Regulatory Toxicology and Pharmacology 69 (2014) 296–303

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Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/yrtph

Ecological risk assessment of the presence of pharmaceutical residues in

a French national water survey
Camille Bouissou-Schurtz a, Paul Houeto a,⇑, Michel Guerbet b,c, Morgane Bachelot e, Claude Casellas d,
Anne-Cécile Mauclaire a, Pascale Panetier e, Cécile Delval a, Dominique Masset a
a
Agence Nationale de Sécurité du Médicament (ANSM), 143/147 Boulevard Anatole France, 93285 Saint-Denis, France
b
ABTE EA 4651, Université de Rouen, 22 Boulevard Gambetta, 76183 Rouen, France
c
UFR Médecine Pharmacie, Université de Rouen, 22 Boulevard Gambetta, 76183 Rouen, France
d
Hydrosciences Montpellier UMR 5569, Faculté de Pharmacie, Université Montpellier 1, Avenue Charles Flahault, 34093 Montpellier Cedex 05, France
e
Agence Nationale de Sécurité Sanitaire (ANSES), 27/31 Avenue du Général Leclerc, F-94701 Maisons-Alfort Cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: In this study, we focused on the list of 33 chemicals that was established through a French national
Received 24 January 2014 prioritisation strategy. Assessing the potential risks to the environment was a step-wise procedure:
Available online 21 April 2014 (i) we determined the Predicted Environmental Concentration (PEC) of all molecules measured in the
national survey based on the highest recommended dose used, (ii) we used the Measured Environmental
Keywords: Concentration (MEC) and the Predicted No-Effect Concentration (PNEC) to establish the Risk Quotient
Risk assessment (RQ) based on either a PEC/PNEC (estimated risk) or MEC/PNEC (real risk) ratio. The risk assessment
Water resource
was performed using a binary ecological classification suggesting that appreciable risk is likely
Environment
Pharmaceuticals
(RQ P 1). Of the 15 molecules quantified in the survey, 12 had a PEC higher than the action limit value
of 0.01 lg/L. According to the EU Guideline, environmental risk was estimated as likely for the following
five compounds: acetaminophen (RQ = 1.6), ibuprofen (RQ = 600), diclofenac (RQ = 15), oxazepam
(RQ = 2.1) and carbamazepine (RQ = 3.2). Only ibuprofen was identified as posing real environmental risk
based on its MEC (RQ = 1.9).
Ó 2014 Published by Elsevier Inc.

1. Introduction physicochemical characteristics and effectiveness of WWTP treat-

As a result of increasingly frequent medical prescriptions and
medication consumption, pharmaceuticals and their metabolites
have been detected in all environmental compartments
(Halling-Sørensen et al., 1998; Kummerer, 2004). This ubiquitous
environmental contamination has been demonstrated worldwide
with a wide range of drugs (e.g., antibiotics, antidepressants. . .)
found in WasteWater Treatment Plant (WWTP) effluents and
surface water at low concentrations, ranging from lg to ng/L.
The improved performance of analytical tools has led to better
descriptions of the presence of such compounds in the influents
and effluents of WWTPs, in the aquatic environment and in water
resources (WRs). These molecules are characterised by consider-
ably diverse chemical structures. More than 3000 human-use and
300 veterinary-use drug substances are currently available on the
French market. The concentration levels of these molecules
vary depending on their chemical stability, biodegradability,
⇑ Corresponding author. Fax: +33 (0)1 55 87 35 82.
E-mail address: paul.houeto@ansm.sante.fr (P. Houeto).
ment and particulate filtration.
Pharmaceutical compounds present in WRs can be biologically
degraded in WWTPs, and subsequently appear in surface water
or be picked up by sludge. Sludge can be used as fertiliser in
agriculture, so substances can penetrate into the soil and reach
groundwater sources. The degradation of such compounds in com-
plex matrices, like soil, is poorly studied. Pharmaceutical analyses
are relatively recent, and data on the environmental fate, behav-
iour and ecological effects of pharmaceuticals are urgently needed.
This situation has led to increased concern and more studies on
issues such as environmental species toxicity, microbial ecology
disturbances and antibiotic resistance. Current WWTP processes
are designed to reduce levels of dissolved organic carbon, as well
as nitrates and phosphates at times, but not pharmaceuticals.
WWTPs can only remove 10% of carbamazepine, an anticonvulsant
and mood stabilizer, but can remove 85% of triclosan, an antibacte-
rial and antifungal agent through biotic or abiotic processes
(Andreozzi et al., 2002). Abiotic environmental factors (e.g., tem-
perature, soil composition, amount, intensity and wavelength of
sunlight, salinity and pH) significantly transform substances in

http://dx.doi.org/10.1016/j.yrtph.2014.04.006
0273-2300/Ó 2014 Published by Elsevier Inc.
 C. Bouissou-Schurtz et al. / Regulatory Toxicology and Pharmacology 69 (2014) 296–303
the environment via photolysis or hydrolysis. Biotic factors
(bacteria, fungi) are the living parts of the ecosystem with which
pharmaceuticals may interact. Biodegradation is also an important
process to be considered in the environment.
We are faced with a problem: the risk associated with these low
concentrations of drug residues in surface water is uncertain for
ecological species. Performing a full ecological risk assessment of
pharmaceuticals is difficult because of the paucity of data on expo-
sure scenarios, target aquatic species and dose–response relation-
ships. Such a widespread occurrence clearly begs the question of
whether or not environmentally realistic concentrations of
pharmaceuticals pose a risk for environmentally exposed biota.
