See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/10807496

Aquatic Ecotoxicity of Pharmaceuticals Including the Assessment of

Combination Effects

Article in Toxicology Letters · June 2003

DOI: 10.1016/S0378-4274(03)00068-7 · Source: PubMed

CITATIONS READS
942 2,726

1 author:

Michael Cleuvers
Dr. Knoell Consult GmbH
15 PUBLICATIONS 2,326 CITATIONS

SEE PROFILE

All content following this page was uploaded by Michael Cleuvers on 08 August 2020.

The user has requested enhancement of the downloaded file.

 Toxicology Letters 142 (2003) 185 /194
www.elsevier.com/locate/toxlet

Short communication

Aquatic ecotoxicity of pharmaceuticals including the

assessment of combination effects
Michael Cleuvers *
Department of General Biology, Aachen University of Technology, Kopernikusstraße 16, D-52056 Aachen, Germany

Received 24 July 2002; received in revised form 31 October 2002; accepted 13 November 2002

Abstract

To evaluate the ecotoxicological potential of ten prescription drugs against aquatic organisms from different
taxonornical classes, a set of biotests were performed using the cladoceran Daphnia magna , the chlorophyte
Desmodesmus subspicatus and the macrophyte Lemna minor . Endpoints were immobilisation for Daphnia and
inhibition of the average growth rate for Desmodesmus and Lemna . For most of the substances, toxicities were
moderate, with EC50s in the range from 10 to 100 mg l
1 or even far above, whereas Lemna was the most sensitive test
species in the majority of all tested compounds. Tests with combinations of various pharmaceuticals revealed stronger
effects than expected from the effects measured singly. Clofibrinic acid and Carbamazepine have been found to act by a
non-specific mode of action (non-polar narcosis), and with Daphnia the combination effect of these substances followed
the concept of concentration addition, while in the algae test the concept of independent action could be used to
calculate the mixture toxicity. The anti-inflammatory drugs Diclofenac and Ibuprofen have also been found to act
unspecific by non-polar narcosis and to follow the concept of concentration addition in the algal test as well as in the
Daphnia test. The measured toxicities of the tested pharmaceuticals shows that acute effect of single substances in the
aquatic environment are very unlikely. But we should keep in mind that considerable combination effects can occur and
that toxicity data from chronic studies are needed to assess the environmental risk of drug residues.
# 2003 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Pharmaceuticals; Ecotoxicity; Mixture toxicity; Concentration addition; Mode of action

1. Introduction increased in scientific publications (Ternes, 1998;

Daughton and Ternes, 1999; Kümmerer, 2001;
1.1. Background Daughton and Jones-Lepp, 2001; Ternes et al.,
2001). Among the detected substances in rivers
In recent years reports about residues of phar- were e.g. beta blocker (e.g. Metoprolol up to 1.54
maceuticals in surface and drinking waters have mg l
1) and beta-sympathomimetics (Hirsch et al.,
1996; Sedlak and Pinkston, 2001), analgesic and
anti-inflammatory drugs (e.g. Diclofenac up to 1.2
* Tel.: /49-241-8026554; fax: /49-241-8022602.
E-mail address: [email protected] mg l
1; Ternes, 1998; Stumpf et al., 1998; Buser et
(M. Cleuvers). al., 1998a, 1999), estrogens (e.g. 17b-estradiol up
0378-4274/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0378-4274(03)00068-7
186 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194

