Review

Illicit drug consumption estimations derived from wastewater analysis:
A critical review

Alexander L.N. van Nuijs
a
, Sara Castiglioni
b
, Isabela Tarcomnicu
a
, Cristina Postigo
c
, Miren Lopez de Alda
c
,
Hugo Neels
a
, Ettore Zuccato
b
, Damia Barcelo
c,d
, Adrian Covaci
a,e,

a
Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium
b
Department of Environmental Health Sciences, Mario Negri Institute for Pharmacological Research, Via La Masa 19, 20156 Milan, Italy
c
Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research, IDAEA-CSIC, C/ Lordi Girona, 18-26, 08034 Barcelona, Spain
d
Catalan Institute for Water Research (ICRA), Emili Grahit 101, Eidifici H
2
O, Parc Cientific i Tecnològic de la Universitat de Girona, 17003, Girona, Spain
e
Ecophysiology, Biochemistry and Toxicology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
a b s t r a c t a r t i c l e i n f o
Article history:
Received 11 December 2009
Received in revised form 21 May 2010
Accepted 21 May 2010
Available online 18 June 2011
Keywords:
Illicit drugs
Wastewater
Analysis
Sewage epidemiology
Drug consumption
Estimates
The consumption of illicit drugs causes indisputable societal and economic damage. Therefore it is necessary
to know their usage levels and trends for undertaking targeted actions to reduce their use. Recently, a new
approach (namely sewage epidemiology) was developed for the estimation of illicit drug use based on
measurements of urinary excreted illicit drugs and their metabolites in untreated wastewater. This review
aims at critically evaluating the published literature and identifying research gaps of sewage epidemiology.
Firstly, the existing analytical procedures for the determination of the four most used classes of illicit drugs
worldwide (cannabis, cocaine, opiates and amphetamine-like stimulants) and their metabolites in
wastewater are summarized and discussed. The focus lies on the sample preparation and on the analysis
with chromatographic techniques coupled to mass spectrometry. Secondly, back-calculations used to
transform measured concentrations in wastewater (in ng/L) into an amount of used illicit drug (in g/day per
1000 inhabitants or doses/day per 1000 inhabitants) are discussed in detail for the four groups of illicit drugs.
Sewage epidemiology data from Spain, Belgium, UK, Italy, Switzerland and USA are summarized and
compared with data from international organisations, such as the European Monitoring Centre for Drug and
Drug Addiction (EMCDDA) and the United Nations Office on Drugs and Crime (UNODC). The results derived
from wastewater analysis show in general good agreement with existing prevalence data (percentage of a
population that uses illicit drugs at a given time) and demonstrate the potential of sewage epidemiology.
However, this review confirms that future work should focus on further optimisation and standardisation of
various important parameters (e.g. sample collection and back-calculations). In the future, sewage
epidemiology could be used in routine drug monitoring campaigns as a valuable tool in addition to the
classical socio-epidemiological studies for the determination of local, national and international illicit drug use.
© 2010 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3565
2. Analysis of wastewater for illicit drugs and metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3566
2.1. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3566
2.2. LC–MS/MS analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3566
3. Sewage epidemiology results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3569
3.1. Cocaine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3569
3.1.1. Back-calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3569
3.1.2. Results and comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3570
3.2. Amphetamine-like stimulants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3571
3.2.1. Back-calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3572
3.2.2. Results and comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3572
Science of the Total Environment 409 (2011) 3564–3577
☆ Submitted to the special issue of Science of the Total Environment dedicated to the INNOVA-MED conference (guest editor: Dr. Mira Petrovic).
⁎ Corresponding author. Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium. Fax: +32 3 265 2722.
E-mail address: adrian.covaci@ua.ac.be (A. Covaci).
0048-9697/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2010.05.030
Contents lists available at ScienceDirect
Science of the Total Environment
j our nal homepage: www. el sevi er. com/ l ocat e/ sci t ot env
3.3. Opiates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3573
3.3.1. Back-calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3573
3.3.2. Results and comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3573
3.4. Cannabis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3574
3.4.1. Back-calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3574
3.4.2. Results and comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3574
4. Critical discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3574
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3576
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3576
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3576
1. Introduction
The use of illicit drugs cannot be ignored in our society. An
important increase in drug consumption, especially for cocaine and
cannabis, was reported in the last decade by the European Monitoring
Centre for Drugs and Drug Addiction (EMCDDA, 2009). The abuse of
illicit drugs has incalculable societal consequences, such as health
treatment costs, higher incidence of criminality and economic damage
(EMCDDA, 2009). For policy makers, it is therefore important to gain
knowledge in trends, usage levels, hot spots and prevalence of illicit
drug consumption to start prevention campaigns or to develop
effective interventions.
Official figures for the prevalence and occurrence of illicit drugs in
different countries are currently obtained from population surveys
integrated with crime statistics, medical records, and drug production
and seizure rates (EMCDDA, 2009; UNODC, 2009). These epidemio-
logical tools give a useful general picture of illicit drug abuse, but
estimates of consumption rates and drug use prevalence may be
inaccurate and have also several limitations, including substantial
time lags that impact negatively the reliability, validity and utility of
such data, self-report bias and limited population coverage. This
biased selection of population may lead not just to an underestima-
tion of rates of illicit drug use, but also to incorrect estimates as most
of the information is obtained from the consumers themselves.
Moreover, since illicit drug abuse is considered primarily an urban
problem (Banta-Green et al., 2009; van Nuijs et al., 2009a), most
major surveillance efforts are focused upon only a few major
metropolitan areas. As recommended by the Commission on Narcotic
Drugs of the United Nations (UNODC, 2007), novel approaches are
needed not only to provide more realistic, objective and comparable
estimates of illicit drug consumption in different communities, but
also to rapidly detect changes in the patterns of illicit drug abuse.
A new concept to assess collective drug consumption, based on
measuring concentrations of illicit drugs and their metabolites in
untreated wastewater, was hypothesized by Daughton in 2001
(Daughton, 2001). Later, the methodological approach, named
sewage epidemiology, was devised and implemented by Zuccato et
al. (2005) using cocaine as a model drug. Consequently, sophisticated
analytical methods for the simultaneous measurement of a cocktail of
illicit drugs have been set up (Jones-Lepp et al., 2004; Hummel et al.,
2006; Castiglioni et al., 2006; Boleda et al., 2007; Bones et al., 2007;
Huerta-Fontela et al., 2007; Gheorghe et al., 2008; Kasprzyk-Hordern
et al., 2008; Postigo et al., 2008a; Chiaia et al., 2008; Mari et al., 2009;
Bijlsma et al., 2009; Bartelt-Hunt et al., 2009; Loganathan et al., 2009;
van Nuijs et al., 2009d; González-Mariño et al., 2009) and reviewed by
Castiglioni et al. (2008) and Postigo et al. (2008b).
Within this approach, human metabolic excretion products
resulting from drug consumption are rapidly collected and pooled
by the sewage systems, providing valuable evidence of the amount
and type of drug consumed by a population (Daughton, 2001). Earlier
work with therapeutic drugs showed a close correspondence between
the known amounts consumed by the population and the amounts
estimated from concentrations of metabolic drug residues measured
in wastewater (Heberer and Feldmann, 2005). If an excretion product
is stable in wastewater and efficiently conveyed to the wastewater
treatment plant (WWTP), it is reasonable to assume that the amount
collectively excreted in a given period should be reflected by the
amount reaching the WWTP in the corresponding interval (Daughton,
2001). This approach provides objective, quantitative, near real-time
profiles of illicit drug consumption by monitoring selected drug
residues entering the municipal sewage system.
Levels of illicit drugs (parent compounds or metabolites) in
wastewater can be used to back-calculate the mass loads of the
parent drugs and/or metabolites. These loads can then be used to
estimate consumption, in g/day or doses/day, with knowledge of the
drug metabolism and excretion patterns. Furthermore, after dividing
the drug consumption by the served population, results can be
expressed as g/day per 1000 inhabitants or doses/day per 1000
inhabitants, and thus results fromdifferent locations can be compared
(Fig. 1). Although this concept is relatively simple, various factors
influencing its reliability need to be fully understood before
implementation on a large scale is possible (Frost et al., 2008; Zuccato
et al., 2008b). Among the most important factors are: understanding
the fate of drugs (e.g. stability, degradation, partitioning, and
sorption), human metabolic patterns of the investigated drugs,
characteristics of sewer systems (population served by a WWTP,
wastewater flowrate) and effects of external parameters (e.g. climatic
conditions) on illicit drugs. Until now, this approach has been applied
to investigate the consumption of various drugs, especially cocaine
(Zuccato et al., 2005; Zuccato et al., 2008a; Huerta-Fontela et al., 2008;
Boleda et al., 2009; Kasprzyk-Hordern et al., 2009; van Nuijs et al.,
2009a,b,c; Postigo et al., 2010).
Fig. 1. Schematic overview of the sewage epidemiology approach.
3565 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
The sewage epidemiology application for illicit drug consumption
monitoringcouldbeusedprospectively ina multi-disciplinary approach
for a) using updated drug profile analyses to rapidly identify emerging
hot spots of drug abuse; b) testing in real time the efficacy of different
counter-measures such as prevention through education, enforcement,
and global concerted actions against illicit drug consumption; c) cross-
validation of population surveys versus wastewater monitoring
programs; and d) assessing the actual amount of illegal money involved
in drug trafficking. If applied to other public health issues, this approach
could potentially extract useful epidemiological data from qualitative
and quantitative profiling of biological indicators entering the sewage
system and might reopen the debate on what type of surveillance
approaches are ethically and politically acceptable.
The overall aims of this review were to explore the potential and
limitations of sewage epidemiology by critically looking to the current
literature. The most used illicit drugs worldwide (cannabis, cocaine,
opiates andamphetamine-likestimulants) havebeendiscussedandboth
analytical methodologies and calculation methods have been critically
reviewed and evaluated. An effort was directed to the investigation of
spatial and temporal trends in consumption of illicit drugs.
2. Analysis of wastewater for illicit drugs and metabolites
The published methodologies to determine illicit drugs and their
metabolites in wastewater are summarized in Tables 1 and 2.
2.1. Sample preparation
Studies on illicit drugs and their metabolites in wastewater or
surface water have been performed in various countries, as reported
in Table 1. Analysis of influent wastewater is used to estimate the
community consumption, while effluent wastewater and surface
water can give an indication about the removal or persistence of illicit
drugs in the environment.
