be found in Table S2 and each
analytical method is thoroughly described in the supplementary material
(Text S1 and Table S2). Briefly:
• Chloroalkanes (C10-C13) were extracted by liquid-liquid extraction (LLE)
and extracts were analysed by gas chromatography with an electron
capture detector (GC-ECD).
• Dioxanes and dioxolanes were extracted by solid phase extraction (SPE)
and analysed by gas chromatography coupled with tandem mass spec-
trometry (GC–MS/MS) (Carrera et al., 2017).
• Bromodiphenyl ethers (BDEs), polycyclic aromatic hydrocarbons (PAHs)
and tributyltin cations were analysed by stir bar solid extraction
(Twister®) and gas chromatography coupled with tandem mass spec-
trometry (GC–MS/MS).
• Some volatile organic compounds (VOCs) and volatile disinfection by-
products (DBPs) such as trihalomethanes (THMs) were analysed by
purge-and-trap and gas chromatography coupled with mass spectrometry
(PT-GC/MS).
• Haloacetic acids (HAAs) were analysed by liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS) with direct injec-
tion (Planas et al., 2019).
• Illicit drugs and related metabolites, caffeine and paraxanthine, and food
additives, such as acesulfame and sucralose, were analysed by off-line SPE
and LC-MS/MS (Boleda et al., 2011).
• A selection of volatile and semi-volatile compounds included in the Water
Framework Directive (WFD) 2000/60/EC were analysed by dispersive
liquid-liquid micro-extraction and gas chromatography coupled with tan-
dem mass spectrometry (DLLME-GC–MS/MS) (Rubirola et al., 2019).
• Perfluoroalkyl substances (PFAs); pharmaceutical compounds, X-ray con-
trast media, antiseptics, and related metabolites; pesticides and biocides;
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Science of the Total Environment 866 (2023) 161339
and miscellaneous compounds included in the WFD 2000/60/EC were
analysed by diverse methods based on on-line SPE-GC–MS/MS (Flores
et al., 2013; Rubirola et al., 2017; Quintana et al., 2019).
• Miscellaneous food additives, rodenticides and pharmaceutical com-
pounds were analysed by liquid chromatography coupled with mass spec-
trometry with a hybrid quadrupole-time of flight analyser (LC-QToF-MS).
• Total metals were determined by inductively coupled plasma – atomic
emission spectrometry (ICP-AES), inductively coupled plasma – mass
spectrometry (ICP-MS) or, in the case of Hg, cold vapour atomic fluores-
cence spectroscopy (CV-AFS), with an acid microwave digestion pre-
treatment.
Regarding microbiological indicators, culturable E.coli were counted
using the most probable number method (MPN) according to UNE EN
ISO 9380:2, somatic coliphages were enumerated according to UNE EN
ISO 10705-2, spores of Clostridium perfringens were enumerated according
to an internal method accredited by ISO 17025, and based on UNE EN
ISO 14189:2017. Methods are thoroughly described in the supplementary
material (Text S2).
3.3. Screening guide values for chemical hazards
Guide values (GV) were set for analysed micropollutants the results of
which surpassed the limit of quantification (LOQ). GV were used to prop-
erly assess chemical hazards on both human health (based on drinking
water) and aquatic biota (ecosystem effects). A chemical guide value is
that for which there is a dose below which no adverse or toxic effects
will occur. Therefore, two different GVs were set for all analysed
micropollutants; a GV indicating a threat to human health based on drink-
ing water risk analysis (human health: HH), and another GV indicating that
the micropollutant can affect aquatic life in rivers (ecosystem effect: EE)
mainly based on PNEC (predicted non-effect concentration) values assessed
on aquatic biota (Table S3 - Supplementary material).
Regarding risk analysis on human health (HH), GVs for pharmaceuticals
were set following the steps explained above according to their acceptable
daily intake (ADI) or their minimum therapeutically effective dose (MTED).
In case of food additives, as well as pesticides or veterinary drugs (residues
of which may be found in food), the guide values were calculated using the
acceptable daily intake (ADI) approach. Additionally, GVs were also calcu-
lated for all remaining selected chemicals using a tolerable daily intake
(TDI) value derived from NOAEL (no observed adverse effect level) or
LOAEL (lowest observed adverse effect level) applying uncertainty factors
as additional safety measure. Thus, in these cases GVs were set as:
NOAEL o LOAELðmg=kg bw=day Þ  bw ðkg Þ  P
Guide Value ðGV Þ ¼
SF x V ðL=dayÞ
where: V = water consumption per person (2 L/day); NOAEL (no observed
adverse effect level); LOAEL (lowest observed adverse effect); bw = body
weight; P = proportion attributable to water consumption (10 or 20 %);
SF = security factor (1500).
When GVs were not able to be set using the above-mentioned methods,
the threshold of toxicological concern (TTC) approach was used. In the
absence of toxicological data, the TTC approach is becoming widely
accepted particularly for food contaminants (Kroes et al., 2005). The TTC
concept is an approach that aims to establish a human exposure threshold
value below which there is a very low probability of an appreciable risk
to human health, applicable to chemicals for which toxicological data are
not available. The TTC approach is based on toxicity data of structurally
related chemicals. Quality standards for drinking water were also taken
into account as GVs on human health (HH) when micropollutants were
included in the EU Directive on the water quality for human consumption
(EU, 2020a) (Table S3 - Supplementary material).