Recent EU pharmacovigilance legislation acknowledges that the
pollution of waters and soils with pharmaceutical residues is an
emerging environmental issue.
The aim of this study was to evaluate the potential environmen-
tal impact of pharmaceuticals according to the current European
Guideline on the Environmental Risk Assessment (ERA) of Medici-
nal Products for Human Use (EMA, 2006). In addition to this
research, a preventive environmental approach should be
examined. Ecological risk is determined by the ratio between the
Predicted Environmental Concentration (PEC) or Measured
Environmental Concentration (MEC) and the Predicted No-Effect
Concentration (PNEC) of the compounds determined during the
national survey. Hence, a tailored risk assessment strategy should
be followed by an evaluation of the PNEC based on relevant ecolog-
ical testing in order to define the PEC/PNEC or MEC/PNEC ratio and
identify ecotoxicity. We focused on the list of chemicals estab-
lished through a national prioritisation survey of pharmaceuticals
in drinking water. In addition, this study discusses the benefits
and limitations of this risk assessment approach as applied to the
individual compounds.
2. French national survey
The French Agency for Food, Environmental and Occupational
Health and Safety (ANSES) performed a national survey on drug
residues in WRs to evaluate their presence in water. Thirty-three
(33) molecules in several chemical and therapeutic classes chosen
according to discriminant criteria such as tonnage, solubility and
activity were tested using a multi-residue method (LC–MSMS).
The national survey was conducted from October 2009 to June
2010. In collaboration with regional agencies, 238 sites were
selected and 280 samples taken from all over France were analysed
(ANSES, 2011).
3. Ecological risk assessment method
3.1. Risk assessment strategy according to the European Guideline on
the Environmental
Risk Assessment of Medicinal Products for Human Use (EMEA/
CHMP/SWP/4447/00) according to the EMA Guideline for the eval-
uation of environmental for medicinal products for human use
(EMA, 2006), the potential risks to the environment is evaluated
in a step-wise procedure involving two phases. The first phase
(Phase I) estimates the exposure of the environment to the drug
substance. In phase I, an assessment of the ability of drugs to move
beyond the aquatic environment and bioaccumulate is requested.
The n-octanol/water partition coefficient indicates the transfer of
a drug substance from the aquatic environment into organisms
and its potential to bioaccumulate (Log KOW > 4.5). Then, a specific
risk assessment should be conducted in a step-wise fashion for
Persistence, Bioaccumulation and Toxicity (PBT index).
297
The PEC, which reflects the exposure of the environment to a
drug, is calculated in surface water according the formula PECsur-
facewater = (DOSEai  Fpen)/(WASTEWinhab  DILUTION) where
WASTEWinhab is the amount of wastewater per inhabitant per
day, Fpen is the percentage of market penetration and DOSEai is
the maximum daily dose consumed per inhabitant (or per patient).
If the PEC value is below 0.01 lg/L and no other environmental
concerns are apparent, it is assumed that the medicinal product
is unlikely to represent a risk for the environment if used as pre-
scribed in patients. So, based on an action limit, the assessment
may be terminated at this level. In contrast, if the PEC is equal to
or greater than 0.01 lg/L, a phase II environmental fate and effect
analysis should be performed. Certain substances, such as highly
lipophilic compounds and potential endocrine disruptors, may
need to be addressed during phase II irrespective of the quantity
released into the environment.
In the second phase (phase II), information about the fate and
effects of a medicinal product in the environment is obtained
and assessed. The recommended phase II assessment is conducted
through an evaluation of the PNEC, which is calculated by applying
an assessment factor (AF) to the NOEC values from relevant stud-
ies. Thus, a tailored risk assessment should be followed by a deter-
mination of the PEC/PNEC or MEC/PNEC. Also, in this phase, all
relevant data such as physicochemical properties, toxicology,
metabolism and excretion, degradability and persistence are taken
into account. If the n-octanol/water partition coefficient indicates
the transfer of the drug substance from the aquatic environment
into organisms and a potential to bioaccumulate (Log KOW > 3 in
phase II), then the PBT index should be considered. The absorption
behaviour of substances in sewage sludge is described through the
adsorption coefficient (KOC), which is defined as the ratio between
the concentration of the substance in sewage sludge’s organic car-
bon and the concentration of the substance in the aqueous phase at
adsorption equilibrium. It is assumed that a substance with a high
KOC value is retained in the WWTP and may reach the terrestrial
compartment through the spreading of sewage sludge over land.
If the KOC is higher than 10,000 L/kg, an environmental assessment
of the drug substance in the terrestrial compartment should be
conducted, unless the substance is readily biodegradable. The ter-
restrial risk assessment complements the aquatic risk assessment,
but does not replace it.
3.2. Ecotoxicity concern evaluation
Firstly, we decided to determine the PEC of molecules quanti-
fied in the national survey (excluding caffeine and two metabo-
lites; epoxycarbamazepine and hydroxyibuprofen) according to
the European Guideline (EMA, 2006). The calculation of the PEC
was based upon the highest recommended dose used for a product.