For sewage epidemiology, composite influent samples were
collected over a period of 24 h, in order to be representative for a
complete day (Zuccato et al., 2005; Castiglioni et al., 2006; Boleda
et al., 2007; Huerta-Fontela et al., 2007; Bones et al., 2007; Gheorghe
et al., 2008; Chiaia et al., 2008; Postigo et al., 2008a; Boleda et al.,
2009; van Nuijs et al., 2009a,b,c,d; Bijlsma et al., 2009; Postigo et al.,
2010). Grab samples have also been used (Hummel et al., 2006;
Kasprzyk-Hordern et al., 2008), as well as polar organic integrative
samplers (POCIS) which were placed for several days in wastewater at
the sampling point (Jones-Lepp et al., 2004; Bartelt-Hunt et al., 2009),
offering a good preconcentration of the analytes.
After collection, samples were either stored in the dark at 4 °C and
analysed within 3 days, or stored at −20 °C until processing. The pHof
the water samples was usually kept as is (approximately pH 7), but
some groups adjusted the samples to acidic pH immediately after
collection and before storage to prevent degradation of compounds
(Gheorghe et al., 2008; Kasprzyk-Hordern et al., 2008; van Nuijs et al.,
2009d).
Since most illicit drugs and their metabolites are found in
wastewater in the ng/L range, a preconcentration step is generally
required prior to the analysis by liquid chromatography coupled to
tandem mass spectrometry (LC–MS/MS). Most literature describes
solid-phase extraction (SPE) for this purpose, and the required sample
volumes are high (between 50 and 1000 mL) (Table 1). New
breakthroughs in instrument development are opening the way for
reduced sample volume due to on-line preconcentration (5 mL)
(Postigo et al., 2008a, 2010) or even direct injection without
preconcentration (1.8 mL) (Chiaia et al., 2008). Sample preparation
is a crucial step to remove matrix components that may compete with
the target analytes in the ionization process in LC–MS analysis and to
obtain good analyte recoveries and low method limits of detection
and/or quantification. When different groups of analytes need to be
measured, a compromise of the experimental conditions must be
done, and usually simple sample pretreatment is applied. This results
in sample extracts which may have a higher load of interferences and
matrix. On the other hand, an intense and long sample preparation
protocol requires much analyst time and may cause losses of the
target analytes resulting in poor method recoveries.
Before the sample processing, a filtering step of the wastewater is
necessary to remove solid particles. Then, the pHis adjusted according
to the SPE procedure. As sorbents Oasis HLB, Oasis MCX, Strata-XC,
PLPR-s, Bond-Elut Certify LRC (most commonly the first two) were
used, and the elution was done with methanol and/or alkaline
methanolic solution or with other solvents such as acetone or ethyl
acetate (Hummel et al., 2006; Bones et al., 2007). Table 1 shows the
various SPE procedures reported in the peer-reviewed literature.
SPE with commercially available amphetamine class-selective
molecular imprinted polymers (MIPs) was also evaluated to extract
and concentrate amphetamine drugs from wastewater samples.
González-Mariño et al. (2009) reported better performance of the
MIPs in terms of selectivity, sensitivity, accuracy and precision
compared to Oasis HLB and MCX cartridges. The main drawbacks of
this approach were a higher time requirement for the sample
preparation step (as vacuum cannot be applied to load the sample
on the MIPs) and a lower capacity compared to Oasis SPE cartridges
(which makes impossible the reduction of the limits of detection).
Postigo et al. (2008a) described an on-line SPE method, with the
benefit of a sensitive instrument, opening the possibility for a full-
automated analysis. Only 5 mL of wastewater was necessary and the
sample was loaded automatically onto the SPE cartridges (Oasis HLB or
PLPR-s, depending on the group of analytes). Elution was performed
directly with the LC mobile phase (water/acetonitrile at a flow rate of
0.3 mL/min). Extraction of a sample in a sequence was carried out in
parallel with the chromatographic run of 35 min of the previously
extracted one, thus obtaining a very short time-frame for both clean-up
andanalysis steps. Still, presence of strong matrix effects combinedwith
low recoveries in wastewater (8–59%) indicated the need for using
deuterated surrogate standards for quantification.
Another alternative to speeding-up analyses by eliminating the
time-consuming SPE was reported by Chiaia et al. (2008). These
authors developed a Large-Volume Injection (LVI) LC–MS/MS proce-
dure using a special injector kit. In this way, a total volume of 1.8 mL of
water was loaded in two cycles of 0.9 mL onto the analytical column
protected by a C
18
-silica guard column (2.0×4.0 mm) using an
autosampler. The technique permitted reaching acceptable quantifi-
cation limits for measuring illicit drugs and metabolites in wastewater
in a 35 min run.
Polar organic integrative samplers (POCIS) used for the analysis of
illicit drugs were first described by Jones-Lepp et al. (2004). In this
technique, the same sorbents used for SPE can be enclosed within a
microporous membrane, allowing penetration of hydrophilic organic
chemicals. These devices are exposed for a longer period of time (e.g.
one week) in ambient water. The sorbent is then transferred into glass
gravity-flow chromatography columns (1 cm internal diameter). The
analytes are generally eluted with methanol, the solvent is evapo-
rated, and the residue reconstituted with mobile phase. Yet,
calibration studies to determine POCIS sampling rates for the
compounds of interest are required, making this approach less
attractive and more time-consuming. Bartelt-Hunt et al. (2009)
used POCIS for the determination of illicit drugs and pharmaceuticals
in US wastewater.
2.2. LC–MS/MS analysis
The occurrence of illicit drugs and their metabolites in wastewa-
ter has been determined so far by means of liquid chromatographic
(LC) separation coupled to mass spectrometry (MS) detection, with
the exception of the methodology reported by Mari et al. (2009), in
3566 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
Table 1
Sample preparation based on solid-phase extraction for illicit drugs and their metabolites in wastewater.
Analytes Sample
volume
Extraction Absolute
Recovery (%)
Comments Reference
Cartridge/Elution
Amphetamine 50 mL Oasis MCX (conditioned: 6 mL MeOH,
3 mL H
2
O, 3 mL H
2
O pH2)
113±8 Spain (Castellón) Bijlsma et al. 2009
Methamphetamine, MDA 116±12, 90±7 Composite samples
MDMA, MDEA Elution: 8 mL ammonia 2% in MeOH 73±5, 75±18 Stored at -20 °C
Cocaine, Norcocaine 87±10, 73±8
Cocaethylene, Benzoylecgonine 85±5, n.c.*
Norbenzoylecgonine 50±15
THC-COOH 72±9
Normorphine, Morphine 200 mL Oasis HLB (conditioned: 5 mL MeOH, 90, 100 North-East Spain Boleda et al. 2007
Norcodeine, Codeine 5 mL H
2
O) 99, 92 Composite samples
6-Acetylmorphine, Heroin Elution: 8 mL MeOH 106, 86 Stored at 4 °C, 3 day max
EDDP, Methadone, THC-COOH 60, 93, 43
MDMA 1000 mL Strata-XC (conditioned: 12 mL MeOH, 52±1 Ireland (5 WWTPs from Bones et al. 2007
Cocaine, Cocaethylene 12 mL H
2
O) 56±2, 65±3 Dublin area)
Benzoylecgonine Elution: 10 mL ammonium hydroxide 53±3 Composite samples
Morphine 5% in acetone / ethyl acetate 1/1 (v/v) 4±0 Stored at 4 °C, 1 day
Methadone, EDDP 55±0, 59±2
Amphetamine 500 mL Oasis MCX (conditioned: 6 mL MeOH, 110±4 WWTPs: Castiglioni et al. 2006
Methamphetamine, MDA 3 mL H
2
O, 3 mL H
2
O pH 2) 112±6, 102±3 Italy (Milan)
MDMA, MDEA Elution: 2 mL MeOH, 2 mL ammonia 104±2, 107±3 Switzerland (Lugano)
Cocaine, Norcocaine 2% in MeOH, 2 mL NaOH 2% in MeOH 96±4, 112±6 Stored at 4 °C, 3 days max
Cocaethylene, Benzoylecgonine 109±4, 107±8 Composite samples
Norbenzoylecgonine 85±4
Morphine, 6-Acetylmorphine 88±6, 106±4
Morphine-3β-D-glucuronide 90±10
Methadone, EDDP 105±3, 88±3
THC-COOH 51±1
Cocaine 100 mL Oasis HLB (conditioned: 3 ml MeOH, 96±6 Belgium (Flanders) Gheorghe et al. 2008
Benzoylecgonine 3 mL H
2
O) 92±2 WWTPs: composite
Ecgonine methyl ester Elution: 8 mL MeOH 73±5 samples
Adjusted to pH 2
Stored at -20 °C
Amphetamine 50 mL SupelMIP-Amphetamine 25 mg 98-105 North-West Spain González-Mariño
et al. 2009 Methamphetamine, MDA (conditioned 1 mL MeOH, 1 mL H
2
O at pH8) 92, 101-102 Grab samples
MDEA 97-99 Stored at 4 °C
MDMA Elution: 2 x 1 mL 1% HCOOH in MeOH 111-114 (1 week maximum)
Amphetamine 100 mL Oasis HLB (conditioned: 10 mL MeOH, 85±3 North-East Spain Huerta-Fontela
et al. 2007 Methamphetamine, MDA 10 mL H
2
O) 87±2, 80±1 Composite samples
MDMA, MDEA Elution: 6 mL MeOH 94±3, 101±2 Stored at 4 °C, 3 days max
Cocaine, Benzoylecgonine 90±2, 100±4
Benzoylecgonine 100 mL Oasis HLB (conditioned: 2 mL heptane, 40±5 German WWTPs Hummel et al. 2006
Codeine, Dihydrocodeine 2 mL acetone, 6 mL MeOH, 48±2, 36±1 Grab samples
Oxycodone, Hydrocodone 8 mL H
2
O (pH 7) 50±5, 49±1 Stored at 4 °C, 1 day
Morphine, Methadone Elution: 4 x 2 mL acetone 29±1, 44±2
Amphetamine 1000 mL Oasis MCX (conditioned: 6 mL MeOH, 91 United Kingdom (Wales) Kasprzyk-Hordern
et al. 2008 Cocaine 2 mL HCOOH 0.2% in H
2
O) 70 Grab samples, adjusted to
Benzoylecgonine Elution: 2 mL MeOH 131 pH 2, stored at -20 °C
2 mL ammonia 5% in MeOH
Cocaine 1500 mL Bond-Elut Certify LRC 97-103 Italy (Florence) Mari et al. 2009
Benzoylecgonine (manufacturer procedure for basic drugs) 91-103 Stored at 4 °C, 3 days max
Morphine 96-109
Ephedrine, Amphetamine 5 mL Oasis HLB for cannabinoids; PLRP-s for others 15, 15 Spain (WWTPs: Barcelona, Postigo et al. 2008a
Methamphetamine, MDMA (conditioned: 1 mL AcN, 1 mL H
2
O) 20, 27 Valencia, Benicassim, Gandia)
Cocaine, Cocaethylene Elution: LC mobile phase 59, 52 Composite samples
Benzoylecgonine (AcN/ H
2
O, with gradient from 10% to 8
Heroin, Morphine 100% organic) 22, 14
6-Acetylmorphine On-line SPE 21 Stored at -20 °C
THC, 11-OH-THC, THC-COOH 9, 37, 13
LSD, nor-LSD, OH-LSD 17, 22, 11
Cocaine, Benzoyecgonine 50 mL Oasis MCX (conditioned: 5 mL MeOH, 5 mL H
2
O) 102±6, 87±3 Belgium (11 WWTPs from van Nuijs et al. 2009d
Ecgonine Methyl Esther Elution: 8 mL MeOH 35±3 Flanders)
Amphetamine 102±6 Composite samples
Methamphetamine, MDMA 99±4, 100±4 Adjusted to pH 2
6-Acetylmorphine 92±4 Stored at -20 °C
Methadone, EDDP 103±3, 61±8
Cocaine 500 mL Oasis MCX (conditioned: 6 mL MeOH, N90 Italy (4 WWTPs: Cagliari, Zuccato et al. 2005
Benzoylecgonine 3 mL H
2
O, 3 mL H
2
O pH2) N90 Latina, Cuneo, Varese)
Elution: 2 mL MeOH Composite samples
2 mL ammonia 2% in MeOH Stored at 4 °C, 3 days max
n.c.* =not calculated due to the high concentration in the “blank” sample.