to 0.013 mg l
1; Kuch and Ballschmitter, 2000; assessment for complex mixtures of various sub-
Adler et al., 2001; Huang and Sedlak, 2001) and stances since more than 20 years and a lot of
also antibiotics (e.g. Erythromycin up to 1.7 excellent studies have been performed in this topic
mg l
1; Hirsch et al., 1999; Lindsey et al., 2001; (e.g. Altenburger et al., 2000; Backhaus et al.,
Adler et al., 2001) as well as lipid lowering agents 2000; Faust et al., 2001). Basically, two different
(e.g. Clofibrinic acid up to 0.2 mg l
1; Stan et al., concepts are in use for the prediction of mixture
1994; Buser et al., 1998b; Ahrer et al., 2001; .Öllers toxicity, and generally they are termed concentra-
et al., 2001) and anti-epileptic drugs (e.g. Carba- tion addition and independent action.
mazepine up to 2.1 mg l
1; Ternes, 1998; Möhle et The concept of concentration addition can be
al., 1999; Seiler et al., 1999). Particularly, some traced back to the early works of the pharmacol-
small streams receiving a relatively large amount ogists, Loewe and Muischnek (1926) (Loewe,
of their water from sewage water treatment plants 1927, 1953) and it can be described mathematically
are found to be considerably polluted, with peak for a mixture of n substances by Eq. (1) (Beren-
concentrations of several pharmaceuticals of more baum, 1985):
than 1 mg l
1. Due to their specific mode of action
and the fact that these compounds are intention- X
n
ci
1 (1)
ally designed to exert an effect on humans, ECxi
i1
mammals or other vertebrates, residues of phar-
maceuticals could be as or even more important In this equation, ci are the individual concentra-
for human health than those of pesticides, which tions of the single substances present in a mixture
are created to affect weeds, fungi and invertebrate with a total effect of x %, and ECxi are those
varmints. However, until now the mode of action concentrations of the single substances that would
of pharmaceuticals is not well enough understood alone cause the same effect x as observed for the
to make general statements about potential envir- mixture. As an important point, concentration
onmental effects caused by these substances. addition means, that substances applied below
Drinking water treatment diminishes residues, their individual no effect concentration (NOEC)
but is not able to remove these substances can nevertheless contribute to the total effect of
completely. Thus, even in tap water some phar- the mixture. Concentration addition is based on
maceuticals like Clofibrinic acid could be detected the idea of a similar action of chemicals, whereas
in concentrations up to 270 ng l
1 (Heberer et al., interpretations of this term can differ considerably.
1997, 2001a,b; Ternes et al., 2001). In contrast to From a mechanistic point of view, similar action
the amount of analytical data, information about means in a strict sense that substances should have
the ecotoxicological effects of drug residues are the same specific interaction with a molecular
sparse (Webb, 2001; Cleuvers, 2002). To create a target site in the observed test organism (Pöch,
broader basis for the evaluation of the ecotoxico- 1993). In contrast, used in a more common sense, a
logical relevance of pharmaceutical compounds similar action could be observed for all substances
and mixtures, biotests with Desmodesmus subspi- which are able to cause the same toxicological
catus , Daphnia magna and Lemna minor were response under consideration, e.g. death of the test
performed. organisms. For example, concentration addition is
able to predict mixture toxicities of inert chemi-
1.2. Concepts for the prediction of mixture toxicity cals, i.e. chemicals that are not reactive when
considering overall acute effects, and that do not
Drug residues found in the aquatic environment interact with specific receptors in the organism
usually occur as mixtures, not as single contami- (Broderius et al., 1995; Van Loon et al., 1997). The
nants. Thus, scientific assessment of risk to aquatic mode of action of such compounds is called
life should consider this complex exposure situa- narcosis (Van Leeuwen et al., 1992; Verhaar et
tion. By analyzing combination effects ecotoxicol- al., 1992). Narcosis type toxicity is considered to
ogists try to elucidate the problem of risk be caused by an absolutely non-specific mode of
 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194 187

action, in that the potency of a chemical to induce 2. Methods

narcosis is entirely dependent on its hydrophobi-
city, which is generally expressed by its n -octanol/
water partition coefficient (log Kow). As a result,
2.1. Daphnia acute immobilisation test
in the absence of any specific mechanism of
toxicity, a chemical will, within certain boundaries,
Daphnia tests were conducted following the
always be as toxic as its log Kow indicates. Thus,
European Guideline: ‘Methods for determination
the narcosis type of action is also called baseline
of ecotoxicity; Annex V, C.2, Daphnia acute
toxicity (Verhaar et al., 1992).
The alternative concept of independent action immobilisation test’ (Commission of the European
was already formulated by Bliss (1939). It is based Communities, 1992) using the water flea D. magna
on the idea of dissimilar action of compounds in a Strauss. Daphnids were bred in ADaM, a culture
mixture, i.e. that the compounds have different medium imitating natural fresh water (Klüttgen et
molecular target sites and modes of action. As a al., 1994). Experiments were run at temperatures
result of such a dissimilar action, the relative effect of 209/1 8C and photoperiods of LD 16:8 ( /20
of one of the toxicants in a mixture should remain mEs
1 m
2). Twenty daphnids younger than 24 h
unchanged in the presence of another one. For a were used for the controls and each treatment
binary mixture the combination effect can be subdivided in four replicates each containing five
calculated by the equation daphnids. Culture volume was 50 ml. Immobility
was observed after 24 and 48 h with the latter
being the endpoint for effect calculation.
E(cmix )1
[(1
E(c1 ))(1
E(c2 ))] (2)

