The Presence of Stimulant Drugs in Wastewater from Krakow (Poland): A Snapshot
The Presence of Stimulant Drugs in Wastewater from Krakow (Poland): A Snapshot
Katarzyna Styszko 0 1 2
Agnieszka Dudarska 0 1 2
Dariusz Zuba 0 1 2
0 Institute of Forensic Research , Westerplatte 9, 31-033 Krakow , Poland
1 Department of Coal Chemistry and Environmental Sciences, AGH University of Science and Technology , Al. Mickiewicza 30, 30-059 Krakow , Poland
2 & Katarzyna Styszko
An analysis of wastewater from Krakow (Poland) for the presence of controlled and uncontrolled stimulant drugs of abuse was performed. Samples were collected from the Plaszow wastewater treatment plant, Krakow, Poland, and prepared by solid phase extraction. The LC-QTOFMS method was applied for identification and quantification of popular stimulants: MDMA, mephedrone, 4-MEC, MDPV and mCPP. Environmental loads of illicit drugs were calculated; the WWTP discharged loads ranging from 3.6 to 6.7 mg day-1 1000 inhabitants-1 of MDMA, 3.6 to 7.1 mg day-1 1000 inhabitants-1 of mephedrone and 4.8 to 5.8 mg day-1 1000 inhabitants-1 of 4-MEC. The results confirmed the growing popularity of new psychoactive substances in Poland.
LC-ESI-QTOF-MS; Wastewater analysis; Solid phase extraction; Illicit drugs
In recent years, a large number of new psychoactive
substances (NPS) have been marketed
(Adamowicz et al.
2013; Zuba et al. 2013; Reid et al. 2014; Castiglioni et al.
2015; Report 2015)
. The first ‘head shops’ offering NPS
were opened in Poland in 2008 and in 2010, around 1500
stores selling NPS without any control were in operation
. Up to May 2009, the most popular class of
substances sold as ‘legal highs’ were piperazines, including
1-[3-trifluoromethyl)phenyl]piperazine (TFMPP), 1-(4-fluorophenyl)piperazine (pFPP), and
(Byrska et al. 2010)
BZP and mCPP seemed to be the most popular and easy
accessible substances for users, therefore the European
Monitoring Centre for Drugs and Drug Addiction
(EMCDDA) carried out a risk assessment on these
substances and noted drug addiction, many intoxications,
including lethal poisonings. Based on their reports, BZP was
banned in almost every European country (including Poland),
whereas mCPP has been actively monitored (Annual Report
2012). After the ban for BZP, the market moved to a new
direction; the first derivatives of cathinone were marketed
(Zuba and Byrska 2013)
(4-methylmethcathinone, MPD) became the substance of preference by users and
its popularity has been growing month by month
The popularity of mephedrone was reflected in the increase in
the number of drug addicts. Due to the growing popularity of
NPS, monitoring of drugs of abuse in wastewater had to be
expanded in order to cover a broader range of substances. A
number of papers on quantification of opioids, cannabis
derivatives, codeine, methadone, BZP and mCPP in wastewater
(Zuccato et al. 2008; Baker et al. 2012; Thomas
et al. 2012; Andres-Costa et al. 2014; Bijlsma et al. 2014)
. It was
shown that due to the poor degree of purification in treatment
plants, illicit drugs are still present in effluents being discharged
to surface water
(Kasprzyk-Hordern et al. 2009; van Nuijs et al.
2009; Zuccato and Castiglioni 2009; Mendoza et al. 2014)
to their properties, they can be toxic to aquatic organisms
(Pomati et al. 2007; Rosi-Marshall et al. 2015)
substances have been also identified in drinking water, even
after the treatment process
(Castiglioni et al. 2011; Mendoza
et al. 2014)
. Therefore, monitoring of their presence in different
kinds of water is an important issue.
The aims of this pilot study were to investigate the
profile of stimulant drugs taken by users in Krakow, and to
estimate the environmental loads and consumption. The
study covered ‘traditional’ drug of abuse, MDMA, and
common novel psychoactive substances, that is mCPP,
mephedrone, 4-MEC and MDPV. This is the first study
based on the prevalence of stimulant drugs in the Krakow
Materials and Methods
Standard solutions of mephedrone and MDPV were
purchased from the Australian Government National
Measurement Institute (North Ryde, Australia), MDMA from
Cerilliant (Round Rock, TX, USA), mCPP from Lipomed
AG (Arlesheim, Switzerland), while 4-MEC from LGC
Standards Sp. z o.o. (Dziekano´w Les´ny, Poland). The
molecular formulas, physical and chemical properties of
the compounds are summarized in Table 1. The isotope
labelled standard MDMA-D5 (1.0 mg mL-1 in methanol)
was purchased from Cerilliant (Round Rock, TX, USA).
HPLC supergradient grade methanol and ammonia (25 %)
were obtained from POCH (Gliwice, Poland).