3567 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
Table 2
LC-MS methodologies to determine illicit drugs and metabolites in wastewater.
Liquid chromatography Interface
b
(ionization mode)
Mass spectrometry
c
References
Type
a
Column Mobile phase MS detector Acquisition mode
RP-HPLC Betabasic-18 (250×2.1 mm, 5 µm) A: MeOH;
B: H
2
O: 0.1% HCOOH
ESI (+) QqQ 1 SRM Bartelt-Hunt et al. (2009)
RP-HPLC Phenomenex Onyx monolithic C18 (200×3 mm) A: MeOH;
B: H
2
O:5 mM CH
3
COONH
4
(pH=4.5)
ESI (+) ITMS 1 SRM Bones et al. (2007)
RP-HPLC X-Terra MS C18 (100×2.1 mm, 3.5 µm) A: AcN; B1: H
2
O:0.05% CH
3
COOH; B2:
H
2
O:0.05% TEA (for cannabinoids)
ESI (+/–) QqQ 2 SRM Castiglioni et al. (2006)
RP-HPLC Atlantis T3 C18 (150×4.6 mm, 5 µm) A:AcN;
B: H
2
O: MeOH:CH
3
COOH (94.9:5:0.1)
ESI (+) QqQ 1 or 2 SRM Chiaia et al. (2008)
RP-HPLC Zorbax Extended C18 (50×2.1 mm, 3.5 µm) A: AcN
B: H
2
O:AcN (98:2) 10 mM HCOONH
4
(pH=3)
ESI (+) ITMS 1 SRM Gheorghe et al. (2008)
RP-HPLC Halo C18 (100×2.1 mm, 2.7 µm) A: MeOH: 5 mM CH
3
COONH
4
B: H
2
O: 5 mM CH
3
COONH
4
ESI (+) QqQ 2 SRM González-Mariño et al. (2009)
RP-HPLC Synergi Polar-RP (150×3 mm, 4 µm) A:AcN; B: H
2
O:10 mM HCOONH
4
(pH=4, HCOOH) ESI (+) QqQ 2 SRM Hummel et al. (2006)
RP-HPLC Restek Allure C18 (150×3.2 mm, 5 µm) A: MeOH:H
2
O (98:2), 0.1% CH
3
COOH, 1 mM CH
3
COONH
4
;
B: H
2
O:MeOH (99:1), 0.1% CH
3
COOH, 1 mM CH
3
COONH
4
ESI (+) ITMS Full scan and CID(MS
2
) Jones-Lepp et al. (2004)
RP-HPLC Varian Pursuit XRS C18 (100×2 mm,3 µm) A: MeOH: AcN: 0.5 HCOOH (82:18:0.5%);
B: H
2
O:0.5% HCOOH
ESI (+) ITMS Full scan and CID(MS
2
) Loganathan et al. (2009)
RP-HPLC Purospher Star RP18 (125×2 mm, 5 µm) A: AcN;
B: H
2
O
ESI (+/–) QLIT 2 SRM Postigo et al. (2008a,b)
RP-HPLC Luna C8 (50×2 mm, 3 µm) A: AcN;
B: H
2
O: 0.1% HCOOH
ESI (+) QqQ /
ITMS
2 SRM -quantitative analysis/
Full scan and MS
2
- qualitative analysis
Zuccato et al. (2005)
RP-UPLC Acquity BEH C18 (50×2.1 mm, 1.7 µm) A: MeOH
B: H
2
O:5 mM CH
3
COONH
4
: 0.1% FAc
ESI (+) QqQ 3 SRM Bijlsma et al. (2009)
RP-UPLC Acquity BEH C18 (100×2.1 mm, 1.7 µm) A: MeOH
B: H
2
O:50 mM HCOONH
4
(pH=3.8 with HCOOH)
ESI (+) QqQ 2 SRM Boleda et al. (2007)
RP-UPLC Acquity BEH C18 (100×2.1 mm, 1.7 µm) A: AcN: 0.1% HCOOH
B: H
2
O: 30 mM HCOONH
4
(ph=3.5)
ESI (+) QqQ 2 SRM Huerta-Fontela et al. (2007)
RP-UPLC Acquity BEH C18 (100×2.1 mm, 1.7 µm) A: MeOH:0.5% CH
3
COOH (pH=3.2) B:
H
2
O:MeOH: CH
3
COOH (94.5:5:0.5) (pH=2.8)
ESI (+) QqQ 2 SRM Kasprzyk-Hordern et al. (2008)
HILIC Zorbax Rx-SIL (150×2.1 mm, 5 µm) A: AcN
B: H
2
O:2 mM CH
3
COONH
4
(pH=4.5 with CH
3
COOH)
ESI (+) ITMS 1 SRM Gheorghe et al. (2008)
HILIC Phenomenex Luna HILIC (150×3 mm, 5 µm) A: AcN
B: H
2
O:5 mM CH
3
COONH
4
ESI (+) QqQ 2 SRM van Nuijs et al. (2009d)
RP-HPLC* Omnisphere C18 (100×2 mm, 3 µm) A: AcN
B (PI mode): H
2
O:0.05% HCOOH
B (NI mode): H
2
O:0.05% HCOONH
4
ESI (+/–) LIT-FT Orbitrap Full Scan and MS
n
Hogenboom et al. (2009)
*not implemented in wastewater analysis yet.
AcN — acetonitrile; MeOH — methanol, CH
3
COONH
4
— ammonium acetate; CH
3
COOH — acetic acid, HCOONH
4
— ammonium formate; HCOOH — formic acid.
a
RP: reverse phase, HPLC: high performance liquid chromatography, UPLC: ultra performance liquid chromatography, HILIC: hydrophilic interaction liquid chromatography.
b
ESI: electrospray.
c
ITMS: ion trap mass spectrometer, QqQ: triple quadrupole mass spectrometer, LIT-FT Orbitrap: Linear ion trap-Fourier transformation orbitrap mass spectrometer, SRM: selected reaction monitoring mode, CID: collision induced
dissociation.
3
5
6
8
A
.
L
.
N
.
v
a
n
N
u
i
j
s
e
t
a
l
.
/
S
c
i
e
n
c
e
o
f
t
h
e
T
o
t
a
l
E
n
v
i
r
o
n
m
e
n
t
4
0
9
(
2
0
1
1
)
3
5
6
4

3
5
7
7
which cocaine, its metabolite benzoylecgonine, and morphine were
analysed by gas chromatography–mass spectrometry (Mari et al.,
2009).
High performance liquid chromatography (HPLC) (Jones-Lepp
et al., 2004; Castiglioni et al., 2006; Hummel et al., 2006; Bones et al.,
2007; Chiaia et al., 2008; Gheorghe et al., 2008; Postigo et al., 2008a,
2010; Hogenboom et al., 2009; Loganathan et al., 2009; Bartelt-Hunt
et al., 2009; González-Mariño et al., 2009; van Nuijs et al., 2009d) and
ultra performance liquid chromatography (UPLC) (Boleda et al., 2007;
Huerta-Fontela et al., 2007; Kasprzyk-Hordern et al., 2008; Bijlsma
et al., 2009) have been used to separate the target analytes. In the
reported HPLC and UPLC methods (Table 2), chromatographic
separation was usually carried out on reversed-phase columns,
using a moderately polar mobile phase consisting of a mixture of
water and an organic solvent. The organic solvents commonly applied
to elute the analytes from the chromatographic columns have been
methanol and acetonitrile. The aqueous mobile phases consisted of
water or ammonium formate or acetate (1–50 mM) and have been
mostly acidified with formic or acetic acids (0.05–0.1%) to improve
the ionization of the compounds monitored in the positive ionization
mode. Cannabinoids have been analysed in most studies in the
negative ionization mode using different aqueous mobile phases, such
as a basic solution of triethylamine 0.05% (TEA) (Castiglioni et al.,
2006), no additive (Postigo et al., 2008a) or a slightly acidic solution of
ammonium formate 0.05% (Hogenboom et al., 2009).