or in general 2.2. Algal growth inhibition test

Y
n Algae tests were conducted following the Eur-
E(cmix )1
(1
E(ci )) (3) opean Guideline: ‘Methods for determination of
i1 ecotoxicity; Annex V, C.3, Algal inhibition test’
(Commission of the European Communities,
1993). We used the planktonic chlorococcale green
in which E (c1), E (c2), are the effects of the single
alga Desmodesmus subspicatus (Chodat) Hegewald
substances and E (cmix) is the total effect of the
et Schmidt (formerly Scenedesmus subspicatus
mixture. Following this equation, a substance
applied in a concentration below its individual Chodat, SAG 86.81 /UTEX 2594) obtained
NOEC will not contribute to the total effect of the from the ‘SAG */Sammlung von Algenkulturen’
mixture, i.e., there will be no mixture toxicity if the at the University of Göttingen, Germany. Initial
concentrations of all used single substances are cell densities were adjusted at 104 cells ml
1 using
below their NOEC. a calibration curve of chlorophyll fluorescence
At given concentrations of the single com- (Ex: 460, Em: 685 nm) versus cell number and
pounds in a mixture, the combination effect will appropriate dilution of preculture. Medium was
in general be higher if the substances follow the prepared according to the protocol using deionized
concept of concentration addition. Thus, mislead- water and analytical grade chemicals. Algae were
ingly the different concepts were sometimes incubated at 239/2 8C under continuous white
brought in correlation to the terms synergism light (120 mEs
1 m
2) and were kept in suspen-
and antagonism . But synergisms or antagonisms sion by continuous shaking (:/80 rpm). Results
between the used substances and their effects can were quantified in terms of average growth rates
occur independently of a similar or dissimilar calculated from cell numbers based on measure-
mode of action. ments of chlorophyll fluorescence.
188 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194

2.3. Lemna growth inhibition test effect concentrations (EC5/2, EC10/2, EC20/2,
EC50/2 and EC80/2 or EC90/2) were used. If the
Lemna tests with the duckweed L. minor were substances follow the concept of concentration
performed according to the standard ISO/WD addition, theoretically the combination effect of
20079 (ISO, 2001) using the Steinberg-medium the mixtures would add up to a total effect of
(Steinberg, 1943, 1946) with the modification that about 5, 10, 20, 50 and 80 or 90%, respectively.
two species of phosphate were added. Biotests
were carried out in 400 ml beakers with an outer 2.5. Analysis of the mode of action
diameter of 80 mm filled with 150-ml medium.
Inoculum for each beaker was 12 fronds. Only To find out something about the mode of action
plants with two or three fronds were chosen. Six of the observed substances, we used quantitative
control replicates and three treatment replicates structure activity relationships (QSARs; Verhaar
were used. Tests were carried out in a climatic et al., 1992; Lipnick, 1995; Schüürmann and
exposure test cabinet, calibrated at 259/2 8C, with Markert, 1998) for non-polar narcotic chemicals
fluorescent tubes mounted on the top. Light proposed by Verhaar et al. (1995) for D. magna
intensity was adjusted at 100 mEs
1 m
2. Test (Eq. (4)) and by Van Leeuwen et al. (1992) for the
duration was 7 days (168 h). Number and area of chlorophyte Pseudokirchneriella subcapitata (Eq.
the fronds were determined at days 0, 3, 5 and 7. (5)), which sensitivity is seen to be comparable to
Measurements and evaluations were performed by D. subspicatus .
means of the digital image analysis system Scana-
lyzer produced by LemnaTec, Wuerselen, Ger- log EC50 (mmol l
1 )
0:95 log Kow
1:32 (4)
many. At days 0, 3, 5 and 7 images of the beakers log EC50 (mmol l
1 )
1:00 log Kow
1:32 (5)
were taken for analysis. Based on total frond area
the specific average growth rates were calculated
following the ISO Standard (ISO, 2001).

2.4. Test substances and calculation of effect 3. Results

concentrations
The EU-Directive 93/67/EEC (Commission of
Substances used for the biotests were Clofibrinic the European Communities, 1996) classifies sub-
acid (metabolite of several lipid lowering drugs), stances according to their EC50-value in different
Carbamazepine (anti-epileptic), Propranolol and classes; B/1 mg l
1 (very toxic to aquatic organ-
Metoprolol (b-blocker), Ibuprofen sodium, Diclo- isms); 1 /10 mg l
1 (toxic to aquatic organisms)
fenac sodium and Naproxen sodium (analgesics/ and 10/100 mg l
1 (harmful to aquatic organ-
anti-inflammatory drugs), Captopril (anti-hyper- isms). Substances with an EC50 above 100 mg l
1
tensive), and Metformin (anti-diabetic); all sup- would not be classified. The toxicity of the tested
plied in analytical grade by Sigma Aldrich Chemie pharmaceuticals was very heterogeneous, with
Gmbh, Taufkirchen, Germany. Generally, sub- EC50 values ranging from 7.5 mg l
1 (Proprano-
stances were applied in at least five concentrations lol) to 174 mg l
1 (Naproxen) in the Daphnia test;
(1, 3.2 10, 32, 100 mg l
1). Depending on the from 5.8 mg l
1 (Propranolol) to /320 mg l
1
results, some tests were repeated including lower (Metformin and Naproxen) in the algal test and
(0.1 and 0.32 mg l
1) or higher (320 mg l
1) from 7.5 mg l
1 (Diclofenac) to /320 mg l
1
concentrations. (Metoprolol) in the Lemna test (Table 1). Most
Effective concentrations (ECx ) were determined EC50s were in the range between 10 and 100
with the program SIGMA PLOT 2000 using non- mg l
1 or even above. Values below 10 mg l
1
linear curve fitting based on a sigmoid model were measured merely for Propranolol (Daphnia
(four-parameter logistic function). For the assess- and Desmodesmus ), Metoprolol (Desmodesmus )
ment of mixture toxicities, half the calculated and Diclofenac (Lemna ). With the exception of
 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194 189