Secondly, we used the MEC found in the context of the national
survey in order to compare it to the calculated PEC values. Finally,
concerning the ecotoxicity, we opted to search the available PNEC
values from the literature to establish the Risk Quotient (RQ) based
on either a PEC/PNEC (estimated risk) or MEC/PNEC (real risk)
ratio. When the PNEC value was not available in the literature at
all, we used the NOEC from chronological studies standardised
using an assessment factor (AF). This AF is an expression of the
degree of uncertainty in the extrapolation of the test data to a lim-
ited number of species in the actual environment. This analysis
helps predict the concentration of a substance for which adverse
effects are not expected to occur. There is potential risk to the
environment when the RQ is >1. It is important to note that PEC/
PNEC represents a theoretical or estimated RQ based on calculated
exposure, in contrast with the MEC/PNEC ratio, which is consid-
ered a real risk based on exposure measured during this national
survey. According to the European Guideline (EMA, 2006), the risk
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assessment is based on a binary ecological classification suggesting 4.2. Ecotoxicity concern evaluation
that there is likely (RQ P 1) or unlikely (RQ < 1) appreciable risk to
the environment. For every the 12 molecules for which a MEC has been determi-
nated, we calculated the PEC according to the European Guideline
(EMA, 2006) (Fig. 1). We also determinated the available PNEC val-
4. Results analysis ues from the literature to establish the Risk Quotient (RQ) based on
either a PEC/PNEC (estimated risk) or MEC/PNEC (real risk) ratio.
4.1. Measured Environmental Concentrations (MEC) Results are expressed according to a binary risk classification: risk
likely or unlikely to occur (Table 2).
This paper focuses on medicinal products for human use.
Despite the fact that caffeine is found in large concentrations in
WRs, it was excluded from this study since it is both a medicinal 4.2.1. Acetaminophen
and a food product, and thus, may be regarded as an anthropogenic Acetaminophen is highly soluble (solubility 14,000 mg/L). An
marker compound in the aquatic environment. estimated Log KOW of 0.46 and a bioconcentration factor (BCF) of
The molecules tested in the national survey (ANSES, 2011) are 3 suggest that the potential for bioconcentration in aquatic organ-
summarised in Table 1, ranked by detection frequency and quan- isms is low. KOC and Kd were 42 L/kg and 46 L/kg respectively, indi-
tity (maximal measured concentration). Molecules found at high cating that acetaminophen has low mobility and is not expected to
concentrations in this national survey are acetaminophen adsorb to suspended solids and sediment when released into
(0.443 lg/L) and ketoprofen (0.258 lg/L). In this survey, molecule water. Acetaminophen has been categorised as readily biodegrad-
detection frequency varied from 0.7% (for ramiprilate) to 49.6% able with a reported half-life of 20 days. Thus, it is not considered
(for caffeine). Twenty-four (24) molecules out of the 33 analysed persistent according to REACH and the OSPAR Commission
were detected at least once. Fifteen (15) of them were found at a (Moermond et al., 2012).
level higher than the limit of quantification (LOQ). The other mol- According to the EMA Guideline for Environmental Risk Assess-
ecules were estimated as existing in trace quantities (between the ment, the PEC calculated with a maximum dosage of 4 g per day is
LOD and the LOQ) or immeasurable quantities (less than the LOD). 20 lg/L. Acetaminophen is found at low concentrations in French
Of the 15 molecules found in quantifiable amounts, excluding caf- WRs (maximal MEC = 0.443 lg/L). Acute studies conducted on
feine and two metabolites (epoxycarbamazepine and hydroxyibu- Daphnia magna demonstrated an EC50 ranging from 0.367 mg/L
profen), there was finally 12 molecules for which we decided to to 50 mg/L (Kuhn and Pattard, 1989; Calleja et al., 1994a,b;
conduct an environmental risk assessment as long as ecotoxicity Henschel et al., 1997; Kim et al., 2012). In an acute study on
data were available in literature. Daphnia microcopa and a chronic study on D. magna, NOECs of
12.88 mg/L and 5.72 mg/L, respectively were evaluated (Kim
et al., 2012). Galus et al. (2013a,b) reported increased mortality
Table 1
and developmental abnormalities, such as spinal malformations
Frequency of detection and quantification of different molecules in water resources
(ANSES, 2011). or pericardial oedema, in zebrafish after embryonic exposure to
acetaminophen at a level of 0.5 lg/L, which is close to the environ-
Molecules Frequency Limit of Maximal
mental concentrations found in the national survey (0.443 lg/L)
of quantification environmental
quantified (LOQ) (ng/L) concentration (Galus et al., 2013a,b). Furthermore, in the Galus study, low con-
results (%) (MEC) (ng/L) centrations of acetaminophen resulted in a significant increase in
1 Caffeine 49.6 25 856 the incidence of regressive changes in kidney tubule structure
2 Oxazepam 27.1 5 161 and in the liver. If we consider the NOEC from the acute study,
3 Paracetamol 23.6 25 443 the RQ (PEC/PNEC = 1.6) is greater than 1, suggesting that there
4 Carbamazepine 29.8 5 48 is a risk for aquatic organisms. In the chronic study, the RQ (PEC/
5 Epoxycarbamazepine 20.8 1 8
6 Losartan 10 5 11
PNEC = 0.3) was less than 1, ruling out potential environmental
7 Gadolinium 2.8 5 19 risk. In the worst-case scenario with the highest RQ (1.6), acetami-
8 Hydrochlorothiazide 8.1 25 48 nophen is likely to present an environmental risk. Using the results
9 Ketoprofen 3.9 10 258 of the national survey, the resultant acetaminophen RQ (MEC/
10 Salicylic acid 2.1 25 57
PNEC = 0.04) is less than 1, suggesting that there is no appreciable
11 Trimetazidine 0.8 25 31
12 Ibuprofen 2.5 10 19
13 Hydroxyibuprofen 5 50 83
14 Diclofenac 2.6 10 16
15 Naftidrofuryl 1.8 1 2
16 Amlodipine 0.4 50 Traces
17 Atenolol 4.8 25 Traces
18 Doxycycline 0.5 50 Traces
19 Ofloxacine 2 10 Traces
20 Erythromycin 1.7 50 Traces
21 Fluvoxamine 0.4 50 Traces
22 Amoxicillin 0.9 50 Traces
23 Ramipril 7.4 5 Traces
24 Ramiprilate 0.7 10 Traces
25 17-Beta-oestradiol 0 50 <LOQ
26 Cyclophosphamide 0 5 <LOQ
27 Oestrone 0 50 <LOQ
28 Furosemide 0 50 <LOQ
29 Ifosfamide 0 10 <LOQ
30 Levonorgestrel 0 10 <LOQ
31 Pravastatin 0 50 <LOQ
32 Progesterone 0 10 <LOQ
33 Ranitidine 0 25 <LOQ Fig. 1. MEC and PEC of the 10 molecules with their RQ (noted + when RQ > 1 and
noted – when RQ < 1).