The performance of hydrophilic interaction liquid chromatography
(HILIC) has also been evaluated for the separation of cocaine and its
metabolites benzoylecgonine and the very polar ecgonine methyl ester
(Gheorghe et al., 2008; van Nuijs et al., 2009d), and of the
amphetamine-like compounds amphetamine, methamphetamine and
3,4-methylenedioxymethamphetamine (MDMA or ecstasy) and the
opioids 6-monoacetylmorphine, methadone and its metabolite 2-
ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (van Nuijs et al.,
2009d). The HILIC separation involves a polar stationary phase (e.g.
porous silica microspheres) and a highly organic mobile phase
consisting of methanol or acetonitrile in which water is introduced as
the eluting solvent. This type of chromatography has been shown to
resolve better the polar ecgonine methyl ester that is poorly retained in
reversed-phase columns (Gheorghe et al., 2008; vanNuijs et al., 2009d).
Ionization of illicit drugs and their metabolites has always been
performed by means of electrospray (ESI). Overall, these compounds
are best ionized in the positive mode, although the cannabinoid class
has shown good response in both positive (Boleda et al., 2007; Bijlsma
et al., 2009) and negative ionization modes (Castiglioni et al., 2006;
Postigo et al., 2008a; Hogenboom et al., 2009). The main drawback of
the ESI interface is its susceptibility to matrix effects that results in
suppression or enhancement of the analyte ionization signal.
Ionization of illicit drugs and their metabolites in aqueous environ-
mental matrices has been observed to decrease notably as the
complexity of the matrix increases e.g. in raw or influent wastewater
samples. Co-eluting matrix constituents of rawwastewater have been
observed to reduce analyte ionization between 30 and 94% (Hummel
et al., 2006; Gheorghe et al., 2008; Postigo et al., 2008a). In addition to
a selective sample extraction process, most of the reported methodo-
logies include isotopically labeled analogues as surrogate standards to
compensate for matrix effects in wastewater matrices (Hummel et al.,
2006; Castiglioni et al., 2006; Boleda et al., 2007; Bones et al., 2007;
Huerta-Fontela et al., 2007; Chiaia et al., 2008; Gheorghe et al., 2008;
Kasprzyk-Hordern et al., 2008; Postigo et al., 2008a; Bijlsma et al.,
2009; González-Mariño et al., 2009; van Nuijs et al., 2009d).
Triple quadrupole (QqQ) (Hummel et al., 2006; Castiglioni et al.,
2006; Boleda et al., 2007; Huerta-Fontela et al., 2007; Chiaia et al.,
2008; Kasprzyk-Hordern et al., 2008; Bijlsma et al., 2009; Bartelt-Hunt
et al., 2009; González-Mariño et al., 2009; van Nuijs et al., 2009d),
hybrid instruments based on quadrupole-linear ion trap (QLIT)
(Postigo et al., 2008a), and ion trap (IT) (Bones et al., 2007; Gheorghe
et al., 2008) mass spectrometers have been used for the quantitative
determination of drugs of abuse and their metabolites. QqQ and QLIT
have been operated in the selected reaction monitoring (SRM) mode.
This mode of acquisition provides good sensitivity and selectivity and
guarantees reliability of results if at least two specific SRM transitions
for each target analyte are monitored (Pozo et al., 2006). Target
analyte identification by means of IT mass spectrometers has been
achieved by recording structural information of the target analyte
through collision induced dissociation (CID) analyses or MS
n
spectra
of the molecular ions.
Although LC-high resolution MS/MS (using Orbitrap) has not been
yet implemented for identification of illicit drugs in untreated sewage
waters, this technique has also been used by Hogenboomet al. (2009)
to determine and confirm the presence of these substances in treated
wastewater, surface, ground and drinking waters. In addition to
producing high sensitivity full-scans, the combination of ion trap and
Fourier transform cyclotron resonance technologies allows recording
accurate mass MS
n
spectra of high quality.
3. Sewage epidemiology results
3.1. Cocaine
The existing literature describes the presence of the parent
compound cocaine and its metabolites, benzoylecgonine, norbenzoyl-
ecgonine, norcocaine, cocaethylene and ecgonine methyl ester, in
influent wastewater (Zuccato et al., 2005; Hummel et al., 2006;
Castiglioni et al., 2006; Bones et al., 2007; Huerta-Fontela et al., 2007;
Gheorghe et al., 2008; Kasprzyk-Hordern et al., 2008; Postigo et al.,
2008a; Huerta-Fontela et al., 2008; Chiaia et al., 2008; Zuccato et al.,
2008a; van Nuijs et al., 2009a,b,c,d; Mari et al., 2009; Bijlsma et al.,
2009; Banta-Green et al., 2009; Kasprzyk-Hordern et al., 2009; Postigo
et al., 2010).
3.1.1. Back-calculations
Only 1–9% of a cocaine dose is excreted in urine unchanged, while
35–54% and 32–49% is excreted as benzoylecgonine and ecgonine
methyl ester, respectively (Baselt, 2008). Norbenzoylecgonine, nor-
cocaine and cocaethylene are minor metabolites and account for only
traces of the urinary excretion pattern (Postigo et al., 2008b). In the
back-calculations a mean excretion rate of 45% for benzoylecgonine
and of 10% for cocaine will be taken into account. It is however
important to mention that the current available literature reports
rather broad ranges for the excretion of benzoylecgonine (15%–55%)
and cocaine (1%–15%) which could lead to uncertainties in the
calculations (Ambre et al., 1984; Ambre et al., 1988; Cone et al., 1998;
Jufer et al., 2000; Baselt, 2008). Later in this manuscript this will be
discussed more in detail.
Two different methods were used to back-calculate the amount of
consumed cocaine from concentrations of cocaine and its metabolites
in influent wastewater. Zuccato et al. (2005) first proposed a formula
based on concentrations of benzoylecgonine, the major metabolite of
cocaine, which is present in wastewater at relatively high concentra-
tions. From concentrations of benzoylecgonine (ng/L) and with the
flowrate (L/day) of the wastewater stream, benzoylecgonine loads (g/
day) can be calculated. These loads are then further transformed into
an amount of cocaine consumed (g/day) taking into account the
molecular mass ratio of cocaine to benzoylecgonine (1.05) and the
mid-range excretion percentage of 45% as benzoylecgonine (Baselt,
2008). This leads to the following formula:
cocaine
g
day

= concentration BE
ng
L

* flow rate
L
day

* 2:33
A second approach by Bones et al. (2007) suggested using
concentrations of the parent compound, cocaine, for the back-
3569 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
calculation. Similarly, cocaine loads are calculated by multiplying
cocaine concentrations with the wastewater flow rate. Cocaine
consumption is then calculated, assuming that an upward limit of
10% of a cocaine dose is excreted unchanged in the urine (Bones et al.,
2007).
When comparing the two calculation methods, the formula
proposed by Zuccato et al. (2005) is more advantageous and probably
more reliable than that suggested by Bones et al. (2007). First, it has
been observed that benzoylecgonine is stable in water, while cocaine
shows a significant degradation (80% in 24 h at 20 °C and pH 6)
(Gheorghe et al., 2008). Secondly, by employing only a metabolite
(benzoylecgonine) for calculations, the calculated cocaine consump-
tion more likely originates only from human intake, while cocaine in
wastewater can also be present from disposal of unused cocaine.
Kasprzyk-Hordern et al. (2009) compared these two approaches and
observed discrepancies between the results. Calculations based on
concentrations of the unchanged compounds led to significantly
higher cocaine consumption results than calculations based on its
metabolite, benzoylecgonine. The authors conclude that the latter
calculation method is the preferable, while the one based on the
parent compound can lead to overestimations of cocaine use. Only
Bones et al. (2007) use the parent compound for back-calculations,
while all other papers on sewage epidemiology (Huerta-Fontela et al.,
2008; Zuccato et al., 2008a; Kasprzyk-Hordern et al., 2009; van Nuijs
et al., 2009a,b,c; Postigo et al., 2010) adopted the formula of Zuccato et
al. (2005), which uses benzoylecgonine.
3.1.2. Results and comparison
3.1.2.1. a. Local cocaine consumption. Sewage epidemiology work has
been carried out in Italy, Spain, United Kingdom, Ireland, Switzerland
and Belgium. Table 3 gives an overview of the results and
characteristics of each study.
In Italy, samples have been collected from four medium-sized
Italian cities (Cagliari, Cuneo, Latina and Varese) and from one large-
sized city (Milan) (Zuccato et al., 2005; Zuccato et al., 2008a). The
mean cocaine consumption for the medium-sized cities was 0.44 g/
day per 1000 inhabitants, while the large-sized city of Milan showed a
mean cocaine use of 0.91 g/day per 1000 inhabitants. A logical
conclusion of these findings might be that cocaine use in large cities is
higher than in smaller cities.
In Switzerland, Zuccato et al. (2008a,b) observed a mean cocaine
consumption of 0.62 g/day per 1000 inhabitants for a medium-sized
city (Lugano). This finding is comparable with the results for the
medium-sized cities in Italy and is clearly lower than the cocaine
consumption calculated for the large city of Milan.
In Ireland, a wastewater sample was collected from the largest
WWTP receiving water from the city of Dublin (Bones et al., 2007). As
mentioned earlier, the cocaine consumption in this study had been
calculated using the concentrations of the parent compound instead
of benzoylecgonine. A consumption of 1.44 g/day per 1000 inhabi-
tants is reported, but this has to be interpreted with caution. When
calculating the cocaine use based on the benzoylecgonine concentra-
tions, the wastewater flow rate and the number of inhabitants served
by the WWTP, much lower amounts (0.2 g/day per 1000 inhabitants)
are found. Moreover, the data reported by Bones et al. (2007) indicate
higher concentrations of cocaine than benzoylecgonine in wastewa-
ter, which is in contradiction with the metabolism of cocaine (Baselt,
2008) and with all other papers, which report higher concentrations
of benzoylecgonine compared with cocaine in wastewater.
In Belgium, an extensive sampling campaign was executed for 41
WWTPs (van Nuijs et al., 2009a,b,c). Each WWTP was sampled on a
Wednesday and a Sunday to compare week and weekend variations.
Results ranged from 0.06 g/day to 1.83 g/day per 1000 inhabitants
with a mean of 0.43 g/day per 1000 inhabitants. The highest results
were obtained from wastewater originating from large cities such as
Antwerp, Charleroi and Brussels. Further, higher cocaine consumption
was statistically observed for weekend-days (Sunday) compared with
week-days (Wednesday). This finding confirmed the earlier reported
higher weekend cocaine use from wastewater analysed in Italy, Spain
and Belgium (Zuccato et al., 2008a; Huerta-Fontela et al., 2008; van
Nuijs et al., 2009b).