Table 1
EC50s (mg l
1) of the tested pharmaceuticals as obtained in the
biotests with D. magna , D. subspicatus and Lemna minor

Test substance EC50 (mg l
1)

Daphnia Desmodesmus Lemna

Clofibrinic acid 72 115 12.5

Carbamazepine /100 74 25.5
Ibuprofen /Na 108 315 22
Diclofenac /Na 68 72 7.5
Naproxen /Na 174 /320 24.2
Captopril /100 168 25
Metformin 64 /320 110
Propranolol 7.5 5.8 114
Metoprolol /100 7.3 /320
Fig. 2. Measured mixture toxicity of Clofibrinic acid and
Endpoint for Daphnia was immobilization after 48 h, and for Carbamazepine as obtained in the algal test in comparison to
Desmodesmus and Lemna inhibition of the average growth rate the singly measured toxicities and the mixture toxicity predicted
after 3 and 7 days, respectively. by the concepts of concentration addition and independent
action. For the assessment of mixture toxicities, half the
Metformin, Metoprolol and Propranolol, Lemna calculated effect concentrations (EC5/2, EC10/2, EC20/2, EC50/
was the most sensitive species to all tested sub- 2 and EC80/2) were used.
stances.
To evaluate the mixture toxicity we performed compound Carbamazepine. In the Daphnia test
Daphnia and algal tests with combinations of two these substances follow the concept of concentra-
substances. Figs. 1 and 2 show the results of a test tion addition. As a result, the measured effects
were much stronger than expected from the very
with the two frequently detected drugs Clofibrinic
weak effects measured singly. For example, at the
acid (lipid lowering agent) and the anti-epileptic
EC90/2 level, the concentrations responsible for the
singly measured effects of Clofibrinic acid (1%
immobilized daphnids) and Carbamazepine (16%
immobilized daphnids) cause in the mixture a
strong effect of about 95% immobilization of the
daphnids. In the algal tests (Fig. 2), the mixture
effect could be well calculated using the concept of
independent action, which predicts much lower
combination effects than concentration addition.
Regarding the mode of action it could be found,
that for both substances the measured EC50 is
higher than predicted by the QSAR for non-polar
narcosis (Table 2), i.e. that no specific mechanism
is present.
In the second mixture test Ibuprofen and
Diclofenac were used, both belonging to the
Fig. 1. Measured mixture toxicity of Clofibrinic acid and nonsteroidal anti-inflammatory drugs (NSAID).
Carbamazepine as obtained in the acute Daphnia test in In the Daphnia test, the mixture effect was even
comparison to the singly measured toxicities and the mixture somewhat stronger than predicted by concentra-
toxicity predicted by the concepts of concentration addition and
independent action. For the assessment of mixture toxicities,
tion addition (Fig. 3). The combination effect in
half the calculated effect concentrations (EC5/2, EC10/2, EC20/2, the algal test could be predicted well by that
EC50/2 and EC90/2) were used. concept, which one would expect for substances
190 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194

Table 2
Log Kow, molecular weight and the predicted and measured EC50s for the substances used in the tests with Daphnia and Desmodesmus
with binary mixtures of chemicals

Test substance Log Kow Mol. weight (g mol
1) EC50 (mg l
1)

Predicted for narcosis Measured

Daphnia Algae Daphnia Algae

Clofibrinic acid 3.1 214.63 11.7 10.0 72 115

Carbamazepine 2.45 236.27 53.2 49.4 /100 74
Diclofenac /Na 1.56 318.1 50.2 51.6 68 72
Ibuprofen /Na /1 218.3 1.2 1.3 108 315

The log Kow of Ibuprofen /Na was not available in literature, but because of its free solubility in water it was estimated to be not
higher than 1.