Regarding the environmental risk for aquatic biota (ecosystem effect: EE),
the PNEC (predicted no effect concentration) values were considered as a GV.
A. Munné et al.
The lowest PNEC value was obtained from the NORMAN Ecotoxicology
Database (2019). The NORMAN Ecotoxicology Database contains experi-
mental endpoints from ecotox tests as well as quality targets from different
regulatory contexts. An assessment factor for each chemical according to
biological species was considered (Table S4 - Supplementary material).
Additionally, environmental quality standards (EQS) from the EU Directive
for priority substances (EU, 2013, 2018) expressed as annual average values
(AA-EQS) were also considered as GVs (see Table S3 - Supplementary
material).
3.4. Data analysis
The most restrictive GVs (Table S3 - Supplementary material) were used
to compare the results of measured concentration (MEC) for each chemical
compound considering both human health (HH thresholds) and aquatic
ecosystem effects (EE thresholds). Thus, the GVs of all the analysed chemi-
cal compounds were compared to their results at the effluent of the WWTP
before and after the WRTP (sampling sites W1 and W2), just before the
water reuse discharge into the river (sampling site W3) considering both
the chlorinated and non-chlorinated treatments, in the river before and
after the water reuse discharge (sites R0 and R1), and after the DWTP,
where potable water is supplied to Barcelona (site D1). Furthermore, the
risk of the microcontaminants dissolved in the potable water samples was
estimated using a quantitative approach adapted from López-Roldán et al.
(2016): Briefly, individual risk indexes (Wi) were calculated for the i
detected analytes as their MECi to GVi ratios. These parameters were used
for tentative health risk assessment (W > 1 suggests potential risks, whereas
W ≤ 1 indicates no foreseeable risk for the component i) and for compari-
son among campaigns only for drinking water, after the DWTP (site D1).
The list of analytes included a wide variety of micropollutants, presenting
very different natures and modes of action. A global risk index assuming
additive effects was not calculated as that assumption would imply that
chemicals that produce the same adverse health outcome act in the same
way, which may not be the case (NRMMC-EPHC-NHMRC, 2008; EFSA,
2019). Chlorination and non-chlorination of reclaimed water was analysed
comparing campaigns 1, 2, 3 and 4, with campaigns 5 and 6. Microbiolog-
ical abatement was assessed taking into account both, the treatment
processes from site W0 to site W3 (with and without chlorination after
W2); and also considering the complete water conduction from site W0
until the output of the drinking water treatment plant (DWTP) (site D1)
to obtain the whole abatement data. Chemical abatement was assessed
from W1 to W3 sampling sites (considering chlorination and non-
chlorination after W2), and from R0 to R1 to analyse changes in the river
composition and their possible effects on aquatic life after the water reuse
discharge. Data were also compared from R2 to D1 to analyse changes in
drinking water treatment for human health. Chlorination by-products
were carefully checked from W3 to D1 sampling sites.
4. Results
4.1. Chemical compounds
A total of 102 out of 376 analysed micropollutants (pharmaceuticals,
food additives, flame retardants, industrial solvents, cosmetics, household
related products, pesticides, biocides, cytostatic compounds, etc.) were
detected above the limit of quantification (LOQ) considering in any sample
(Table S5 - Supplementary material), that accounts for 27 % of the total
amount of analysed chemical compounds. Regarding the preliminary list
of micropollutants originally considered as candidates to be found at the
Barcelona WWTP influent, the detected micropollutants (>LOQ) account
for 12 %. Thus, a high amount of pre-selected chemicals (73 %) was unde-
tectable or considered not relevant since they always showed concentra-
tions below the limit of quantification (LOQ), or their occurrence was
considered not relevant due to previous selection analysis.
Regarding micropollutants with values above their LOQs, only 39 out of
the 102 were detected at some sampling site above their GVs (HH or EE
7
Science of the Total Environment 866 (2023) 161339
thresholds). However, 14 out of these 39 compounds (sucralose, diuron,
mecoprop, N-nitrosodimethylamine (NDMA), azithromycin, gemfibrozil,
salbutamol, ciprofloflaxin, doxycycline, furosemide, triclosan, chlorpyrifos,
clarithomycin and lorazepam) only exceeded their GV threshold within the
wastewater treatment train (sewage treatment plant and reclaimed water:
sites W1, W2 or W3), never in the river (sites R0 or R1) nor after the
water potabilization process for the drinking supply (site D1); and 22
micropollutants were detected above their GVs (HH or EE) outside the
wastewater treatment and reclaimed water train, in the river or in the
drinking water supply network (sites R0, R1 or D1). A total of 1 compound
and 2 compound families (carboxylosartan; perfluoroctanosulfonic acid,
PFOS; and bromodiphenyl ethers, BDEs) had LOQs higher than their GVs
(HH or EE), so even though all samples showed concentrations below
their LOQs, it could not be possible to conclude if they could pose a risk
or not to aquatic life or human health.