 C. Bouissou-Schurtz et al. / Regulatory Toxicology and Pharmacology 69 (2014) 296–303 299

Table 2
Risk assessment and classification.

Substance PNEC (lg/L<) (species) PEC (lg/L) RQ (estimated risk) PEC/PNEC Conclusion MEC (lg/L) RQ (real risk) MEC/PNEC Conclusion
Paracetamol 12.9 20 1.6 Likely 0.443 0.04 Unlikely
D. magna
Ibuprofen 0.01 6 600.0 Likely 0.019 1.9 Likely
O. latipes
Ketoprofen 15.6 1.5 0.1 Unlikely 0.258 0.0 Unlikely
V. fischeri
Diclofenac 0.05 0.75 15.0 Likely 0.016 0.0 Unlikely
O. mykiss
Salicylic acid 37 30 0.8 Unlikely 0.057 0.0 Unlikely
D. rerio
Oxazepam 0.481 1 2.1 Likely 0.161 0.3 Unlikely
D. magna
Carbamazepine 2.5 8 3.2 Likely 0.048 0.0 Unlikely
D. rerio
Losartan 14300 0.5 0.0 Unlikely 0.011 0.0 Unlikely
S. capricornutum
Hydrochlorothiazide 100 0.25 0.0 Unlikely 0.048 0.0 Unlikely
D. magna
Gadolinium 100 16 0.2 Unlikely 0.019 0.0 Unlikely
Algae

risk to aquatic organisms. There are some discrepancies between
the PEC and the MEC.
Kim et al. (2012) recently reported that acetaminophen could
alter the steroidogenic pathway, and hence, sex hormone balance,
leading to increased vitellogenesis in male fish. Indeed, acetamino-
phen exposure resulted in concentration-dependent increases of
hepatic vitellogenin protein, confirming endocrine disruption
(Kim et al., 2012). Working doses used in this study ranged from
0.095 lg/L to 9500 lg/L. The observed effect on vitellogenesis did
not occur at the lowest concentration (0.095 lg/L) but rather at
the second concentration (95 lg/L), which is approximately
200 times higher than the acetaminophen concentration measured
in WRs (0.443 lg/L). It is important to note that there is a signifi-
cant gap between the first dose (0.095 lg/L) and the second dose
(95 lg/L) (a 1000-fold difference between the two doses) used
for the vitellogenesis test. None of these doses comes close to the
concentration measured in this survey (0.443 lg/L). Subsequently,
it is not possible to evaluate acetaminophen’s capacity for endo-
crine disruption at environmental concentrations in the context
of the national survey.
4.2.2. Ibuprofen
According the European Pharmacopeia, ibuprofen has a low sol-
ubility in water of 21 mg/L. Log KOW is 3.97 which is higher than
the threshold of 3 (EMA, 2006) and a BCF of 3, suggesting that ibu-
profen has a low ability to bioconcentrate in aquatic organisms. If
released in water, ibuprofen has slight mobility based upon an esti-
mated KOC of 3400 L/kg, suggesting that it is expected to adsorb to
suspended solids and sediment. Ibuprofen is resistant to hydrolysis
and has been shown to be inherently biodegradable during sewage
treatment. Ibuprofen was determined to have a half-life of 20 days,
and thus it is not considered persistent.
Ibuprofen was found in French WRs with one of its metabolites,
the hydroxyibuprofen, which is inactive. Despite the presence of
hydroxyibuprofen, we evaluated only ibuprofen. Given that there
is no dosage for metabolites, it is difficult to calculate the PEC. Fur-
thermore, only parent compounds are addressed in phase I of the
ERA. The maximal MEC for ibuprofen and its metabolite are
0.019 lg/L and 0.083 lg/L, respectively.
The PEC of ibuprofen based on a maximum dosage of 1.2 g per
day is equal to 6 lg/L. In terms of ecotoxicology, a recent study on
carp described ibuprofen’s profound influence on haematological,
biochemical and enzymatic profiles, suggesting the potential
toxicity of this drug in aquatic organisms (Saravanan et al.,
2012). Acute and chronic toxicological studies on daphnia and fish
report a NOEC of 0.1 lg/L, which was used in the PNEC calculation
applying the lowest possible assessment factor of 10 (Han et al.,
2006, 2010). Both the PEC/PNEC ratio (=600) and the MEC/PNEC
ratio (=1.9) were over 1, suggesting a probable environmental risk.
More work is required to elucidate the ecological consequences
of ibuprofen contamination in water and the effects on reproduc-
tion (Saravanan et al., 2012). Ibuprofen affects several endpoints
related to fish reproduction, including inducing vitellogenin in
male fish, fewer offspring per spawning and more eggs per spawn-
ing. As suggested by observations of vitellogenin and sex hormone
balance modification, ibuprofen appears to be linked to endocrine
disruption (Han et al., 2010). This effect occurs at concentrations
(200 lg/L) around 10 times higher than maximal environmental
concentrations measured in this survey.