with a similar mode of action (Fig. 4). But as 4. Discussion

shown in Table 2, that similarity is not due to a
specific toxic action, but as well as in the first The measured toxicity of the substances was
combination study a result of non-polar narcosis. heterogeneous and for most substances moderate
The log Kow of Ibuprofen /Na was not available with EC50s between 10 and 100 mg l
1 or even
in literature. Due to the fact, that Ibuprofen /Na is above. In the acute Daphnia and algal test, solely
freely soluble in water, its log Kow was estimated the tested b-blockers showed EC50s below 10
to be not higher than 1. But in any case, the mg l
1; Propranolol in both tests and Metoprolol
measured EC50 is so high, that any specific toxic in the algal test only. Bearing in mind that
action can be excluded. environmental concentrations of pharmaceuticals
are in most cases below or only little above 1
mg l
1, acute effects of such compounds in the

Fig. 3. Measured mixture toxicity of Diclofenac and Ibuprofen Fig. 4. Measured mixture toxicity of Diclofenac and Ibuprofen
as obtained in the acute Daphnia test in comparison to the as obtained in the algal test in comparison to the singly
singly measured toxicities and the mixture toxicity predicted by measured toxicities and the mixture toxicity predicted by the
the concepts of concentration addition and independent action. concepts of concentration addition and independent action. For
For the assessment of mixture toxicities, half the calculated the assessment of mixture toxicities, half the calculated effect
effect concentrations (EC5/2, EC10/2, EC20/2, EC50/2 and EC80/ concentrations (EC5/2, EC10/2, EC20/2, EC50/2 and EC80/2)
2) were used. were used.
 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194 191

aquatic environment are very unlikely. Neverthe- ciated with the log Kow of the drugs rather than
less, in any case further studies with other test with any specific toxic action. As a result, the
species, e.g. fish and benthic macroinvertebrates as combination effect of Diclofenac and Ibuprofen
well as chronic studies with daphnids should be could be calculated with the concept of concentra-
performed to evaluate the toxic potential of these tion addition, whereas in the Daphnia test, the
compounds. In the Lemna -test, merely the EC50 of observed mixture toxicity was somewhat stronger
Diclofenac was lower than 10 mg l
1, but in six than predicted. As a consequence, we should keep
out of nine cases, Lemna was the most sensitive in mind that toxicity of a single substance could
organism. This is not surprising, because this is a increase strongly in combination with other, and
chronic test with the possibility to record several particularly with similar acting substances. For
sensitive, sub-lethal parameters, such as frond size example, in the Daphnia test the used concentra-
(Cleuvers and Ratte, 2002). In the case of Diclo- tions EC20/2 and EC50/2 were below the individual
fenac, the EC50 was nearly 10 times lower than NOEC, but showed together an effect of about 50
measured in the algae and Daphnia test. Thus, and 80% immobilization of daphnids, respectively.
Lemna growth inhibition tests are very useful and Additionally, synergisms cannot be excluded be-
serve as an additional source of information about tween various substances independent of a similar
phytotoxicity for higher plants, which may differ or dissimilar mode of action. This shows that the
greatly from that to algae (Lewis, 1995). Unfortu- use of individual NOECs is inapplicable regarding
nately, no special QSARs for narcosis are available the assessment of mixture toxicities.
for the Lemna test like it was performed in this In the combination test with Clofibrinic acid
study, but due to the lower measured EC50s it is and Carbamazepine I obtained species dependent
not unlikely, that specific toxic actions of some differing results regarding the best concept to
tested pharmaceuticals have occurred in the calculate the combination effect of these com-
Lemna tests. pounds. In the Daphnia test, these substances
Due to the fact that the selected compounds follow the concept of concentration addition,
belong to different classes of pharmaceuticals with which is generally seen to be appropriate only for
diverse modes of action, no general trend exists in similar acting substances (Pöch, 1993). This of
sensitivity of Daphnia and Desmodesmus . But the course raises the question, which similar mode of
three studied anti-inflammatory drugs Diclofenac, action could be found for a lipid lowering agent
Ibuprofen and Naproxen, interestingly show the and an anti-epileptic drug belonging to utterly
same descending order of sensitivity Lemna / different classes of chemicals. Again it could be
Daphnia /Desmodesmus . Moreover, with all used shown that in both used test species the measured
test organisms, Diclofenac was the most and toxicity was not higher than the baseline toxicity
Naproxen the least toxic compound. This leads predicted by the QSAR for non-polar narcosis.
to the question of the mode of action of these But in contrast to the result found with Daphnia ,
substances, which is normally known only for the in the algal test the combination effect was lower
target organisms, namely humans, and a few than predicted by concentration addition and
laboratory mammals. Diclofenac, Ibuprofen and could be predicted well using the concept of
Naproxen all inhibit the cyclooxygenases, the key independent action, which is untypical for sub-
enzymes catalyzing the biosynthesis of prostaglan- stances acting by non-polar narcosis.
dins, which are inter alia responsible for the
genesis of pain and inflammations (Vane and
Botting, 1998; Vane, 1971). This inhibition is 5. Conclusions
responsible for the analgesic and anti-inflamma-
tory effect of these drugs. But as shown above, in The toxicity of the tested pharmaceuticals was
the tests with Daphnia as well as with Desmodes- very heterogeneous. In the majority of cases, based
mus these compounds act unspecifically by non- upon the obtained EC50s, Lemna was the most
polar narcosis, Thus, the toxicity may be asso- sensitive species, and it is recommended to per-
192 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194