Regarding the possible effect on aquatic life, a total of 15 micropollutants
exceeded their VGs (EE thresholds) in the Llobregat River (sites R0 and/or
R1) (Fig. 3). Most of them were drugs or radiological contrast agents
(iomeprol, diclofenac, lamotrigine, iopamidol, ioprimid, ofloxacin, valsartan,
losartan and diazinon), the major metabolite of methadone (EDDP, that is 2-
ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine), the neonicotinoid insecti-
cide imidacloprid, a herbicide (terbutryn), and some chlorinated by-
products (DBPs) or industrial compounds such as bromodichloromethane,
dibromochloromethane and chloroform, especially when reclaimed water
was chlorinated. Out of the 15 micropollutants that exceeded any EE
threshold in the river, a total of 10 were not detected above EE threshold in
site R0 (upstream) but after the reclaimed water discharge in the Llobregat
River (site R1) (Fig. 3). Thus, the occurrence of these 10 micropollutants,
mostly pharmaceuticals as well as some industrial compounds and
DBPs (bromodichloromethane, dibromochloromethane, chloroform, EDDP,
diclofenac, iopamidol, ioprimid, lamotrigine, ofloxacin and valsartan),
which could pose a risk for aquatic biota, can be considered to have been
caused by the reclaimed water discharge into the river. On the other hand,
diazinon, iomeprol, losartan and terbutryn were already found above the
EE threshold upstream before the reclaimed water discharge (site R0). The
Llobregat River already showed high micropollutants concentration from
human activity located upstream. Terbutryn increased its concentrations
after the reclaimed water discharge into the river, even though the EE thresh-
old was already slightly exceeded upstream; and diazinon, iomeprol and
losartan showed similar concentrations before and after the reclaimed
water discharge (R0 and R1 sites), always above the EE threshold.
Regarding the chlorination effect on reclaimed water and its subsequent
discharge into the river, broadly, concentrations of pharmaceutical
compounds and illicit drugs were higher when reclaimed water was not
chlorinated rather than chlorinated. Conversely, concentrations of DBPs
such as bromodichloromethane and dibromochloromethane were higher
when reclaimed water was chlorinated and below their LOQs when non-
chlorinated. Surprisingly, chloroform also appeared over the EE threshold
in some samples when reclaimed water was not chlorinated. Therefore,
chloroform could be attributed to some industrial activity instead of being
a chlorinated by-product.
Regarding the possible impact on human health through the drinking
water supply, only 6 micropollutants were found above their HH threshold
in the river (sites R0 or R1) or after the DWTP (site D1) (Fig. 4). Most of
them overcame their HH only in the river (sites R0 and R1) before the
water intake to the drinking water treatment. So, the herbicide glyphosate
and its metabolite AMPA (aminomethylphosphonic acid), the insecticide
imidacloprid and the illicit drug MDMA (3,4-methylenedioxyamphetamine)
were detected above the HH thresholds only in river sampling sites (R0 or
R1) but not after the drinking water treatment plant (site D1). Additionally,
the insect repellent DEET (N,N-diethyltoluamide) was detected above the
HH threshold mostly in the river (sites R0 and R1), though the HH threshold
was also slightly exceeded in one sample after the drinking water treatment
(site D1). Imidacloprid already exceeded the EE threshold in some samples
before the reclaimed water discharge (R0) but not the HH threshold, and
after the reclaimed water discharge (R1) it exceeded both HH and EE
A. Munné et al. Science of the Total Environment 866 (2023) 161339

Fig. 3. Micropollutants that exceed VGs for aquatic life (EE thresholds) in the Llobregat River (sites R0 and/or R1). Samples are shown in different colours (blue, samples
collected in the river; red, samples collected throughout the wastewater and reclaimed water treatment train; purple, samples collected after the drinking water
treatment). Circles indicate when reclaimed water was not chlorinated (campaigns 1, 2, 3 and 4), and asterisks indicate samples collected when reclaimed water was
chlorinated after W2 sampling site (campaigns 5 and 6). Guide values (GVs) for aquatic life (EE threshold) and human health (HH threshold) are also shown. A solid line
indicates that the GV comes from regulated requirements (quality standards from the European Union) whereas dashed lines indicate GVs assessed from not regulated sources
(see Table S3 – Supplementary material).

thresholds. MDMA only exceeded the HH threshold after the reclaimed water
discharge (site R1). Both MDMA and imidacloprid increased their occurrence
and surpassed their HHs in the river due to the reclaimed water discharge,
whereas glyphosate, AMPA and DEET already exceeded their HH thresholds
upstream (sites R0 and R1).
Special attention must be paid to the organic solvent 1,4-dioxane. It was
detected in the river over its HH threshold after the reclaimed water
discharge (site R1), not before (R0), and also slightly exceeded its HH
threshold after the drinking water treatment (site D1) in a single sample
when reclaimed water was not chlorinated. That did not pose any risk to
human health since water was rapidly mixed with other water sources
and concentrations were diluted to meet drinking water quality standards.
However, 1,4-dioxane was proven to be the main micropollutant that can
pose a highest risk on human health through the drinking water supply
due to the reclaimed water discharge (indirect potable reuse). The quantita-
tive risk assessment of potable waters also confirmed this observation. As it
can be seen in Table 3, 1,4-dioxane presented the highest risk for the human
health every week. In week 4, a high concentration of DEET was registered
(151 ng/L) at the D1 site, which posed a high quantitative risk as well.