4.2.3. Diclofenac
As for chemical and physical properties, even if the Log KOW of
diclofenac is 4.5, its BCF is under 2000 suggesting that diclofenac
has a low ability to bioconcentrate. According to its KOC (245 L/kg),
diclofenac shows low mobility in migrating to the terrestrial
compartment if released in water. Diclofenac is not expected to
be hydrolysed, but direct photolysis is the predominant removal
process in freshwater. With its half-life of 8 days, it is not consid-
ered persistent.
Within the context of this national survey, diclofenac has been
measured in WRs at levels of up to 0.016 lg/L. The PEC determined
based on the maximum dosage of 150 mg/day was 0.75 lg/L. There
were several ecotoxicological studies performed on daphnia and
fish. It appears that Rainbow trout (Oncorhynchus mykiss) is the
most sensitive species, with a NOEC of 0.5 lg/L reported in a
chronic study (Triebskorn et al., 2004, 2007). Triebskorn et al.
(2004) found cytological effects at 1 lg/L, the lowest concentration
tested, in the liver, kidney, gills and intestine of O. mykiss after
28 days of exposure. These results were confirmed in an ultrastruc-
tural analysis in a second publication leading to a NOEC at 0.5 lg/L
(Triebskorn et al., 2007). We used an AF of 10 to derive the PNEC.
The resultant RQ (PEC/PNEC = 15) was greater than 1, predicting
that environmental risk is likely. However, the RQ evaluated by
the MEC/PNEC ratio was lower than 1.
None of the reproduction-related endpoints evaluate the poten-
tial of these drugs to produce endocrine disruption in aquatic
300 C. Bouissou-Schurtz et al. / Regulatory Toxicology and Pharmacology 69 (2014) 296–303
organisms such as fish or D. magna. However, these drugs act on
the prostaglandin pathway, which seems to be related to endocrine
disruption. Some authors have demonstrated that a number of
putative endocrine-disrupting compounds are potent inhibitors
of prostaglandin synthesis in both rodent and human cells
(Kristensen et al., 2011, 2012).
4.2.4. Oxazepam
Oxazepam shows a very particular profile because it is both an
active molecule and a metabolite of several benzodiazepines
(Besse et al., 2008). Oxazepam undergoes direct glucuronidation
before its excretion. Therefore, oxazepam is the benzodiazepine
to search for in the environment, and could be used as an indicator
of benzodiazepine contamination in the aquatic environment
(Besse and Garric, 2008).
Oxazepam is highly soluble (solubility of 179 mg/L). An esti-
mated Log KOW of 2.24 suggests that the potential for bioconcentra-
tion in aquatic organisms is low. If released into water, oxazepam
is not expected to adsorb to suspended solids and sediment based
upon the estimated KOC (390 L/kg). Significant hydrolysis is not
expected since this compound lacks functional groups that
hydrolyse under environmental conditions.
Oxazepam is measured at levels of up to 0.161 lg/L in WR. Since
oxazepam is both a drug and a metabolite, exposure is difficult to
evaluate. The PEC (1 lg/L) is thus calculated under the assumption
of a maximum dosage of 200 mg per day. The ecotoxicological
behaviour of oxazepam is poorly described in the literature. Only
Calleja et al. report an EC50 resulting from an acute study in
D. magna (Calleja et al., 1994a,b). The PNEC is thus calculated with
an assessment factor of 1000 (PNEC = 0.481 lg/L). The resulting
theoretical RQ (PEC/PNEC = 2.1) is greater than 1, suggesting that
environmental risk is likely. In contrast, when taking into account
the MEC and determining the RQ (MEC/PNEC = 0.3), environmental
risk is classified as unlikely.
4.2.5. Carbamazepine
Despite the presence of 10,11 epoxycarbamazepine, the active
metabolite of carbamazepine, we evaluated only carbamazepine.
Given that there is no dosage for a metabolite, it was difficult to
calculate its PEC. Furthermore, only parent compounds are
addressed in phase I of the environmental risk assessment.
Carbamazepine is fairly soluble (solubility 17.7 mg/L). Its
Log KOW of 2.45 suggests that its potential for bioconcentration in
aquatic organisms is low. If released into water, carbamazepine
is expected to adsorb to suspended solids and sediment based
upon the estimated KOC (520 L/kg). Carbamazepine is fairly persis-
tent, exhibiting a half-life of 63 days. The compound is susceptible
to direct photolysis in water, with a half-life of approximately
1 day.
As the most commonly used antiepileptic agent, carbamazepine
is found in French WRs with its metabolite, the epoxycarbamaze-
pine, at concentrations of up to 0.048 lg/L and 0.008 lg/L, respec-
tively (ANSES, 2011). This concentration is lower than the PEC,
which is about 8 lg/L based on a maximum dosage of 1.6 g/day.
The NOEC was evaluated in cladocera and fish in acute and chronic
studies, and we applied an AF of 10 to calculate the PNEC (2.5 lg/L)
(Ferrari et al., 2003; Houeto et al., 2012). Carbamazepine increased
mortality and delayed development in Japanese medaka and
caused spinal malformation (upward curvature of the tail) and
growth retardation in zebrafish (Nassef et al., 2010; van den
Brandhof and Montforts, 2010). The PEC/PNEC ratio (3.2) is higher
than 1, suggesting a probable environmental risk. However, based
on the MEC found in the national survey, the RQ is 0.02.