form such tests routinely in addition to the other Buser, H.R., Poiger, T., Müller, M.D., 1998a. Occurence and
standard tests. Regarding the assessment of the fate of the pharmaceutical drug Diclofenac in surface
waters: rapid photodegradation in a lake. Environ. Sci.
environmental risk of pharmaceuticals, acute ef- Technol. 33, 2529 /2535.
fects seems to be unlikely, but we need to bear in Buser, H., Muller, M.D., Theobald, N., 1998b. Occurence of
mind that considerably combination effects of the pharmaceutical drug clofibric acid and the herbicide
substances can occur, even if the toxicity of the mecoprop in various Swiss lakes and in the North Sea.
single substance is low. Thus, the combination Environ. Sci. Technol. 32, 188.
Buser, H.-R., Poiger, T., Müller, M.D., 1999. Occurence and
effect of pharmaceuticals detected together in
experimental behaviour of the pharmaceutical drug ibupro-
water samples should be determined to achieve a fen in surface waters and in wastewater. Environ. Sci.
better assessment of the ecotoxicological potential Technol. 33, 2529 /2535.
of drug residues in the aquatic environment. Cleuvers, M., 2002. Aquatic ecotoxicology of selected pharma-
ceutical agents */algal and acute Daphnia tests. UWSE-Z
Umweltchem Ökotox 14, 85 /89.
Cleuvers, M., Ratte, H.T., 2002. Phytotoxicity of coloured
Acknowledgements substances: is Lemna duckweed an alternative to the algal
growth inhibition test? Chemosphere 49, 9 /15.
The author wants to thank Stephanie Esser for Commission of the European Communities, 1992. Methods for
determination of ecotoxicity; Annex V, C.2, Daphnia , acute
technical support and Arnd Weyers for critically
toxicity to Daphnia . L 383A, 172 /178. EEC Directive 92/
reading the manuscript. 69/EEC.
Commission of the European Communities, 1993. Methods for
determination of ecotoxicity; Annex V, C.3, Algal inhibition
test. L 383A, 179 /186. EEC Directive 92/69/EEC.
References Commission of the European Communities, 1996. Technical
guidance document in support of commission directive 93/
Adler, P., Steger-Hartmann, T., Kalbfus, W., 2001. Distribu- 67/EEC on risk assessment for new notified substances and
tion of natural and synthetic estrogenic steroid hormones in commission regulation (EC) No 1488/94 on risk assessment
water samples from southern and middle Germany. Acta for existing substances. Part II; Environmental Risk Assess-
Hydrochim. Hydrobiol. 29, 227 /241. ment. Office for official publications of the European
Ahrer, W., Scherwenk, E., Buchberger, W., 2001. Occurence Communities, Luxembourg.
and fate of fluoroquinolone, macrolide, and sulfonamide Daughton, C.G., Ternes, T.A., 1999. Pharmaceuticals and
antibiotics during wastewater treatment and in ambient personal care products in the environment: agents of subtle
waters in Switzerland. In: Daughton, C.G., Jones-Lepp, change? Environ. Health Perspect. 107, 907 /938.
T.,(Eds.), Pharmaceuticals and personal care products in the Daughton, C.G., Jones-Lepp, T. (Eds.), 2001. Pharmaceuticals
environment: scientific and regulatory issues. Symposium and personal care products in the environment scientific and
Series 791, American Chemical Society, Washington DC,
regulatory issues. Symposium Series 791, American Chemi-
pp. 56 /69.
cal Society, Washington DC, pp. 56 /69.
Altenburger, R., Backhaus, T., Boedeker, W., Faust, M.,
Faust, M., Altenburger, R., Backhaus, T., Blanck, H., Boede-
Scholze, M., Grimme, L.H., 2000. Predictability of the
ker, W., Gramatica, P., Hamer, V., Scholze, M., Vighi, M.,
toxicity of multiple chemical mixtures to Vibrio fischeri :
Grimme, L.H., 2001. Predicting the joint algal toxicity of
mixtures composed of similarly acting chemicals. Environ.
multicomponent s -triazine mixtures at low-effect concen-
Toxicol. Chem. 19, 2341 /2347.
Backhaus, T., Altenburger, R., Boedeker, W., Faust, M., trations of individual toxicants. Aquatic Toxicol. 56, 13 /32.
Scholze, M., Grimme, L.H., 2000. Predictability of the Heberer, Th., Dünnbier, U., Reilich, Ch., Stan, H.J., 1997.
toxicity of a multiple mixture of dissimilarly acting chemi- Detection of drugs and drug metabolites in ground water
cals to Vibrio fischeri , Environ. Toxicol. Chem. 19, 2348 / samples of a drinking water treatment plant. Fresenius
2356. Environ. Bull. 6, 438 /443.
Berenbaum, M.C., 1985. The expected effect of a combination Heberer, Th., Fuhrmann, B., Schmidt-Bäumler, K., Tsipi, D.,
of agents: the general solution. J. Theor. Biol. 114, 413 /431. Koutsouba, V., Hiskia, A., 2001a. Occurence of pharma-
Bliss, C.I., 1939. The toxicity of poisons applied jointly. Annu. ceutical residues in sewage, river, ground and drinking water
Rev. Appl. Biol. 26, 585 /615. in Greece and Germany. In: Daughton, C.G., Jones-Lepp,
Broderius, S.J., Kahl, M.D., Hoglund, M.D., 1995. Use of joint T., (Eds.), Pharmaceuticals and personal care products in
toxic response to define the primary mode of action for the environment: Scientific and regulatory issues. Sympo-
diverse industrial organic chemicals. Environ. Toxicol. sium Series 791, American Chemical Society, Washington
Chem. 14, 1591 /1605. DC, pp. 56 /69.
 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194 193