However, the quantitative risk assessment carried out for DEET must be
considered carefully since we used 100 ng/L as its HH threshold, as for
any pesticide according to the EU Drinking Water Directive (EU Directive

8
A. Munné et al. Science of the Total Environment 866 (2023) 161339

Fig. 4. Micropollutants that exceed VGs for human health (HH thresholds) in the Llobregat River (sites R0 and/or R1) or in the drinking water supply (site D1) after the
reclaimed water discharge. Samples are shown in different colours (blue, samples collected in the river; red, samples collected throughout the wastewater and reclaimed
water treatment train; purple, samples collected after the drinking water treatment). Circles indicate when reclaimed water was not chlorinated (campaigns 1, 2, 3
and 4), and asterisks indicate samples collected when reclaimed water was chlorinated after W2 sampling site (campaigns 5 and 6). Guide values (GVs) for aquatic life
(EE threshold) and human health (HH threshold) are also shown. Dashed lines indicate GVs (see Table S3 – Supplementary material).

2020/218/EU), but some health authorities have set the threshold of
concern for DEET up to 200 μg/L for drinking water (MDH, 2013). DEET
actually is an insect repellent which makes it not relevant to pose a risk
on human health when using 200 μg/L as the HH threshold for drinking
water. Therefore, the future implementation of water reuse schemes
in the area should involve effective measures to attenuate the levels of
1,4-dioxane in the discharged wastewater and/or to improve its removal
in the drinking water treatment plant. DEET should also be reduced and
monitored carefully. Other micropollutants with a non-negligible risk index
(>0,1) included an illicit drug, MDMA; two insecticides, imidacloprid and
acetamiprid; a disinfection by-product, bromoform; and the anticonvulsant
gabapentin as contaminants of interest to be reduced or carefully monitored,
even though they showed a low risk index in all campaigns (Table 3).
Finally, the chlorination of reclaimed water did not have a statistically
significant impact on the risk of drinking water according to Mann Whitney
tests conducted with a significance level (α) of 0.05. The concentrations of
the detected DBPs (THMs and NDMA), and hence their risk, were fairly
stable in the potable water during both stages (chlorinated and non-
chlorinated) of the pilot test.
4.2. Microbiological indicators
Microbial load decreased along the two treatment processes, first through-
out the WWTP and WRTP (from W0 to W3 sampling sites) and later through-
out the DWTP, until reaching undetectable values (<1/100 mL) in the
drinking water supply (at the D1 sampling site) in both chlorinated and non-
chlorinated reclaimed water campaigns (Fig. 5). Chlorine was added at
the DWTP effluent in all campaigns as usual.
Non-chlorinated reclaimed water campaigns showed between 4 and 5
log reduction of E. coli, and between 4 and 6 log reduction of somatic
coliphages between the W0 and W3 sites (throughout the wastewater
treatment process), whereas chlorinated reclaimed water campaigns
showed around 6 log reduction for E. coli and somatic coliphages
(Tables 4 and 5). Clostridium perfringens spores presented around 2-3 log
reduction when reclaimed water was non chlorinated, but only 3-4 log
reduction when chlorinated. Therefore, chlorination was effective to signif-
icantly reduce bacteria and virus rather than protozoa after the reclaimed
water treatment. However, the chlorination effect was no longer noticeable
once reclaimed water was delivered into the Llobregat river, where

Table 3
Quantitative risk index (Wi) adapted from López-Roldán et al. (2016) for each i detected analytes. Wi values are based on their MECi and GVi ratios. Wi > 1 suggests potential
risks, whereas Wi ≤ 1 indicates no foreseeable risk for the component i.
Parameter VGi (μg/L) MECi (μg/L) Wi (MEC/VG)

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

1,4-dioxane 50 61.5 48.0 32.6 39.2 36.0 37.4 1.23 0.96 0.65 0.78 0.72 0.75
2,6-ditert-buthyl-4-methylphenol 2 n.d. 0.015 n.d. 0.017 n.d. n.d. <0.01 0.01 <0.01 0.01 <0.01 <0.01
Acesulfame 28,000 0.075 0.125 0.107 0.144 0.121 0.084 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Acetamiprid 0.1 0.016 n.d. n.d. n.d. n.d. n.d. 0.16 <0.01 <0.01 <0.01 <0.01 <0.01
Cotinine 10 n.d. n.d. n.d. n.d. 0.021 0.011 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
DEET 0.1 n.d. n.d. n.d. 0.151 n.d. 0.038 <0.01 <0.01 <0.01 1.51 <0.01 0.38
Desmethyltramadol 25 0.190 n.d. n.d. n.d. n.d. n.d. 0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Gabapentin 10 1.15 n.d. n.d. n.d. n.d. n.d. 0.12 <0.01 <0.01 <0.01 <0.01 <0.01
Imidacloprid 0.1 0.012 0.007 0.007 0.006 0.006 0.006 0.12 0.07 0.07 0.06 0.06 0.06
Iomeprol 6,700 0.079 0.266 0.089 0.182 0.193 0.092 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
MDMA 0.35 0.036 0.076 0.038 0.023 n.d. 0.031 0.10 0.22 0.11 0.06 <0.01 0.09
NDMA 0.1 0.0011 0.0019 0.0014 n.d. 0.0073 n.d. 0.01 0.02 0.01 <0.01 0.07 <0.01
Sucralose 52,500 0.527 0.836 0.659 1.44 1.05 0.888 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Tetrachloroethylene 10 n.d. n.d. n.d. n.d. 0.51 n.d. <0.01 <0.01 <0.01 <0.01 0.05 <0.01
Tolytriazole 20 0.390 0.180 n.d. 0.170 0.120 0.210 0.02 0.01 <0.01 0.01 0.01 0.01
ΣTHMs 100 6.95 10.5 14.0 17.4 21.6 12.6 0.07 0.11 0.14 0.17 0.22 0.13