In the United Kingdom, samples were collected from South Wales
(Kasprzyk-Hordern et al., 2009) and London (Zuccato et al., 2008a).
Cocaine consumption calculated from wastewater from two WWTPs
in South Wales was on average 0.6 and 1.2 g/day per 1000 inhabitants,
respectively. It is difficult to compare the results from South Wales
with the estimations in other countries because it is not known from
which cities (small to large-size) these WWTPs receive wastewater.
However, results are in the same order of magnitude as other reported
values. The city of London shows cocaine consumption of 0.69 g/day
per 1000 inhabitants, which is lower compared with other large cities
such as Milan and Antwerp or Brussels.
In Spain, two large monitoring campaigns have been carried out,
both in the North-East of Spain (Huerta-Fontela et al., 2008; Postigo
et al., 2010). In the first study, samples from 42 WWTPs were
collected; the amount of inhabitants served ranged from 200 to
317400. The results ranged from not detected to 5.4 g/day per 1000
inhabitants with a mean of 1.4 g/day per 1000 inhabitants (Huerta-
Fontela et al., 2008). One has to be cautious to generalize from these
results, since very small WWTPs (b5000 inhabitants served) were
included in the sampling campaign and only grab samples were
collected which could lead to high variability in the concentrations of
Table 3
Cocaine use calculated using sewage epidemiology for different countries.
Country
a
WWTPs included Sampling procedure Cocaine use (g/day per 1000 inhabitants) Reference
Belgium 41 24-h composite sampling; 4 samples per
WWTP on Wednesday and Sunday
Range: 0.06–1.86
Mean: 0.43
van Nuijs et al. (2009a,b,c)
Ireland 1 24-h composite sampling; 1.44 Bones et al. (2007)
Italy 5 24-h composite sampling; one
(Varese, Cagliari, Cuneo, Latina) or 21
(Milan) samples
Range: 0.21–0.91
Mean: 0.53
Zuccato et al. (2005, 2008a)
Spain 42 grab sampling; 1 sample per WWTP Range: 0–2.3
Mean: 1.4
Huerta-Fontela et al. (2008)
Spain 7 24-h composite sampling; 2 samples per WWTP Range: 0.6–3.1
Mean: 1.8
Postigo et al. (2010)
Switzerland 1 24-h composite sampling; 7 samples 0.62 Zuccato et al. (2008a)
United Kingdom 2 Grab (Cilfynydd) and 24-h composite samples
(Coslech); 2 samples per month for 5 months
Range: 0.1–3.7
Means: 0.6 and 1.2
Kasprzyk-Hordern et al. (2009)
United Kingdom 2 24-h composite samples (London);
2 samples per WWTP (London)
Mean: 0.3 Zuccato et al. (2008a)
a
WWTP: wastewater treatment plant.
3570 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
cocaine and benzoylecgonine. It can be concluded that the mean value
obtained by this extensive sampling campaign (1.4 g/day per 1000
inhabitants) was higher than the studies fromother countries. Postigo
et al. (2010) collected samples from seven different WWTPs, also
located in the North-East of Spain. All WWTPs served more than
35000 inhabitants. The results ranged from 0.6 to 3.1 g/day per 1000
inhabitants aged 15–64 years, with a mean of 1.8 g/day per 1000
inhabitants aged 15–64. To compare these findings with data from
other studies (reporting cocaine consumption only for the total
population), it is necessary to transformthe results for group aged 15–
64 into usage levels for the total population. This is possible since
there is known that approximately 70% of the served inhabitants of
the WWTPs belong to the group aged 15–64. Transformations to the
total population mean that the cocaine consumption for the total
population ranged from 0.4 to 2.2 g/day per 1000 inhabitants with a
mean of 1.3 g/day per 1000 inhabitants. The findings of Huerta-
Fontela et al. (2008) and Postigo et al. (2010) – both investigating the
North-East of Spain – are comparable and the mean of both sampling
campaigns reflects a higher per capita cocaine consumption compared
with the mean cocaine consumption in Belgium, which was calculated
from a large sampling campaign. This would suggest a higher per
capita cocaine use in Spain than in Belgium, which is in good
correlation with the information given by the UNODC and the
EMCDDA. These instances reported a lower prevalence of cocaine
use in Belgium (0.9%) compared to Spain (3.1%), the latter being also
the highest-prevalence country in Europe (EMCDDA, 2009; UNODC,
2009). It is difficult to compare local results from different countries,
because the studies in the UK, Italy, Switzerland and Ireland were
performed only for a small number of WWTPs, making it difficult to
compare it with national prevalence numbers. If we assume that an
average cocaine dose is 100 mg (UNODC, 2004), the overall
conclusion can be made that the calculated cocaine consumption
ranges between 0.6 and 54 doses/day per 1000 inhabitants. The
results in Italy and Belgium suggest higher cocaine use in large and
high urbanised cities, such as Antwerp, Brussels and Milan. This trend
was also observed by Banta-Green et al. (2009) in the United States of
America.
3.1.2.2. b. National cocaine consumption. Four studies extrapolated the
local results, expressed in g/day per 1000 inhabitants, to regional or
national cocaine use, expressed in tonnes/year (Huerta-Fontela et al.,
2008; van Nuijs et al., 2009c; Kasprzyk-Hordern et al., 2009; Postigo
et al., 2010) (Table 4). Huerta-Fontela et al. (2008) extrapolated their
results, originating from 2.5 million inhabitants, to the whole
population of the North-East of Spain (7.1 million inhabitants) and
calculated a daily use of 7 kg of cocaine, corresponding with
2.6 tonnes a year, for this region of Spain. The authors did not further
extrapolate to Spain (47 million inhabitants). Postigo et al. (2010)
extrapolated their results (from 1.4 million inhabitants) to a yearly
cocaine use in Spain and calculated an amount of 21 tonnes of cocaine
used yearly in Spain. Kasprzyk-Hordern et al. (2009) extrapolated the
results of the two sampled WWTPs in Wales to the total Welsh
population (3 million inhabitants) and obtained an estimate of
cocaine use of 0.93 tonnes/year. A further extrapolation to the
whole United Kingdom (60.6 million inhabitants) led to a yearly
cocaine usage of 19 tonnes. As already pointed out (Huerta-Fontela
et al., 2008; Kasprzyk-Hordern et al., 2009; Postigo et al., 2010), the
extrapolations in Spain and the United Kingdom were made from
results representing less than 5% of the total population and therefore
the consumption maybe not be representative for the whole of the
United Kingdom or Spain.
In Belgium, the extrapolation of the results from wastewater
collected from 3.7 million inhabitants to the total Belgian population
(10.5 million inhabitants) led to a yearly cocaine use of 1.88 tonnes
(van Nuijs et al., 2009c). Here, the authors go even further by
calculating a yearly prevalence of 0.8% for the population aged 15–64
based on several assumptions, such as standard dose of cocaine and
the amount consumed by “the average cocaine user”. The authors
stress that these assumptions need to be confirmed in the future and
that the calculated prevalence should be seen only as indicative.
However, the calculated 0.8% prevalence is in very good agreement
with information about cocaine use in Belgium given by the UNODC,
which states 0.9% prevalence (UNODC, 2004). The study of van Nuijs
et al. (2009c) collected samples equivalent to 35% of the total Belgian
population which is a more representative percentage than the
population included in the Spanish and British studies. Moreover, the
Belgian sampling campaign was executed throughout all regions of
Belgium and in small- to large-sized cities in a way to encompass a
representative part of Belgium.
To compare national cocaine use, the calculated yearly amounts
were divided by population numbers for each country (Table 4). In
this way, it can be observed that Spain has the highest calculated
cocaine use (0.46 g/year per inhabitant) followed by the United
Kingdom (0.31 g/year per inhabitant) and Belgium (0.17 g/year per
inhabitant). This means that Spain has 2.7 and 1.5 times higher
cocaine use than Belgium and the United Kingdom, respectively, and
that the United Kingdom has 1.8 times higher cocaine use than
Belgium. The EMCDDA and the UNODC reported the same trend in
cocaine use: Spain has the highest yearly cocaine use prevalence
(3.1%), followed by the United Kingdom (2.3%) and Belgium (0.9%)
(EMCDDA, 2009; UNODC, 2009). The classical socio-epidemiological
studies showed that Spain has 3.4 and 1.3 times higher cocaine use
prevalence than Belgium and the United Kingdom, respectively, and
that the United Kingdom has 2.6 times higher cocaine use than
Belgium. The national results derived from wastewater analysis are
thus in good agreement with the socio-epidemiological information
from the UNODC and the EMCDDA, except for the comparison
between Belgium and the United Kingdom. This is most probably due
to the way of extrapolating for the United Kingdom. These calcula-
tions used only wastewater originating from a small population in
Wales which is not representative for the total UK population.
3.2. Amphetamine-like stimulants
Various authors have investigated the occurrence of amphe-
tamine-like stimulants (ALS), such as α-methylphenylethylamine
(amphetamine) (Castiglioni et al., 2006; Chiaia et al., 2008; Huerta-
Fontela et al., 2008; Kasprzyk-Hordern et al., 2008; Postigo et al.,
2008a, 2010; Bijlsma et al., 2009; Bartelt-Hunt et al., 2009; van Nuijs
et al., 2009d), methamphetamine (Jones-Lepp et al., 2004; Castiglioni
et al., 2006; Chiaia et al., 2008; Huerta-Fontela et al., 2008; Postigo et
al., 2008a, 2010; Loganathan et al., 2009; Bijlsma et al., 2009; Bartelt-
Table 4
National cocaine use estimated via sewage epidemiology and comparison with reported official prevalence data.
Country Sampled
population
Total
population
Calculated cocaine use
(tonnes/year)
Grams/year per
inhabitant
Yearly
prevalence (%)
a
Reference
Belgium 3.7 million 10.5 million 1.88 0.17 0.9 van Nuijs et al. (2009c)
Spain 1.4 million 46.6 million 21 0.46 3.1 Postigo et al. (2010)
United Kingdom 141000 60.6 million 19 0.31 2.3 Kasprzyk-Hordern et al. (2009)
a
From EMCDDA (2009) and UNODC (2009).