Heberer, Th., Verstraten, I.M., Meyer, M.T., Mechlinski, A., ceuticals and pesticides at the low-ng/l level in surface and
Reddersen, K., 2001b. Occurence and fate of pharmaceu- waste water. J. Chromatogr. A 911, 225 /234.
ticals during bank filtration /preliminary results from in- Pöch, G., 1993. Combined effects of drugs and toxic agents.
vestigations in Germany and the United States. Water Modern evaluation in theory and practice. Springer, Berlin,
Resour. Update 120, 4 /17. Germany.
Hirsch, R., Ternes, T.A., Haberer, K., Kratz, K.L., 1999. Schüürmann, G., Markert, B., 1998. Ecotoxicology: Ecological
Occurence of antibiotics in the aquatic environment. Sci. Fundamentals, Chemical Exposure, and Biological Effects.
Total Environ. 225, 109 /118. Wiley and Spektrum Akademischer Verlag, New York and
Hirsch, R., Ternes, T.A., Haberer, K., Kratz, K.L., 1996. Heidelberg.
Nachweis von Betablockern und Bronchospasmolytika in Sedlak, D.L., Pinkston, K.E., 2001. Factors affecting the
der aquatischen Umwelt. Vom Wasser 87, 263 /274. concentrations of pharmaceuticals released to the aquatic
Huang, C.H., Sedlak, D.L., 2001. Analysis of estrogenic environment. Water Resources Update 120, 56 /64.
hormones in municipal wastewater effluent and surface Seiler, R.L., Zaugg, S.D., Thomas, J.M., Howcroft, D.L., 1999.
water using enzyme-linked immunoadsorbent assay and Caffeine and pharmaceuticals as indicators of waste water
gas chromatography/tandem mass spectrometry. Environ. contamination in wells. Ground Water 37, 405 /410.
Toxicol. Chem. 20, 133 /139. Stan, H.J., Heberer, T., Linkerhägner, M., 1994. Vorkommen
ISO (International Standards Organisation), 2001. Standard von Clofibrinsäure im aquatischen System */führt die
ISO/WD 20079. Water quality-Duckweed growth inhibi- therapeutische Anwendung zu einer Belastung von Ober-
tion; determination of the toxic effect of water constituents flächen-, Grund- und Trinkwasser? Vom Wasser 83, 57 /68.
and waste water to duckweed (Lemna minor ). Draft Steinberg, R.A., 1943. Use of Lemna as test organism.
document. Chronica Bot. 7, 420.
Klüttgen, B., Dülmer, U., Engels, M., Ratte, H.T., 1994. Steinberg, R.A., 1946. Mineral requirements of Lemna minor .
ADaM, an artificial freshwater for the culture of zooplank- Plant Physiol. 21, 42 /48.
ton. Water Res. 28, 743 /746. Stumpf, M., Ternes, T.A., Haberer, K., Baumann, W., 1998.
Kuch, H.M., Ballschmitter, K., 2000. Determination of en- Isolicrung von Ibuprofen */Metaboliten und deren Bed-
dogenous and exogenous estrogens in effluents from sewage cutung als Kontaminanten der aquatischen Umwelt. Vom
treatment plants at the ng/l-level. Fresenius J. Anal. Chem. Wasser 91, 291 /303.
366, 392 /395. Ternes, T., 1998. Occurence of drugs in German sewage
Kümmerer, K. (Ed.), Pharmaceuticals in the Environment. treatment plants and rivers. Water Res. 32, 3245 /3260.
Sources, Fate, Effects and Risk. Springer, Berlin, Germany Ternes, T.A., Bonerz, M., Schmidt, T., 2001. Determination of
2001. neutral pharmaceuticals in wastewater and rivers by liquid