4.2.6. Other miscellaneous drugs
Ketoprofen and salicylic acid demonstrate a limited ability to
bioaccumulate and low mobility to the terrestrial compartment if
released in water based upon their Log KOW (3.12 and 2.26, respec-
tively) and KOC (404 for salicylic acid). Aspirin, the parent
compound of salicylic acid, is susceptible to direct photolysis by
sunlight, and both are hydrolysed, with hydrolysis half-lives
ranging from 1.2 h to 12.5 days.
Like for oxazepam, salicylic acid is both a metabolite (of aspirin)
and a drug. Aspirin is the compound mainly used in humans; sal-
icylic acid is used topically. Thus, we use the maximal dosage of
aspirin (6000 mg per day) to calculate the PEC of salicylic acid.
For all these drugs, the PEC is over 0.01 lg/L suggesting the need
for ecotoxicological data. The acute toxicity of ketoprofen and sal-
icylic acid were assessed in Vibrio fischeri (Henschel et al., 1997;
Farré et al., 2001). The PEC/PNEC ratio is less than one, ruling out
environmental risk.
Like for diclofenac, there are no studies evaluating reproduction
related endpoints and the potential for endocrine disruption. How-
ever, these drugs act on the prostaglandin pathway, which seems
to be related to endocrine disruption.
Losartan and hydrochlorothiazide are used in cardiology. The
Log KOW for losartan is greater than 3 (the threshold level for phase
II products according to the EMA Guideline), suggesting a low abil-
ity to bioaccumulate. This Log KOW is low for hydrochlorothiazide
(–0.07). We did not find a KOC for either of these drugs. Losartan
and hydrochlorothiazide are found in French WRs at concentra-
tions of up to 0.011 lg/L and 0.048 lg/L, respectively. There are
few data related to their toxicity in the literature. We only found
some acute studies on algae (Carlsson et al., 2006). For all these
drugs, PEC (0.5 lg/L and 0.25 lg/L, respectively) is greater than
0.01, placing all of them in phase II for risk assessment. The NOEC
or EC50 of these drugs were found in the literature. For losartan, the
NOEC resulted from a chronic study, and we applied an AF of 10 to
the NOEC (PNEC = 14,300 lg/L). As hydrochlorothiazide is poorly
described in the literature, we only found an EC50 from an acute
study performed in invertebrates, and we applied an AF of 1000
to this EC50 (PNEC 100 lg/L) (Carlsson et al., 2006). For all these
drugs, both theoretical and measured RQ values were under 1,
suggesting that environmental risk is unlikely.
Gadolinium, a lanthanide, is a contrast product used for imag-
ing. Gadolinium is found in concentrations of up to 0.019 lg/L in
French WRs. Since its PEC (16 lg/L) is higher than the action limit
threshold, this drug undergoes a phase II risk assessment. Gadolin-
ium is poorly described in the literature: we only found an EC50
from an acute study performed in invertebrates, and an AF of
1000 was applied to this value (PNEC 100 lg/L) (Carlsson et al.,
2006). Both theoretical and measured RQs were less than 1,
suggesting that environmental risk is unlikely.
Trimetazidine and naftidrofuryl were the last 2 molecules for
which a MEC has been determinated during the national survey,
respectively 0.031 and 0.002 lg/L in WRs. The PEC of each
(0.35 lg/L and 3 lg/L, respectively) was over 0.01 lg/L. However
we could not evaluate the PNEC or NOEC due to a lack of informa-
tion for either of these drugs. Descriptions of risk were not possible
for trimetazidine and naftidrofuryl due to a lack of ecotoxicity data.
5. Discussion
Of the 15 molecules evaluated in the national survey, we further
analysed the drugs for which we could calculate exposure based on
a maximum daily dosage. We thus excluded caffeine and the two
metabolites examined from this study. It is important to note that
we do not emphasise caffeine, which is identified as an anthropo-
genic marker for WR surface water contamination. The two
 C. Bouissou-Schurtz et al. / Regulatory Toxicology and Pharmacology 69 (2014) 296–303
metabolites found in WRs were 10,11 epoxycarbamazepine and
hydroxyibuprofen. The first is the active metabolite of carbamaze-
pine, present at lower concentrations than carbamazepine. Our risk
assessment did not take into account the presence of metabolites.
We mean to emphasise that WRs are polluted by both carbamaze-
pine and its metabolite, and both compounds are still active when
released into the environment, even though the ecotoxicological
impact remains unknown (Li et al., 2011; Fenet et al., 2012).
Furthermore, Donner et al. (2013) described that acridine and
acridone, the products of photodegradation, are considerably more
toxic than the parent compounds (Donner et al., 2013). The second
metabolite is the inactive hydroxyibuprofen, measured at much
higher concentrations (fourfold) than the parent compound
ibuprofen.
The drugs most frequently detected in this national ANSES sur-
vey were oxazepam, acetaminophen, carbamazepine and losartan
(10–30%). Except for losartan, these results are consistent with
those of other studies published in the literature (Andreozzi
et al., 2003; Tixier et al., 2003; Stackelberg et al., 2004; Carlsson
et al., 2006; Santos et al., 2007).
Pharmaceutical residues found at highest concentrations in
WRs during the national survey were oxazepam, acetaminophen,
and ketoprofen. For these molecules, concentrations varied from
0.161 to 0.443 lg/L. In addition, the total amount of oxazepam
found in WRs is questionable because this anxiolytic drug is both
an active molecule and metabolite of four benzodiazepines
(Besse et al., 2008). Several studies have stipulated that the occur-
rence of acetaminophen in WRs varies from 0.002 lg/L to 0.161 lg/L
(Stackelberg et al., 2004; Bound and Voulvoulis, 2006; Choi et al.,
2008; Conley et al., 2008; Focazio et al., 2008; Yu et al., 2013).