9
A. Munné et al.
Fig. 5. Concentrations of microbiological indicators (log10 units per 100 mL). Samples are shown in different colours: blue, samples collected in the river (R0 and R1); red,
samples collected throughout the wastewater and reclaimed water treatment train (W0, W1, W2 and W3); purple, samples collected after the drinking water treatment (D1).
Circles indicate when reclaimed water was not chlorinated (campaigns 1, 2, 3 and 4) and asterisks indicate samples collected when reclaimed water was chlorinated after W2
(campaigns 5 and 6).
microbiological concentrations went back to non-chlorinated levels
(Fig. 5). Concentrations of microbiological indicators was restored to the
same concentration measured upstream (R0) after reclaimed water was
discharged into the river (R1), even when the reclaimed water was chlori-
nated. Therefore, chlorination of reclaimed water did not affect the microbio-
logical quality at the R1 site where the DWTP takes water to be treated, since
raw water from upstream showed microbiological concentrations close to
non-chlorinated reclaimed water. Neither chlorinated nor non-chlorinated
reclaimed water did worsen the microbiological quality of the Llobregat River.
The drinking water treatment plant (DWTP) always adds chlorine at its
effluent to meet the drinking quality standards. Thus, microbiological
concentration was always undetectable (<1/100 mL) at the D1 sampling
site. The total log reduction throughout the whole water treatment train
(WWTP-WRTP-DWTP) was around 5-7 log reduction for E. coli, 7-9 log
reduction for total coliphages, and 6 log reduction for Clostridium perfringens
spores when reclaimed water was not chlorinated. Campaigns in which
reclaimed water was chlorinated presented a higher total log reduction,
around 9-10 log reduction for E. coli and total coliphages, and 6 log reduc-
tion for Clostridium perfringens spores (in this case similar to non-chlorinated
reclaimed water) (Table 6). Nevertheless, the indirect water reuse causes
that disinfection of reclaimed water be masked by the microbiological
load that the river contains upstream, getting similar microbiological load
to non-chlorinated.
All the results of the microbiological indicators measured in the treated
drinking water were below the limit of quantification. Therefore, the
analytical validation of the logarithmic reduction that could actually be
achieved through the DWTP is limited by this fact, and by the relatively
low quantification of the microbial load at the DWTP inlet. Only can be an-
alytically validated the minimum logarithmic reduction that DWTP can
achieve with respect to the microbial load of the raw water to be treated.
In this sense, the log reductions listed in Table 6 are the reductions that
can be validated by analytical control, although the log reduction potential
is expected to be higher. Despite this, studies of analytical control at the
DWTP performed with a concentration system of large sample volumes
confirm that the whole treatment train (WWTP-WRTP-DWTP) is capable
of achieving log reductions of up to 13.3–14.2 log for E.coli and
Table 4
Concentration of microbiological quality indicators throughout the WWTP and WRTP (from W0 to W3 sampling sites) when reclaimed water was non-chlorinated
(campaigns 1, 2, 3 and 4) and chlorinated (campaigns 5 and 6).
WWTP affluent WRP effluent Reclaimed water discharge without chlorination
(W0) (W2) (W3)
E. coli (NMP/100 mL) 5.2–10 × 106 190–4900 1100–6100
C. perfringens spores (PFU/100 mL) 2.2–5.8 × 105 70–840 420–1900
Somatic coliphages (PFU/100 mL) 3.8–6.1 × 106 2–1300 400–600
10
Science of the Total Environment 866 (2023) 161339
11.8–11.9 log for somatic coliphages. This allow the validation that the
whole water treatment train can achieve the minimum log reduction
(performance targets) for microbiological indicators E.coli and somatic
coliphages required for indirect potable water reuse in accordance with
performance targets criteria (log reductions) established by different inter-
national organizations or environmental and health agencies (NRMMC–
EPHC–AHMC, 2008; US Environmental Protection Agency, 2012).
For Clostridium perfringens spores, since it was not technically possible to
use a large sample volume concentration system to analyse this parameter,
the most accurate calculation of the log reduction achieved by the treat-
ment train was not set. Clostridium perfringens spores were not detected in
sufficient quantity in the water abstraction site to allow the log reduction
achievement validation. Given the limitation that exists on validation of
compliance with the performance target (log reduction) for Clostridium
perfringens spores by analytical control, validation can be done by adding
the performance attributed to each of the treatment step/stage based on
scientific evidence, published data from trial reports, or testing in a labora-
tory under controlled conditions. In the case of the whole treatment train
(WWTP-WRTP-DWTP), the addition of performance granted to individual
treatment steps ensures the achievement of the performance target (log
reduction) also for Clostridium perfringens spores. The DWTP can achieve a
log reduction in the count of microbiological indicators sufficient to meet
the criteria established for water intended for human consumption.