3571 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
Hunt et al., 2009; van Nuijs et al., 2009d), methylenedioxymetham-
phetamine (MDMA) (Jones-Lepp et al., 2004; Castiglioni et al., 2006;
Chiaia et al., 2008; Huerta-Fontela et al., 2008; Postigo et al., 2008a,
2010; Loganathan et al., 2009; Bijlsma et al., 2009; van Nuijs et al.,
2009d), 3,4-methylenedioxyethamphetamine (MDEA) (Castiglioni et
al., 2006; Chiaia et al., 2008; Huerta-Fontela et al., 2008; Bijlsma et al.,
2009), 3,4-methylenedioxyamphetamine (MDA) (Castiglioni et al.,
2006; Chiaia et al., 2008; Huerta-Fontela et al., 2008; Bijlsma et al.,
2009) and ephedrine (Chiaia et al., 2008; Postigo et al., 2008a; Postigo
et al., 2010) in wastewater. However, only amphetamine, metham-
phetamine, MDMA and ephedrine consumption has been estimated
from ALS residues present in wastewaters in a few of the peer-
reviewed works (Huerta-Fontela et al., 2008; Zuccato et al., 2008a,b;
Kasprzyk-Hordern et al., 2009; Banta-Green et al., 2009; Postigo et al.,
2010).
3.2.1. Back-calculations
In the case of ALS, amphetamine is one of the major excretion
products and it has been usually selected as a consumption indicator.
After ingestion, amphetamine, methamphetamine, MDMA and
ephedrine are excreted unchanged in urine and faeces in percentages
up to 30, 43, 65 and 75%, respectively (Baselt, 2008; Postigo et al.,
2008b; Zuccato et al., 2008a). Taking into account these excretion
rates and the molecular mass ratio of each ALS to its corresponding
consumption indicator (1.00), the correction factors used to convert
the loads found in influent wastewaters into the drug amounts
consumed are 3.3 for amphetamine, 2.3 for methamphetamine, 1.5 for
MDMA and 1.3 for ephedrine. It has to be mentioned however that the
excretion rates of amphetamine, methamphetamine and ephedrine
are changing dramatically when the urine of a user is highly acidic or
alkaline (Baselt, 2008) and can influence the back-calculations.
Average doses usually applied to express consumption estimates are
100 mg for MDMA, 25 mg for ephedrine, and between 30 to 50 mg for
amphetamine and methamphetamine (Couper and Logan, 2004;
UNODC, 2004; Baselt, 2008).
The use of amphetamine as a consumption indicator to estimate
ALS use may lead to overestimation. Different sources, other than
amphetamine use, may contribute to amphetamine levels in waste-
water since amphetamine also results from the metabolism of
methamphetamine (up to 7% excreted as amphetamine), and other
drugs such as the stimulant prodrug fenethylline (27%), the appetite
suppressant fenproporex (38%) and the anti-Parkinson drug selegiline
(Maurer and Kraemer, 1992; Baselt, 2008). Similarly, metamphe-
tamine is not only an indicator of methamphetamine consumption,
but its various isomers are also metabolic by-products of selegeline
and the analgesic and antipyretic agent famprofazone (up to 15% of
the dose is excreted as methamphetamine and in trace amounts as
amphetamine and ephedrine) and is further a minor metabolite of the
anorectic pharmaceutical benzphetamine (Maurer and Kraemer,
1992; Baselt, 2008). On the other hand, the reported analytical
methodologies do not differentiate between enantiomeric forms of
the investigated ALS and thus between legal ((R)-(−)-enantiomer,
with a low stimulating activity) and the illegal ((S)-(+)-enantiomer,
with 4–5 times higher stimulating activity) usage (Kraemer and
Maurer, 2002). Therefore, consumption of illicit ALS based on
undifferentiated concentrations measured in wastewater may be
overestimated.
3.2.2. Results and comparison
Estimates of ALS consumption reported in the different countries
through the above described methodology are summarized in Table 5.
Amphetamine use has been observed to vary substantially within the
geographical areas where it has been studied. In the North-East of
Spain, Postigo et al. (2010) calculated an average use of amphetamine
in the Ebro river basin area of 276 mg/day per 1000 inhabitants aged
15–64 (corresponding with 193 mg/day per 1000 inhabitants for the
total population). This exceeds the average amphetamine use that
would result fromthe amphetamine loads reported by Huerta-Fontela
et al. (2008) in the Catalonia region (76 mg/day per 1000 inhabitants
on average). In the study carried out by Zuccato et al. (2008a) in
several European cities, amphetamine was not detected in the
wastewater from Lugano (Switzerland) whereas the levels found in
Milan (Italy) and London (UK) resulted in an amphetamine use
estimation of 9 and 80 mg/day per 1000 inhabitants, respectively. The
highest amphetamine use (2500 mg/day per 1000 inhabitants),
which surpasses the previous estimates by one to three orders of
magnitude, has been reported for South Wales (Kasprzyk-Hordern
et al., 2009).
Methamphetamine was rarely detected in the Ebro River basin
area, where it only reached 1.5 mg/day per 1000 young adults aged
15–34 years (Postigo et al., 2010). This figure is lower than the one
resulting fromthe analyses carried out by Huerta-Fontela et al. (2008)
in the Catalonia region (24 mg/day per 1000 inhabitants). In the study
performed by Zuccato et al., methamphetamine was also found to be
Table 5
Amphetamine-like stimulants use calculated using sewage epidemiology for different countries and comparison with reported official prevalence data.
Country Location Sampled
population
Average doses/day per
1000 inhabitants
a
Yearly
prevalence (%)
b
Reference
Italy Milan 1.25 million AM: 0.3
MA: 0.4
MDMA: 0.06
AM+MA: 0.2
MDMA: 0.4
Zuccato et al. (2008a)
Spain Catalonia 2.0 million AM: 2.5
MA: 0.8
MDMA: 4.0
c
AM+MA: 0.9
MDMA: 2.3
c
Huerta-Fontela et al. (2008)
Spain Ebro River basin 1.4 million AM: 9.2
MA: 0.05
c
MDMA: 0.6
c
AM+MA: 0.9
MDMA: 2.3
c
Postigo et al. (2010)
Switzerland Lugano 0.12 million AM: n.d.
MA: n.d.
MDMA: 0.1
Not available Zuccato et al. (2008a)
United Kingdom London 5.5 million AM: 2.6
MA:0.2
MDMA: 0.05
AM+MA: 1.0
MDMA: 1.5
Zuccato et al. (2008a)
United Kingdom South Wales 0.14 million AM: 83.3 AM+MA: 1.0
MDMA: 1.5
Kasprzyk-Hordern et al. (2009)
AM = amphetamine; MA = methamphetamine; MDMA = methylenedioxymethamphetamine.
a
Average dose of amphetamine and/or methamphetamine: 30 mg; average dose of MDMA: 100 mg (UNODC, 2004).
b
Last year prevalence of drug use among adult population(aging 15-64) for eachcountry. Values reportedfor UnitedKingdomrefer only toEngland and SouthWales (EMCDDA, 2009).
c
On the basis of young adults (aging 15–34).
3572 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
absent in the wastewater from Switzerland, whereas it reached about
10 mg/day per 1000 inhabitants in the city of Milan (Italy) and it was
below 6 mg/day per 1000 inhabitants in the city of London (UK)
(Zuccato et al., 2008a).
According to official estimations (EMCDDA, 2009), the prevalence
of amphetamine and methamphetamine use among the adult
population aged 15–64 years in 2008 reached 1% in England and
South Wales, 0.9% in Spain and 0.2% in Italy. If we assume that a typical
daily intake is one 30 mg pill (UNODC, 2004), the prevalence of
amphetamine use would be 0.3% in London and 8% in South Wales,
between 0.3 and 0.9 % in the North-East of Spain, and 0.1% in Milan.
Considering that these values are representative of their respective
countries, sewage epidemiology results in Spain and Italy do not
deviate very much from the EMCDDA official estimations, whereas
results from wastewater analysis in the UK suggest a higher
consumption compared to the values reported by the EMCDDA.
Ecstasy or MDMA consumption in the North-East of Spain was on
average 60 mg/day per 1000 young adults (aged 15–34) in the Ebro
river basin area (Postigo et al., 2010), and 400 mg/day per 1000 young
adults in the Catalonia region (Huerta-Fontela et al., 2008). Average
estimations reported for the cities of Milan, London and Lugano were
below 10 mg/day per 1000 inhabitants (Zuccato et al., 2008a). It is
difficult to compare the sewage epidemiology results from Spain and
Italy, because both studies report values for very different age classes
(in Spain: young adults; in Italy: total population).
The prevalence of MDMA use among young adult population (15–
34 years) in 2008 was reported to be 2.3, 1.5 and 0.4% in Spain, in
England and South Wales and in Italy, respectively (EMCDDA, 2009).
If we assume that the daily intake is one 100-mg pill (UNODC, 2004),
MDMA prevalence data resulting from the sewage epidemiology
approach (between 0.06 and 0.4% of young adults in North-East of
Spain, and less than 0.01% adults in Italy and in England and South
Wales) are well below those reported by classical social-epidemio-
logical methods. The differences would be reduced if the actual
consumption of an average MDMA user were lower than that
considered for the calculations. On the other hand, the sewage
epidemiology data for MDMA were normalized among different age
segments in the different countries, making inter-comparison
difficult.
In Oregon (USA), Banta-Green et al. (2009) estimated consump-
tion for methamphetamine and MDMA (mg/inhabitant per day, not
corrected for excretion rates) and related it to the degree of urbanism
of the investigated areas. Changes in methamphetamine use within
the areas of study were not significant, whereas higher consumption
of MDMA was linked to more urbanised areas. Yet, the highest per
capita consumption of these substances was observed in one of the
smallest urban areas investigated in the Ebro River basin (Spain)
(Postigo et al., 2010).
Estimates of ephedrine consumption have been performed in the
Ebro River basin area (North-East of Spain) (Postigo et al., 2010). An
average of 300 mg/day per 1000 inhabitants aged 15–64 was obtained
for this drug which, apart from illegal abuse, is also therapeutically
applied to treat asthma and bronchitis.