The amount of acetaminophen found in WRs in this national sur-
vey was 0.443 lg/L. This quantity of acetaminophen is 3–222
times greater than quantities described in several studies.
Perhaps this is due to changes in drug consumption or medical pre-
scriptions. This survey was performed in winter (October to June),
when acetaminophen consumption in France is high (ANSES,
2011). Yu et al. (2013) report major seasonal variations in the high-
est acetaminophen concentrations. This concentration varies from
5 lg/L in the summer to 218 lg/L in the winter (Yu et al., 2013).
Other drugs, such as the NSAID ibuprofen, are subject to seasonal
changes in consumption that may be explained by a higher inci-
dence of the flu or other illnesses in the winter. Ketoprofen was
rarely found in French WRs (<10%) in the context of this survey,
and its occurrence was poorly described in studies compared
with that of other drugs. Nevertheless, ketoprofen was measured
in large amounts in this survey (0.258 lg/L, which is half the
concentration measured in Korean effluents (Yu et al., 2013).
In terms of ecological impact, this survey highlights the follow-
ing five drugs: acetaminophen, ibuprofen, diclofenac, oxazepam
and carbamazepine, as being the most problematic compounds,
because the environmental risk evaluation resulting from PEC/
PNEC ratio is considered as likely.
The ecotoxicological impact of oxazepam is poorly described. It
has been related to altered feeding rates in fish but at concentra-
tions 10 times higher than environmental concentrations (Brodin
et al., 2013). Diclofenac affects organ histology, gene expression
and enzymatic parameters in fish at approximately 1 lg/L, which
is 100 times higher than environmental concentrations (Cuklev
et al., 2012; Saravanan et al., 2013). However, since diclofenac acts
on the prostaglandin pathway, potential endocrine disruption can-
not be excluded. With carbamazepine, Galus et al. (2013a,b)
described a significant increase in the incidence of regressive
changes in kidney tubule structure and the liver of the zebrafish.
In addition, exposure to carbamazepine increased the occurrence
of atreitic oocytes in female fish and decreased levels of ketotestos-
terone, a sex steroid hormone, in both male and female fish (Galus
301
et al., 2013a,b). These effects occurred at 0.5 lg/L, which is approx-
imately 10 times higher than the concentration measured in this
survey. Nevertheless, the concentration of carbamazepine reported
in the literature varies from 0.011 lg/L to 0.870 lg/L (Ferrari et al.,
2003). Paracetamol is widely used as a therapeutic and has been
shown to affect reproduction in daphnia or fish embryonic devel-
opment, even at low concentrations (0.5 lg/L) (Kim et al., 2012;
Galus et al., 2013a,b). At this low acetaminophen concentration,
which is close to the measured concentration in the environment,
ecological risk does exist. However, according to the Guideline, and
taking into account the MEC, we would be wrong to conclude that
environmental risk for acetaminophen is unlikely. Ibuprofen may
reach the terrestrial compartment, leading to an underestimation
of its measured concentration on the one hand and contamination
of a compartment other than water on the other hand. Also, the
PEC should be assessed in sludge and soil. Ibuprofen has major tox-
icological affects on reproduction as well as on haematological,
biochemical and enzymatic profiles (sublethal concentration of
8.5 lg/L) as toxicological endpoints that are widely used to assess
toxic stress, immune system integrity and tissue damage (Talas
and Gulhan, 2009; Kavitha et al., 2010; Saravanan et al., 2012).
Moreover, ibuprofen poses a major risk to the environment since
it is the only molecule for which both theoretical and real RQ,
assessed by PEC/PNEC and MEC/PNEC respectively, are greater than
one.
Although the concentrations of pharmaceuticals found in WRs
are much lower than therapeutic doses, chronic exposure and the
potential by some medicinal products to cause endocrine disrup-
tion may lead to deleterious effects in aquatic organisms. Some
studies show a reduction in egg production in female zebrafish
treated with WR for 7 days (Lister et al., 2009). Some intersex
and elevated vitellogenin have been reported in wild fish from
WRs (Woodling et al., 2006; Vajda et al., 2008). Oestrogen present
in environmental waters has been widely blamed for these effects
on fish reproduction and gonadal development (Vajda et al., 2008).
This is not only due to the presence of oestrogens in water. Phar-
maceuticals have multiple mechanisms of action that may lead
to endocrine disruption (Mennigen et al., 2008; David and
Pancharatna, 2009a,b). One of them is on the prostaglandin path-
way through the inhibition of cyclooxygenase, which in turn
reduces prostaglandins levels. It has been reported that prostaglan-
dins are important reproduction regulators and that their inhibi-
tion leads to endocrine disruption (Hayashi et al., 2008). This is
notably the case for acetaminophen and all NSAIDs, for which
reproductive effects start at 10 lg/L (David and Pancharatna,
2009a,b; van den Brandhof and Montforts, 2010).