5. Discussion and conclusions
5.1. Comparison with other worldwide case studies
Indirect and direct potable water reuse is becoming a powerful and
feasible solution to cope with the world's growing water shortage, so
several cities and regions, such as Windhoek in Namibia, Orange County
in California, Perth and New South Wales in Australia, or Singapore, have
already started good and fruitful experiences on water reuse for direct or
indirect drinking uses (Ormerod and Silvia, 2017; Khan and Anderson,
2018; Tortajada and van Rensburg, 2020). As a result of several droughts
suffered in Catalonia (NE Spain), with expected increasing frequencies
Reclaimed water discharge with chlorination
(W3)
< 1–2
80–360
< 1–70
A. Munné et al.
Table 5
Removal logarithms concentration (log10 reduction) of microbiological quality
indicators throughout the WWTP and WRTP (from W0 to W3 sampling sites) when
reclaimed water was not chlorinated (campaigns 1, 2, 3 and 4) and chlorinated
(campaigns 5 and 6).
Log reduction without Log reduction with
chlorination chlorination
E. coli 3.8–4.7 6.4–7.2
C. perfringens spores 2.8–3.6 3.4–3.5
Somatic coliphages 4.5–6.4 6.7–6.8
and duration according to the climate change scenarios (Barrera-Escoda
and Cunillera, 2011; Sherwood and Fu, 2014), the Catalan Water Agency
(the public water authority in Catalonia) is launching new measures to
increase water availability to preserve environmental flows in rivers and
to assure water supply (i.e. indirect potable water reuse). These measures
are especially relevant for urban wastewater treatment plants located
close to the shoreline, which are currently discharging treated water
directly into the sea. Over 340 hm3 of treated urban wastewaters are
currently discharged to the sea every year from Catalan urban areas located
close to the Mediterranean coast (e.g. Barcelona WWTP), while it could be
reused to restore water flow in rivers, coastal lagoons and for groundwater
replenishment, thus enhancing water availability in urban areas and pre-
serving aquatic ecosystems, which would be especially useful in drought
periods. At the moment, direct potable water reuse is not allowed in
Spain and in many other countries due to tight health restrictions to
avoid any human health risk. Therefore, planned indirect potable water
reuse becomes as a powerful alternative to ensure drinking water supply
availability in highly populated areas located in dry regions such as Catalo-
nia. However, indirect potable water reuse needs to be in accordance with
both environmental quality standards for aquatic life and tight human
health requirements to make it safe and reliable. In our work we tackled
both challenges equally to carry out an indirect potable water reuse safely,
and we provided a useful monitoring procedure to boost coming indirect
water reuse initiatives. The novelty brought by our work is the inclusion
of an ecological line of risk assessment in the evaluation of indirect water
reuse, which was formerly almost exclusively based on human health. On
one hand, we analysed the possible effect on aquatic life when reclaimed
water was discharged into the Llobregat River, paying special attention to
micropollutants of emerging concern and disinfection by-products when
reused water was chlorinated. On the other hand, human health require-
ments were also considered when water was withdrawn downstream
from the Llobregat River to the Barcelona urban area through a drinking
water treatment plant. In our opinion, both approaches (effect on aquatic
ecosystems and human health risk) must be considered equally in indirect
water reuse initiatives, as we did in our pilot test.
Similarly, the Australian guidelines for water recycling (NRMMC-
EPHC-NHMRC, 2008) suggest that, besides the health criteria of the final
drinking water, evaluating the potential effects on aquatic ecosystems is a
critical component of any proposed water reuse project, and it states that
discharges of wastes should not be considered unless the quality is consis-
tent with the receiving environment. To this end, the analyses of final pota-
ble water must be complemented with the monitoring of the reclaimed
water and the receiving environmental compartments, as we did in our
Table 6
Logarithms (log10 units per 100 mL) of removal throughout the WWTP-WRP-DWTP train.
Logs reduction WWTP-WRTP-DWTP Logs reduction WWTP-WRTP-DWTP
(without chlorination at WRP) (with chlorination at WRP)a
E. coli > 5.6–7.1 > 9.0–9.9
C. perfringens spores > 6.1–6.7 > 6.5–6.7
Somatic coliphages > 7.0–9.2 > 9.6–9.7
a
The logarithmic reduction was calculated considering the whole treatment train WWTP-WRP-DWTP without considering river transport, which increases microbial
concentration between WRTP and DWTP.
11
Science of the Total Environment 866 (2023) 161339
pilot test. We identified up to 10 chemical compounds as recycled water
constituents that may pose a potential risk to aquatic life in the Llobregat
River, which are fairly in accordance with the reference chemicals provided
by Australian guidelines (NRMMC-EPHC-NHMRC, 2008). On the other
hand, in the United States, water reuse schemes combine mandatory output
quality standards (i.e. drinking water contaminant list) with regulations
and guidelines largely focusing on drinking water quality and human
health protection (Mukherjee and Jensen, 2020). To this end, microbial
and case-specific chemical indicators are established and controlled. As
an example, the California's State Water Board requires treatment plants
which discharge recycled water for the purposes of groundwater recharge
to analyse 17β-estradiol, gemfibrozil, and iopromid, whereas the Orange
County Water District (OCWD) voluntarily expands this list to up to 27
pharmaceuticals and hormones in the Groundwater Replenishment System
(GWRS) project (Miarov et al., 2020). The environmental protection of
indirect water reuse is covered by the Clean Water Act (Mukherjee and
Jensen, 2020) and by the EPA water reuse guidelines (US Environmental
Protection Agency and CDM Smith, 2017).