Hydrochloric acid (32 %) and formic acid (89–91 %) were purchased
from Merck (Darmstadt, Germany). HPLC-grade
acetonitrile was purchased from J.T. Baker (Phillipsburg, NJ,
USA). Deionized water was obtained by reverse diffusion
in a Millipore system (Warsaw, Poland).
Effluent samples were collected from the Plaszow
WWTP, Krakow, Poland. It treats approximately
165,000 m3 of urban wastewater per day, which is over
70 % of the total volume of the city’s wastewater. Effluent
water was collected after the secondary treatment, which
involves primary settling, biological treatment and
Effluent samples were collected in May 2012. Four
wastewater samples (5 L each) were collected once a week,
on Sunday. Equal aliquots of wastewater were taken every
hour over a 24 h period, collected in pre-cleaned
polyethylene containers with UV protection and stored at 4 C
until the collection process was finished. Then, samples
were transported to the laboratory and processed within
12 h. In the first step, before the solid phase extraction,
wastewater was filtered using MN GF-4 and MN GF-1
glass fibre filters from Macherey–Nagel (Du¨ren, Germany).
Afterwards, samples were acidified to pH 4.5 with 2 M
a values obtained from ChemSpider Database
Product 1 main product ions (quantifier), Product 2 secondary product ions (qualifier)
RT retention time in min, precursor and product mass fragments
Oasis HLB 3 cc (60 mg/3 mL) extraction cartridges
from Waters (Milford, MA, USA) were used in the
analytical procedure. SPE was performed using a 12-port
vacuum extraction manifold (J.T. Baker, Philipsburg,
USA). The extraction cartridges were conditioned by 2 mL
MeOH/NH4OH (v/v, 95:5) and 2 mL deionised water
adjusted with hydrochloric acid solution to pH 4.5. 500 mL
of samples adjusted to pH 4.5 were spiked with 4 lL
(200 ng) internal standard solution of concentration
50 lg mL-1 before being passed through SPE cartridges.
A flow of 5 mL min-1 water through the cartridge was
achieved by applying a mild vacuum to the extraction
manifold. The respective cartridges were then washed by
10 mL deionised water (pH 4.5) and completely dried
under vacuum. The analytes were eluted twice from the
cartridges with 2 mL of MeOH/NH4OH (v/v, 95:5).
Extracts were pooled to 5 mL vials and stored in a freezer
in temperature -24 C for 24/48 h until analysis. Directly
prior to analysis 100 lL (0.025 M) hydrochloric acid were
added to extracts. Subsequently, extracts were evaporated
to 100 lL by nitrogen stream in a Pierce Reacti-Vap III
evaporator and were finally reconstituted with mobile
phase to a volume of 1 mL. The supernatants were
successively transferred into 2 mL auto sampler vials for
analysis by means of LC–ESI-QTOF-MS. In the
experiments, all calibration solutions as well as extracts were
dissolved in starting mobile phase (0.1 % formic acid in
95 % water/5 % acetonitrile).
LC/MS analyses were carried out using an Agilent
Technologies 1200 Series liquid chromatography
instrument coupled with a 6520 Accurate-Mass Q-TOF LC/MS
detector equipped with electrospray ionization (ESI)
manufactured by the same company. The mobile phase was
composed of a mixture of 0.1 % (v/v) formic acid in water
(A) and in acetonitrile (B). The separation was performed
at 35 C using an Ascentic Express C18 column
(7.5 cm 9 2.1 mm 9 2.7 lm) from Supelco. The flow rate
was 0.3 mL min-1, and the volume of injection was 10 lL.
All analyses were carried out in a gradient mode (shown in
relation to content of phase B): 0 min—5 %, 16 min—
20 %, 16.2 min—5 %, 21 min—5 %.
The instrument operated with an electrospray ion source
(ESI) in positive ionization mode. Nitrogen was used as the
drying gas at a temperature of 300 C and a flow rate of
10 L min-1 and the nebulizing gas at a pressure of 45 psi.
Capillary voltage was set at 3000 V and skimmer voltage
at 65 V. Fragmentor voltage was set at 120 V. The
quadrupole was used as an ion guide in MS mode, and for
selection of precursor ions with Dm/z = 1.3 in MS/MS
mode. Nitrogen was used as the collision gas in MS/MS
mode and collision energy was set at 4 V for MDMA and
MDMA D5, 8 V for mephedrone and 4-MEC, 16 V for
mCPP, 24 V for MDPV. Collision energy for compounds
was obtained by using Mass Hunter Optimizer Software,
Version B.02.00 (Agilent Technologies).
The mass range was 50–1000 m/z, in both MS and MS/MS
modes. Spectra were internally mass-corrected in real time
using an automatically introduced reference mass solution
containing two compounds: purine ([M ? H]? = 121.0509)
HP-921—hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine ([M ? H]? = 922.0098).