3.3. Opiates
Untreated wastewater of WWTPs in Italy, Spain, Switzerland, the
UK, Belgium, Germany and Ireland contained morphine and 6-
acetylmorphine (a metabolite specific for heroin). Morphine median
concentrations were lower than 100 ng/L in Italy and Spain
(Castiglioni et al., 2006; Boleda et al., 2007; Postigo et al., 2008a),
but higher in Germany, Switzerland, and England (approx. 300, 200
and 600 ng/L, respectively), probably in relation to higher consump-
tion of therapeutic morphine in these countries (Hummel et al., 2006;
Zuccato et al., 2008a). Levels of 6-acetylmorphine were in the range
8.6–14 ng/L. Trace amounts of morphine-3-glucuronide (up to 5.1 ng/
L) were also found in some WWTPs (Zuccato et al., 2008a). Opiate
pharmaceuticals such as codeine, methadone and its major metabolite
2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine were also mea-
sured in Italy, Switzerland, Germany and Spain (Castiglioni et al.,
2006; Hummel et al., 2006; Boleda et al., 2007).
3.3.1. Back-calculations
Morphine is excreted mainly as the glucuronide conjugate,
morphine-3-glucuronide. However, it is promptly hydrolysed to
morphine by the beta-glucuronidases of faecal bacteria present in
untreated wastewater (D'Ascenzo et al., 2003) and during wastewater
treatment (Ternes et al., 1999). Morphine can therefore be used as a
consumption indicator either of heroin or morphine. Unfortunately,
several other sources may account for the presence of morphine in
wastewater, including therapeutic use of morphine and codeine.
However, results from a study carried out in Milan and Lugano suggest
that the relative contribution of codeine use to wastewater morphine
levels can be considered likely negligible (Zuccato et al., 2008a). Hence,
when back-calculating heroin consumption based on wastewater
morphine, corrections must be only applied to compensate for the
contribution from therapeutic morphine. In a recent study carried out
in Italy, Switzerland and UK(Zuccato et al., 2008a), the daily amounts of
wastewater morphine expected to originate from therapeutic mor-
phine use were subtracted from the total daily amounts of wastewater
morphine. The remaining wastewater morphine was assumed to
originate exclusively from heroin. Accurate updated information
about local morphine use is therefore necessary to estimate for actual
heroin consumption. Since 42.5% of a dose of intravenous heroin is
excreted in the urine as morphine (Baselt, 2008) and that the heroin/
morphine molecular mass ratio is 1.29, the following formula has been
used to back-calculate heroin consumption:
heroin
g
day

=
ð
concentration morphine
ng
L

* flow rate
L
day

−therapeutic morphine
g
day

Þ
*3:04
Alternatively 6-acetylmorphine, a minor but exclusive metabolite
of heroin (excretion rate 1.3% (Baselt, 2008)), could also be used to
estimate consumption of this illicit drug, but until now it is not used
because of the lowconcentrations found in wastewater. However, the
presence of 6-acetylmorphine in the wastewater samples from Milan
and Lugano, at levels that averaged about 5% of measured morphine,
proved that heroin is a constant contributor to wastewater morphine
(Zuccato et al., 2008a).
3.3.2. Results and comparison
Sampling campaigns have been carried out in Milan, Lugano,
London in 2005-2006 (Zuccato et al., 2008a), and in North-East of
Spain in 2007–2008 (Boleda et al., 2009; Postigo et al., 2010). These
results are summarized in Table 6.
In Milan, Lugano, and London, heroin consumption has been
estimated from the total wastewater morphine, after subtracting the
fraction presumably originating fromlocal use of therapeutic morphine
(Zuccato et al., 2008a). After subtraction, wastewater morphine from
heroin consumption accounted for 23, 32 and 68 mg/day per 1000
inhabitants in Milan, Lugano, and London, respectively. Since 42.5% of a
dose of intravenous heroin is excreted in the urine as morphine (Baselt,
2008) and that the heroin/morphine molar ratio is 1.29, this
corresponded to a mean of 70, 100 and 210 mg heroin/day per 1000
inhabitants in Milan, Lugano, and London, respectively. Boleda et al.
(2009) measured morphine in 15 WWTPs from Catalonia (NE Spain)
and Postigo et al. (2010) monitored this compound in 7 WWTPs in the
Ebro River basin (NE Spain). They estimated heroin consumption of
138 mg/day per 1000 inhabitants and 61 mg/day per 1000 inhabitants
aged 15–64 years (corresponding to about 43 mg/day per 1000
3573 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
inhabitants and considering that approximately 70% of the served
inhabitants of the WWTPs are of the 15–64 age class). Consumption
rates in doses/day per 1000 inhabitants were also calculated, consid-
ering an average content of pure active drug in a typical dose of
30 mg for intravenous heroin (UNODC, 2004). Means±SD were 2.3±
0.2, 3.3±0.5 and 7.0±1.2 doses/day per 1000 inhabitants in Milan,
Lugano, and London, respectively (Zuccato et al., 2008a) and 4.6 and 1.4
doses/day per 1000 inhabitants in the North-East of Spain (Boleda et al.,
2009; Postigo et al., 2010). Extrapolation to the whole countries would
suggests that heroin consumption was higher in the UK than in Italy,
Switzerland and Spain, which is in line with information from the
UNODC (2006), which reported an annual prevalence of heroin use for
the age group 15–64 years in England and Wales (0.9%), greater than
those in the other countries. However, consumption estimates by
wastewater analysis in Milan were lower than in Lugano and in the
North-East of Spain, while the corresponding prevalence of use were
higher in Italy (0.8%), than in Switzerland (0.6%) (UNODOC, 2006) and
Spain (0.4%) (EMCDDA, 2009). This discrepancy might result from
spatial and temporal differences in consumption and difficulties in
extrapolating data from single cities or regions to the whole country.
A further study was carried out in Milan one year later. Results
showed that heroin consumption estimated in the Milan population
served by the WWTP located in Nosedo (1 250 000 inhabitants)
decreased from a mean of 2800 doses/day in 2006 to 1900 doses/day
in 2007 (Zuccato et al., 2008b).
3.4. Cannabis
Residues of the active principal of cannabis, Δ9-tetrahydrocan-
nabinol (THC), and its metabolites 11-nor-9-carboxy-Δ9-tetrahydro-
cannabinol (THC-COOH) and 11-hydroxy-Δ9-tetrahydrocannabinol
(OH-THC) have been documented in untreated wastewater of several
municipal WWTPs. Average levels of THC-COOH were 62, 91 and
159 ng/L in Milan Lugano and London respectively (Zuccato et al.,
2008a), and 15.3 and 37.8 ng/L in Spain (Boleda et al., 2007; Postigo
et al., 2008a), where the same authors found also measurable levels of
THC (11.3–18 ng/L) and OH-THC (30.7 ng/L).
3.4.1. Back-calculations
Estimates of THC consumption rates have been mainly based on
THC-COOH levels in wastewater, taking into account previous findings
in volunteers that, for each milligram of THC smoked as resin or herbal
cannabis, 6 micrograms (0.6%) of THC-COOH are excreted in the urine
(Huestis et al., 1996) and that the THC/THC-COOH molecular ratio is
0.91. THC-COOH can be excreted as the glucuronide conjugate (Baselt,
2008), but for the reasons explained above for morphine the free
compound alone is used as a target residue.
3.4.2. Results and comparison
Studies have been carried out in Milan, Lugano, London in 2005–
2006 (Zuccato et al., 2008a), and in North-East of Spain in 2007–2008
(Boleda et al., 2009; Postigo et al., 2010) (Table 7). THC consumption
estimates were 3.0, 6.6 and, 7.6 g/day per 1000 inhabitants, in Milan,
Lugano and London, respectively, while in the North-East of Spain,
Boleda et al. (2009) and Postigo et al. (2010) estimated a consumption
3.5 g/day per 1000 inhabitants for the region of Catalonia and 0.5 g/
day per 1000 inhabitants for the Ebro River basin area, respectively.
Cannabis consumption rates expressed as doses were (mean±SD)
24±2, 53±12 and 61±26 doses/day per 1000 inhabitants in Milan,
Lugano and London, respectively (Zuccato et al., 2008a) and 27
(Boleda et al., 2009) or 4 doses/day per 1000 inhabitants (Postigo
et al., 2010) in the North-East of Spain. These figures are in good
agreement with data from the UNODC which in 2006 reported an
annual prevalence for cannabis use in the age group 15–64 years of
11% in UK, 10% in Switzerland and 7% in Italy (UNODC, 2006), while
the annual prevalence estimate for Spain in 2008 was 10.1%
(EMCDDA, 2009). Results of a second sampling campaign carried
out in 2007, indicated a decrease of the cannabis consumption rates in
Milan, were the estimated usage in the population served by the
Nosedo WWTP (1250000 inhabitants) decreased from 3.8±0.6 kg/
day (about 30.400 doses/day) in 2006 to 2.8±0.2 kg/day (22 500
doses/day) in 2007 (Zuccato et al., 2008b).
4. Critical discussion
The above results show the usefulness of the wastewater analysis
for the estimation of local and nation-wide illicit drug consumption.
The reported results of the different sewage epidemiology studies are
comparable for the most used illicit drugs (cocaine, amphetamine-like
stimulants, cannabis and opiates) and draw similar conclusions. The
agreement between wastewater results and socio-epidemiological
information from internationally-acknowledged authorities, such as
Table 6
Heroin use calculated using sewage epidemiology for different countries and comparison with reported official prevalence data.
Country Location Sampled
population
Average doses/day
per 1000 inhabitants
Yearly
prevalence (%)
a
Reference
Italy Milan 1.25 million 2.3 0.8 Zuccato et al. (2008a)
Spain Catalonia 2.0 million 4.6 0.4 Boleda et al. (2009)
Spain Ebro River basin 1.4 million 1.4 0.4 Postigo et al. (2010)
Switzerland Lugano 0.12 million 3.3 0.6 Zuccato et al. (2008a)
United Kingdom London 5.5 million 7 0.9 Zuccato et al. (2008a)
a
Last year prevalence of drug use amongadult population(aging15–64) for eachcountry. Values reportedfor UnitedKingdomrefer only toEnglandandSouthWales (EMCDDA, 2009).
Table 7
Cannabis use calculated using sewage epidemiology for different countries and comparison with reported official prevalence data.