In practice, this double health and environmental approach is somehow
uncommon in the literature, even in the environmental monitoring per-
formed in relevant water catchment locations. Lin et al. (2020) conducted
a dual approach in a water reuse scenario located in south-eastern China
analysing 5 persistent organic pollutants and 18 contaminants of emerging
concern. However, the micropollutants that were observed to pose a
relevant risk in Chinese coastal rivers (e.g. atrazine, sulfamethoxazole,
oxytetracycline, hormones E1, E2, EE2 and E3, diethyl hexyl phthalate
and bisphenol A) are of little relevance in the present case of study. Also,
Miarov et al. (2020) recommended the monitoring of four pharmaceutical
compounds and two estrogens in Israelian water reuse exercises, of which
only diclofenac was observed to pose a potential risk in our case. These dif-
ferences indicate that the relevance of micropollutants varies significantly
depending on the local environmental pressures and regional wastewater
epidemiological patterns, and the profile of relevant micropollutants
should be prioritised on a case-by-case basis. This difficulty was tackled in
the present work by comprehensively screening local data from health,
urban and agricultural institutions. Alternatively, some authors have pro-
posed to complement the conventional chemical indicators analyses with
deliberately unspecific bioanalytical tools (i.e. in vitro assays to test the cy-
totoxicity, genotoxicity, mutagenicity, endocrine disruption, neurotoxicity,
adaptative stress response and xenobiotic metabolism; Leusch and Snyder
(2015)). Non-target analysis is another non-specific tool that has gained
increasing attention and has proven to be helpful in recent water reuse-
related studies (McEachran et al., 2018; Backe, 2021; Ishida et al., 2021).
5.2. Comparison with previous water reuse exercises carried out in the Llobregat
River
Some previous analysis and trials regarding indirect water reuse from
the Barcelona WWTP to the lower Llobregat River had already been
performed during the last severe drought from 2008, when some chemicals
and microbiological and ecological effects were already monitored (López-
Serna et al., 2012; Rubiano et al., 2012; Prat et al., 2013). In those previous
studies, the authors concluded that, in general, the discharge of reclaimed
water in the river influenced the occurrence of pharmaceuticals and, in
less extent, some illicit drugs. Nevertheless, it was not very significant in
Logs reduction WWTP-WRTP-DWTP (analytical control performed with a
concentration system of large sample volumes)
> 13.3–14.2
–
> 11.8–11.9
A. Munné et al.
any case. Additionally, discharging reclaimed water into the river did not
increment the load of pathogens in the Llobregat River, and some biomark-
ers indicated a potential further deterioration of the ecological status of the
river, although changes in applied biological quality indices were not
detected. Nevertheless, the monitoring carried out during the last severe
drought (in 2008) was not based on an initial approach taking into account
all potential threats and contaminants that could be present in reclaimed
water as other initiatives did take into account later (i.e. NRMMC-EPHC-
NHMRC, 2008; US Environmental Protection Agency and CDM Smith,
2017). Therefore, unlike the study performed in 2018, in the present
work we previously planned a procedure to properly identify the main
threats that could be present in reclaimed water. Therefore, we took into
account the industrial activity and prescribed drugs in the Barcelona
urban area in order to get a reliable initial list of candidates that could be
found in the sewer network and could drain to the WWTP. Thus, in the pres-
ent work, a total of 835 micropollutants were included in a preliminary list
as candidates that could be present in the Barcelona WWTP affluent after a
thorough selection, in which a screening procedure was applied to choose
those that could pose any negative effect on aquatic life or human health
(see Fig. 2). We consider that this previous planned approach provides
useful and reliable information that can help to enhance public perception
and social acceptance of the indirect potable water reuse, and to avoid
opposition from citizens due to a lack of confidence (e.g., Hurlimann and
Dolnicar, 2010).
5.3. Comments on additional effluent disinfection
Some pharmaceuticals were removed when reclaimed water was chlori-
nated hence reducing the chemical cocktails discharged into the river
(before water withdrawal for drinking supply), but DBPs appeared instead,
which can largely affect aquatic biota. Moreover, when reclaimed water
was chlorinated, microbiological load was significantly reduced, mainly E
coli and somatic coliphages, but concentrations were restored again when
reclaimed water was delivered into the river buffer, since the basal micro-
biological indicators of the Llobregat River exceeded those of disinfected
reclaimed water. Therefore, microbial loads showed similar values in the
river at the DWTP withdrawal site whether the reclaimed water was chlori-
nated or not. Chlorination after the reclaimed water treatment seems not to
be a good procedure unless the concentration and number of pharmaceuti-
cals have been reduced before being discharged into the river. Reclaimed
water discharge did not result in additional microbiological health risk
for the drinking water supply, even when it was not chlorinated after the
WRTP (bear in mind that the DWTP effluent was always chlorinated).