Loewe, S., 1927. Die Mischarznei. Versuch einer allgemeinen chromatography /electrospray tandem mass spectrometry.
Pharmakologie der Arzneikombination. Klinische Wo- J. Chromatogr. A 938, 175 /185.
chenschrift 6, 1077 /1085. Vane, J.R., Botting, R.M., 1998. Mechanism of action of
Loewe, S., 1953. The problem of synergism and antagonism of nonsteroidal antiinflammatory drugs. Am. J. Med. 104
combined drugs. Arzneimittel-Forsch. /Drug Res. 3, 285 / (3A), 2S /8S.
290. Vane, J.R., 1971. Inhibition of Prostaglandin synthesis as a
Loewe, S., Muischnek, H., 1926. Über Kombinationswirkun- mechanism of action for aspirin like drugs. Nature 231,
gen. 1. Mitteilung: Hilfsmittel der Fragestellung. Naunyn- 232 /235.
Schmiedebergs Archiv für experimentelle Pathologie and Van Leeuwen, C.J., van der Zandt, P.T.J., Aldenberg, T.,
Pharmakologie 114, 313 /326. Verhaar, H.J.M., Hermens, J.L.M., 1992. Application of
Lewis, M.A., 1995. Use of freshwater plants for phytotoxicity QSARs, extrapolation and equilibrium partitioning in
testing */a review. Environ. Poll. 87, 319 /336. aquatic assessment. 1. Narcotic industrial pollutants. En-
Lindsey, M.E., Meyer, M., Thurman, E.M., 2001. Analysis of viron. Toxicol. Chem. 11, 267 /282.
trace levels of sulfonamide and tetracycline antimicrobials Van Loon, W.M.G.M., Verwoerd, M.E., Eijnker, F.G., van
in groundwater and surface water using solid-phase extrac- Leeuwen, C.J., van Duyn, P., van deGuchte, C., Hermens,
tion and liquid chromatography/mass spectrometry. Anal. J.L.M., 1997. Estimating total body residues and baseline
Chem. 73, 4640 /4646. toxicity of complex organic mixtures in effluents and surface
Lipnick, R.L., 1995. Structure /activity /relationships. In: waters. Environ. Toxicol. Chem. 16, 1358 /1365.
Rand, G.M. (Ed.), Fundamentals of Aquatic Toxicology. Verhaar, H.J.M., van Lecuwen, C.J., Hermens, J.L.M., 1992.
Taylor and Francis, London, UK. Classifying environmental pollutants. 1. Structure /activity
Möhle, E., Horvath, S., Merz, W., Metzger, J.W., 1999. relationships for prediction of aquatic toxicology. Chemo-
Determination of hardly degradable organic compounds sphere 25, 471 /491.
in sewage water */identification of pharmaceutical residues. Verhaar, H.J.M., Mulder, W., Hermens, J.L., 1995. QSARs for
Vom Wasser 92, 207 /223. ecotoxicity. In: Hermens, J.L.M. (Ed.), Overview of
.Öllers, S., Singer, H.P., Fässler, P., Müller, R.S., 2001. structure /activity relationships for environmental end-
Simultaneous quantification of neutral and acidic pharma- points, part 1: General outline and procedure. Report
194 M. Cleuvers / Toxicology Letters 142 (2003) 185 /194

prepared within the framework of the project ‘QSAR for Webb, S.F., 2001. A data-based perspective on the environ-
prediction of fate and effect of chemicals in the mental risk assessment of human pharmaceuticals I-col-
environment’, Environmental Technologies RTD Pro- lation of available ecotoxicity data. In: Kummerer, K. (Ed.),
gramme, European Commission, contract number EV5V- Pharmaceuticals in the Environment. Sources, Fare, Effects
CT92-0211. and Risks. Springer, Berlin, Germany.

View publication stats