There were some substantial limitations within our study and
several points need to be improved in future investigations. Ana-
lytically, it is important to emphasise inevitable uncertainties in
exposure scenarios. These pertain to the methods for sampling
and analysing pharmaceutical residues in WR and the approaches
for assessing risk in other compartments of the environment. In
fact, taking into account that there is no standardised analytical
method for each molecule, ANSES (2011) used a multi-residue
analysis. Consequently, maximal concentrations may be overesti-
mated or underestimated. One critical point is that some mole-
cules, such as 17 beta-oestradiol, cyclophosphamide, oestrone,
furosemide, ifosfamide, levonorgestrel, pravastatin, progesterone
and ranitidine, are found in 0% of the analysed samples. This is
due to a lack of sensitivity in the analytical method, which
warrants further investigation (ANSES, 2011). Another key issue
pertains to the EMA Guideline on the Environmental Risk Assess-
ment of Medicinal Products for Human Use, which is an important
tool in making decisions on individual compounds in environmen-
tal waters. It is clear that this overall ecological risk assessment
process from the prior study was not representative of the
302 C. Bouissou-Schurtz et al. / Regulatory Toxicology and Pharmacology 69 (2014) 296–303
pharmaceutical residues that are most likely to be harmful. In the
national survey, pharmaceuticals were measured only in water and
not in other environmental compartments (e.g., soil, sediment).
This MEC does not represent the exposure of the environment as
a whole. Our results require confirmation in a larger group of
molecules ideally to better cover the overall risk related to
environmental organism system exposure. These findings repre-
sent preliminary work and we think that a properly designed
national survey of the presence of drug residues in WRs is needed
to reinforce this process for assessing overall ecological risk, which
may vary in frequency, concentration or significance (geographical
distribution) with the region. In addition, the seasonal variations of
compounds at different sampling sites should be taken into
account.
There is a paucity of ecotoxicity risk assessment data in market-
ing authorisations (MA). The MAs for these molecules were granted
(from 1973 to 1995) before the implementation of the European
Environmental Guideline in 2006. Consequently, a detailed
literature review was required for these molecules. In addition,
long-term data are preferred, but when lacking, short-term data
may be used. Ideally, to improve the environmental risk
assessment according to the principles set forth in the European
Guideline, we need ecotoxicity data from three trophic levels
(daphnia, algae and fish). Furthermore, we do not address the issue
of multi-exposure and interactions between pharmaceutical resi-
dues. Major studies report the effects of exposure from individual
pharmaceuticals. However, WRs are polluted with multiple drugs
from various therapeutic classes, and the risks to aquatic species
of chronic exposure to low concentrations of several drugs are
unclear. Studies of multiple compounds have been lacking up to
now. Such considerations should be taken into account and dem-
onstrate the importance of testing the effects of pharmaceutical
combinations because drug residues often occur as combinations,
and not as single contaminants, in WRs. Therefore, there should
be greater interest in the effects of exposure to individual or multi-
ple pharmaceuticals in WRs that could act in additive, synergic or
antagonistic ways with the objective of determining whether these
small amounts pose an ecological hazard. Mechanisms for detoxi-
fying one drug may interfere with the metabolism of another one,
leading to a resultant higher or a lower level of stress for the eco-
system. Organisms in the environment can be unintentionally
exposed to trace residues of pharmaceuticals. While this does not
preclude the possibility of subtle or immeasurable biological
effects, very conservative estimates of the synergistic, additive or
antagonistic effects of drug residue combinations do not reveal
the possible hazards. However, recent studies evaluate the impact
of a combination of various pharmaceuticals (acetaminophen,
carbamazepine, gemfibrozil and venlafaxine) on zebrafish at con-
centrations close to those found in the environment (Madureira
et al., 2011; Galus et al., 2013a,b). These studies report reproduc-
tive toxicity and associated kidney tubular effects of low concen-
trations (lower than the PNEC) of individual pharmaceuticals and
combinations. In these studies, the effect of combinations did not
seem to be additive, since all effects were seen with single pharma-
ceuticals and combinations (Madureira et al., 2011; Galus et al.,
2013a,b). These studies reveal that the environmental risk assess-
ment based on the European Guideline (EMEA/CHMP/SWP/
4447/00) was insufficient. The case of acetaminophen illustrates
this weakness, since we could wrongly conclude that a risk is
unlikely, whereas this risk actually exists.
6. Conclusion
This analysis of the national survey performed by the ANSES
confirms the pollutant impact of several medicinal product
residues on environmental water. In addition, our results support
the notion that there is a risk for acute and chronic effects in the
environment inherent to the release of pharmaceuticals in water.
Some of these drugs cause endocrine disruption that is deleterious
for the entire aquatic ecosystem at environmental concentrations.
There is a need to develop more sensitive analytical methods to
improve the detection of medicinal products in the environment.
Perfecting new tools for performing adequate risk and hazard
assessments should enhance relevance of the PEC/PNEC ratio in
characterising environmental risk. For example, biomarkers that
can be easily measured in the aquatic ecosystem should be created
to improve the assessment of ecological toxicity.
It is important to note that pharmaceuticals do not occur as
pure isolated substances in an environmental compartment. Since
a broad range of different substances is used simultaneously in
humans in any given area, pharmaceuticals are present as multi-
component combinations in the environment. Furthermore,
assessing the risk of metabolite and transformation products is
not possible. Since all recent studies reveal that combinations of
pharmaceuticals, even at low concentrations, may be detrimental
to exposed organisms, it seems particularly important to remove
drugs from WR treatment plant effluent before being discharged
into the environment. Therefore, the knowledge of pharmaceutical
degradation and removal is a major issue for WR treatment.
Conflict of interest
None declared.
Acknowledgments
The authors are grateful to Jean François Munoz and Christophe
Rosin (Nancy laboratory of hydrology, ANSES) for providing data on
measured concentrations within the framework of the national
survey. We also want to thank Fabien Lavergne and Alan Sanh
(French National Agency for Medicines and Health Products Safety,
ANSM) for their advice and support throughout the writing of this
paper.
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