The analyses performed during the pilot tests showed that drinking water
broadly complied with the drinking water quality standards for the micro-
biological and chemical parameters (EU Directive 2020/218/EU), and only
the occurrence of 1,4-dioxane was proven to pose a risk to human health
through the drinking water supply due to the reclaimed water discharge
when reclaimed water was not chlorinated. Therefore, even acknowledging
that the treatment used in the pilot test safely meets the current drinking
water quality standards used in this study throughout indirect potable
water reuse, our results suggest that it is still recommended to introduce
advanced treatments to reduce the chemical cocktails that are discharged
into the river to avoid their possible effects on aquatic life and also to reduce
the potential threats that they could pose to human health.
5.4. Final remarks and conclusions
Water scarcity is particularly concerning in regions under Mediterra-
nean influence, where the occurrence of droughts is not only common but
also expected to increase in severity and length in coming years due to cli-
mate change (IPCC, 2013). These circumstances also lead to a shift in some
rivers reducing their water flow, putting some of them at risk of changing
from perennial to intermittent flow with scarce or almost null capacity of
diluting contamination or maintaining suitable habitats for aquatic life
(Döll and Schmied, 2012; Acuña et al., 2014; Stubbington et al., 2017).
12
Science of the Total Environment 866 (2023) 161339
Therefore, indirect water reuse becomes an effective solution which
provides two main benefits, on one hand, a water flow augmentation in
rivers to maintain habitats and aquatic living conditions and, on the other
hand, ensuring water availability downstream for urban uses.
In the present study, the indirect reuse of reclaimed water in the
Llobregat River was proved to be a valid temporary solution to preserve a
minimum river flow and to safely overcome drinking water shortages
during acute droughts. The reclaimed water discharged into the lower
Llobregat River after a conventional secondary wastewater treatment
(activated sludge with nutrient removal) and an additional water reuse
treatment (microfiltration plus UV disinfection) did not significantly affect
the basal river water quality, even in the most extreme drought case (1:1
dilution between river flow and reclaimed water discharged) Only 1,4-
dioxane and, occasionally, DEET were identified as the main potential
risks for human health, while ten other emerging contaminants
(bromodichloromethane, dibromochloromethane, chloroform, EDDP,
diclofenac, iopamidol, ioprimid, lamotrigine, ofloxacin and valsartan)
posed a potential risk for the environment. Active measures should be
taken to control and reduce the presence of these pollutants. The Barcelona
drinking water treatment plant withdraws an average of 2 m3/s from the
lower Llobregat River to provide drinking water to Barcelona's inhabitants
(about 2 million people). That is the reason why the Llobregat River must
be carefully monitored and wastewater effluents properly treated to reduce
micropollutants of concern and microbiological loads. The pilot test
performed in the lower Llobregat River ensured that conventional water
treatments for water reuse were safe enough to preserve aquatic life and
meet the quality standards for the drinking water supply. Nevertheless, ad-
vanced treatments in indirect potable water reuse should be considered to
avoid any risk for human health and aquatic biota, even more considering
that drought scenarios are expected to increase due to climate change. The
public expects to have safe water and suitable sanitation treatments. There-
fore, a suitable monitoring program is essential to make water reuse trust-
worthy. Public and institutional trust can be fragile and, once lost, it is
difficult to regain. Thus, the prioritisation and monitoring method that
we propose in this paper can be adopted in other similar regions to promote
indirect potable water reuse in a safe way.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2022.161339.
CRediT authorship contribution statement
Antoni Munné: Conceptualization, Methodology. Validation, Investi-
gation, Data curation, Original draft preparation, Writing, Reviewing and
Editing, Validation. Carolina Solà, Pere Serra, Irene Corbella, Mercè
Aceves and Miquel Paraira: Conceptualization, Methodology. Validation,
Investigation, Data curation, Writing, Reviewing, Validation. Elisabet
Ejarque, Josep Sanchís, Belen Galofré, M. Rosa Boleda: Data analysis
and curation, Investigation, Reviewing, Visualization. Jordi Molist
Conceptualization, Reviewing, Validation.
Data availability
Data will be made available on request.
Declaration of competing interest
The authors declare that they have no known competing financial inter-
ests or personal relationships that could have appeared to influence the
work reported in this paper.
Acknowledgments
The authors specially appreciate to the expert panel who was deeply
involved in the pilot test and provided useful advice to improve the work
and gave relevant suggestions to get conclusions. The expert panel was
made up of (in alphabetical order): Antoni Ginebreda (IDAEA-CSIC), Damià
A. Munné et al.
Barceló (ICRA & IDAEA-CSIC), Francisco Lucena (MARS-IdRA-UB), Irene
Jubany (CTM-EURECAT), Jesús Gómez-Catalan (GRET-UB), Joan Jofre
(MARS-IdRA-UB), Josep Caixach (IDAEA-CSIC), Lluís Reales (BTV), Narcís
Prat (FEHM-IdRA-UB), Rafael Mujeriego (UPC), Wolfgang Gernjak (ICRA,
ICREA), and Xavier Martínez-Lladó (CTM-EURECAT). Anna Belver, Núria
Juliachs and Dolors Gispert from Barcelona Health Protection Service of
the Public Health Agency of Catalonia, and Lourdes Olivella from Catalan
Water Agency laboratory, provided helpful opinions as well. We also appreci-
ate additional advice from David Sedlak (Berkeley Water Center. University
of California).
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