Country Location Sampled
population
Average doses/day
per 1000 inhabitants
Yearly
prevalence (%)
a
Reference
Italy Milan 1.25 million 24 7 Zuccato et al. (2008a)
Spain Catalonia 2.0 million 27 10.1 Boleda et al. (2009)
Spain Ebro River basin 1.4 million 4 10.1 Postigo et al. (2010)
Switzerland Lugano 0.12 million 53 10 Zuccato et al. (2008a)
United Kingdom London 5.5 million 61 11 Zuccato et al. (2008a)
a
Last year prevalence of drug use among adult population (aging 15-64) for each country. Values reported for United Kingdom refer only to England and South Wales (EMCDDA,
2009).
3574 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
the UNODC and the EMCDDA, highlights the potential value of sewage
epidemiology. However, the sewage epidemiology approach is still in
development and future research is strongly recommended before
sewage epidemiology can be used in routine drug monitoring
campaigns. Table 8 overviews each part of the sewage epidemiology
approach and the possible bias(es) that can be related with that part
of the methodology.
The analytical determination of illicit drugs and metabolites in
wastewater requires a thorough sample preparation due to the low
concentrations (ng/L range) that have to be measured in a difficult
matrix. While most studies apply off-line SPE, two alternative ways –
large volume injection and on-line SPE – have also been proposed
(Chiaia et al., 2008; Postigo et al., 2008a). Both methods are less time-
consuming and easier in handling, but also have disadvantages over off-
line SPE. In LVI, higher limits of quantification and matrix effects are
observedcomparedwithoff-line or on-line SPE. The on-line SPE method
reports rather low recoveries and very high matrix effects. As a result,
both methods require the use of deuterated internal standards for
quantification to correct for matrix effects. With off-line SPE, although
protocols are more elaborate, matrix effects are significantly lower, and
the use of deuterated internal standards is still recommended.
Analytically, efforts should be made in the future to discriminate
between enantiomers, if applicable. Kasprzyk-Hordern et al. (2009)
point out that only one enantiomer of amphetamine is used illegally,
while the other enantiomer is present in pharmaceutical preparations
and is a metabolite of certain legal drugs. If these enantiomers are
chromatographically not separated, an overestimation of the illegal
use of amphetamine is probably made. Concerning the MS/MS
detection, other ionization sources (such as atmospheric pressure
chemical ionization; APCI) should be tested to reduce the above
mentioned matrix effects.
As for the measurement of the concentrations of illicit drugs in
influent wastewater, there is a clear need for stability studies. Most
published studies implicitly assume that the investigated illicit
drugs and metabolites are stable during the transport from the
place of excretion to the WWTP, during the sampling procedure
(24-h composite sampling) and during storage. A few stability
experiments (Castiglioni et al., 2006; Gheorghe et al., 2008) show,
however, that degradation of the studied compounds could be an issue.
As an example, they report that cocaine is readily degraded in
wastewater. It is obvious that for all studied compounds, extensive
and multi-factorial stability experiments have to be conducted
regarding the degradation in wastewater during its transit time to
the WWTP and during sampling and storage. If degradation occurs, the
measured concentrations of illicit drugs and metabolites in influent
wastewater must be corrected for this loss. As a result, the calculated
usage of the illicit drugs would be higher than now calculated.
Standardised protocols for water collection, storage and handling
before SPE (e.g. thawing cycles) are essential. In general, there is a need
for an agreement on which validation guidelines have to be followed
when analytical methods are validated. Moreover, interlaboratory
exercises would ameliorate the quality and comparability of the results.
Back-calculation from the concentration in wastewater to an
imputed amount of consumed illicit drug is an important step in the
sewage epidemiology approach and has a major impact on calculated
illicit drug consumption. The knowledge of the metabolism pattern of
each illicit drug plays here a crucial role. The pharmacokinetic
parameters of the illicit drugs applied in the present studies use
information from rather old studies, executed on limited populations
with non-representative dosages and administration routes of the illicit
drugs andemploying obsolete techniques. Furthermore, the values used
inthe back-calculations are a meanof all studies, but have highstandard
deviation indicating large variations. For example, the reported percent
urinary excretion of cocaine as benzoylecgonine ranges from15%to 55%
(mean 45%) (Ambre et al., 1984, 1988; Cone et al., 1998; Jufer et al.,
2000). To unambiguously knowthe pharmacokinetic parameters of the
illicit drugs following efforts have to be made: 1/ a comprehensive and
critical evaluation of the existing literature and 2/ new clinical research
is needed with representative populations, dosages, administration
routes and the best techniques to confirm or update this information.
Back-calculation is also subject to two important parameters of the
WWTP, namely, the number of inhabitants served by a WWTP and the
Table 8
The sewage epidemiology approach, its possible biases and necessary future research.
Sewage epidemiology methodology part Possible bias Solution + Future research
Concentrations of illicit drugs and/or metabolite(s)
in influent wastewater
1/ Adsorption to particulate; degradation during transport to
WWTP
1/ Performance of stability tests and adsorption
experiments
2/ No distinction in different chiral forms in analytical methods 2/ Include chiral separation in analytical methods if
necessary
3/ Dumping of (large) amounts of illicit drugs via sewage
system
3/ Monitor preferably metabolite(s) originating
from human metabolism
4/ Different applied analytical techniques 4/ Organisation of interlaboratory tetst and ring tests
5/ Leakage/outflow of wastewater in the sewer system 5/ Strong control of wastewater flow at WWTP
Back-calculations from concentrations in wastewater
to an amount of used illicit drug (g/day)
A. Flow rate of wastewater 1/ Errors on measurement of flow rate in WWTP 1/ Estimate standard deviation of measurements
B. Excretion rates of illicit drugs and/or
metabolites after human consumption
1/ Experiments executed on a limited numbers of subjects 1-4/ Further pharmacokinetic studies with larger
number of subjects, different routes of intake,
use patterns,...
2/ Experiments are not discriminating in different routes of
intake
3/ Experiments are executed with rather old techniques
4/ Excretion rates are depending on various factors or are
varying inter-individual (pH of urine, route of intake, dose...)
Amount of inhabitants served by a WWTP to
normalise the calculate amount of used
illicit drug for the amount inhabitants of the
WWTP region (g/day per 1000 inhabitants)
1/ Fluctuating number of inhabitants served by a WWTP
(tourists, commuters, holidays,...)
1/ Monitor number of inhabitants for each collected
sample separately by various indicators (e.g. energy
consumption, other human by-products in wastewater,
calculations based on N, P excretion,...)
Standard dose of illicit drug to calculate
amount of doses consumed in WWTP region
(doses/day per 1000 inhabitants)
1/ Different routes of intake correspond sometimes with
different dosages
1-3/ Detailed socio-epidemiological research
2/ Different drug markets
3/ Different habits in consumption (light users, heavy users,
occasional users)
Consumption pattern of “average user” to
calculate prevalence of illicit drug use in
certain region
1/ Type of drug (used on regular base vs party drug, constant
dose,...)
1-2/ Detailed socio-epidemiological research
2/ Type of users (light users, heavy users, occasional users,...)
WWTP=wastewater treatment plant.
3575 A.L.N. van Nuijs et al. / Science of the Total Environment 409 (2011) 3564–3577
flow rate. Until now, the number of inhabitants served by a WWTP is
generally derived from the design capacity of the WWTP. However, it is
clear that the population served by a WWTP is not stable along time and
that daily variations occur following commuting, holidays, etc.
Therefore, alternative ways should be found to calculate the real
number of inhabitants corresponding to each collected wastewater
sample. The determination of biological oxygen demand, chemical
oxygen demand, total phosphorus and nitrogen in wastewater or the
measurement of a stable indicator of human metabolism could be an
alternative and should be consistently explored in the future (Andreot-
tola et al., 1994; Zessner and Lindtner, 2005; Garnier et al., 2006). As for
the flowrate, the error of the measured value is not known. A high error
for the flowrate will lead to high errors on the measured values of illicit
drug consumption. Discussions with WWTP operators could develop
low-error measurements of the flow rate. Knowledge on the time of
residence of the drug residue in the sewer and on the light and
temperature conditions of the sewer collection–distribution system
would also be important since these factors can modulate degradation.
The calculation of prevalences of illicit drug consumption derived
from wastewater analysis requires several assumptions such as the
standard dose that is ingested (depending on the administration
route), average consumption pattern, etc. These parameters can only
be derived fromother studies (e.g. socio-epidemiological) andare very
important for the calculation of the prevalence by means of
wastewater analysis. Inthis light, it is important that multi-disciplinary
meetings are organised to tune both methodologies to each others
needs. Moreover, standardization of back-calculations is essential for a
high comparability between results from different studies.
Sewage epidemiology results should be viewed with a certain
degree of uncertainty — not as exact numbers. Even the measured
concentrations of illicit drugs have to be interpreted critically. For
example, when wastewater samples are analysed for the parent
compound (ALS, cocaine, and methadone) rather than a unique
metabolite, the possibility exists that a certain portion is present as a
result of direct discharge of the illicit drug to the sewer. The detection
and quantification of unique and specific metabolite(s) in wastewater
can overcome this problem but there are limitations here too. Several
compounds (ALS and cocaine), for example, are also legally used and
several measured metabolites (such as morphine and amphetamine)
can also originate from the ingestion of commercial pharmaceuticals.
This could lead to an overestimation of the illicit drug consumption.
5. Conclusions
This review shows that sewage epidemiology is a promising tool
for the estimation of local and nation-wide illicit drug consumption.
Clearly, the method is still in full development and various issues and
shortcomings need to be addressed with new and extensive research
before it might be used in routine drug monitoring systems. This
approach may never become a stand alone or an alternative to the
classical socio-epidemiological studies, but it could offer useful
additional information − eventually leading to a better knowledge
of illicit drug use and perhaps helping policy makers organise
prevention campaigns and targeted actions. In addition to the socio-
epidemiological studies, this approach can allow the identification of
illicit drug consumption trends in a short period of time and provides
near real-time data and in-field information on illicit drug abuse.
Acknowledgements
Adrian Covaci and Alexander van Nuijs are grateful to the Flanders
Scientific Funds for Research (FWO) for the financial support. Isabela
Tarcomnicu acknowledges the University of Antwerp for funding.
Cristina Postigo, Miren Lopez de Alda and Damia Barcelo acknowledge
the funding of the Spanish Ministry of Science and Innovation through
the projects SCARCE (Consolider-Ingenio 2010 CSD2009-00065) and
CEMAGUA (CGL2007-64551/HID). We greatly acknowledge the
thorough and positive comments of an anonymous reviewer which
have improved very much the manuscript.
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