The concentrations of target compounds were
determined by using the main product ion (quantifier) presented
in Table 2. Concentrations in the samples were calculated
by comparing the peak area ratios of the analytes and
surrogate standard to the corresponding ratios in the
standard solutions. The stimulant drugs stock solution with a
concentration of 10 lg mL-1 in methanol was used for
preparation of calibration solutions. Calibrations curves
were obtained from this stock solution spiked in mobile
phase at 5, 10, 25, 50, 100, 250, 500, 1000 ng mL-1. All
calibration solutions contained 200 ng mL-1 of the
internal standard, MDMA D5.
Results and Discussion
Extraction efficiency was tested by placing 500 mL
deionized water adjusted to pH 4.5 in volumetric flasks and
then spiking them with prepared drugs stock solutions.
After manual shaking, the samples were extracted by SPE.
For each sample three extractions were prepared and each
extract was analysed in triplicates. To check for
concentration dependencies of the recovery rate and to determine
the limit of quantification from the recovery experiment,
deionized water samples were spiked to 40, 200 and
600 ng L-1 of the target compounds. All the samples were
spiked with labelled internal standard as well as
hydrochloric acid to adjust the pH to 4.5. The eluates were
concentrated and reconstituted with mobile phase to 1 mL
and extracts were analysed by LC–ESI–QTOF–MS. The
results presented in Table 3 show that most recovery rates
were between 50 % and 100 %.
The recovery rates were quantitated and the relative
standard deviations (RSD %) were about 10 %. The
applied conditions of the liquid chromatograph allowed for
the successful separation of all analytes within 11 min.
Before analysis of target compounds, blank measurements
were applied for detection of possible impurities. For all
analytes, molecular ions were selected as precursor ions.
Specific and intense product ions of each target analyte
were used for quantification and a secondary product ion
was used as qualifier ion for confirmatory purposes.
Details of the specific parameters for detection of the
analytes are given in Table 2. Delta retention time in MS/
MS mode was 3 min, the time window wherein the
fragmentation occurs, i.e. retention time ±1.5 min.
Calibration curves were produced by a weighted (1/x)
linear least square regression. Relative peak areas (ratios of
the analytes to the surrogate standard) were used for
calculations. Correlation coefficients, R, were in the range of
0.9982–0.9997. Limits of detection (LOD) and
quantification (LOQ) were calculated on the basis of signal to noise
ratios (S/N) of 3 and 10, respectively. All parameters are
shown in Table 4.
The analytical results corrected for the average recovery
(shown in Table 3) are shown in Table 5. The highest
concentration found in wastewater samples corresponds to
mephedrone (42.9 ng L-1). The detected concentrations of
MDMA and 4-MEC were similar, ranging between 21.6
and 41.3 ng L-1. As the analytes concentrations in
wastewater samples were low, the results were recalculated
for recoveries at low concentration level (40 ng L-1), but
the differences were insignificant.
MDPV and mCPP were not detected in the tested
Loads (mg 1000 inhabitants-1 day-1) of illicit drugs
discharged via effluents into the aquatic system were
estimated from concentrations of each compound detected in
the effluents (ng L-1) and the daily flow of effluents
discharged to the aquatic system. Loads of illicit drugs
(MDMA, mephedrone and 4-MEC), discharged by the
WWTP ranged from 3.6 to 6.7 mg day-1 1000
inhabitants-1 of MDMA, 3.6 to 7.1 mg day-1 1000
inhabitants-1 of mephedrone and 4.8 to 5.8 mg day-1 1000
inhabitants-1 of 4-MEC.
The hazard quotient (HQ) method was used to screen the
toxicological risk level
(Mendoza et al. 2014)
. The lack of
aquatic ecotoxicological data for psychoactive compounds
makes it difficult to conduct proper environmental risk
assessment. For MDMA, Mendoza et al.
(Mendoza et al.
published a PNEC of 0.216 lg L-1 based on the
lowest median lethal (effective) concentration (L(E)50) in
algae, cladocerans and fish divided by an assessment factor
of 1000. PNEC derived by the Ecological Structure
Activity Relationships (ECOSAR) modeling. For
mephedrone and 4-MEC, no PNECs could be found in available
literature. The calculated HQs for MDMA, which are well
below 1, up to 0.188, mean that, as far as data are available,
the environmental risk is low but a potential adverse effect
could be expected for MDMA. The values of HQs
estimated in this work for an environmental risk assessment
are in agreement with data reported by Mendoza et al.
(Mendoza et al. 2014)
and Bijlsma et al.
(Bijlsma et al.
. These data were calculated in the effluent of the
WWTP, so this is the worst case scenario because effluents
are diluted further in a surface water.
Acknowledgments This work was supported by AGH University
Grant No 22.214.171.124. The authors thank the staff of ‘‘Municipal
Waterworks and Sewer Enterprise in Krako´w’’ for their support in the
collecting of samples.
Open Access This article is distributed under the terms of the
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link to the Creative Commons license, and indicate if changes were
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