Journal of Antimicrobial Chemotherapy (2000) 46, Suppl.

S1, 53–58
JAC
Environmental risk assessment of antibiotics: comparison of
mecillinam, trimethoprim and ciprofloxacin

B. Halling-Sørensen*, H.-C. Holten Lützhøft, H. R. Andersen and F. Ingerslev

Section of Environmental Chemistry, Department of Analytical and Pharmaceutical Chemistry,

Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen 0, Denmark

The effects of mecillinam, trimethoprim and ciprofloxacin, antibiotics used in the treatment of
urinary tract infections, on the aquatic environment were assessed. Mecillinam and cipro-
floxacin were both readily biodegradable (primary degradation) in activated sludge, whereas
trimethoprim persisted. The toxicity of these antibiotics towards sludge bacteria, a green alga,
a cyanobacterium, a crustacean and a fish were investigated; both mecillinam and cipro-
floxacin were highly toxic to the cyanobacterium Microcystis aeruginosa (EC50 in the range
5–60 µg/L). Risk characterization for the aquatic environment was performed for the three com-
pounds by calculating the predicted environmental concentration (PEC) and the predicted no-
effects concentration (PNEC). A PEC/PNEC ratio of <1 indicates that, with the present pattern
of use, no environmental risk is expected. PEC/PNEC ratios of <1 for present usage in Europe
were found for mecillinam and trimethoprim whereas a PEC/PNEC ratio >1 was found for
ciprofloxacin.

Introduction posed in the EU.4 There are, however, no regulations or

By their nature, medicinal products, such as antibiotics, are
biologically active molecules. Antibiotics are specifically
designed to control pathogenic bacteria in animals and
humans, but very little is known about their ecotoxicology.
After administration of antibiotics to humans, a significant
amount is excreted into waste water. Surplus antibiotics are
also sometimes disposed of into household drains. Anti-
biotics therefore enter municipal sewage and sewage-
treatment plants (STPs). If the drugs are not completely
mineralized in the STP, they are released into surface water
or sludge and, if the sludge is used to fertilize arable land,
they may enter the topsoil of fields.1,2
Medicinal products are licensed for use by regulatory
authorities if they comply with scientific criteria on quality,
efficacy and safety. The authorities consider safety to the
consumer and to the individuals handling the product dur-
ing treatment. In addition to these criteria, the environ-
mental risk has recently become a matter of increasing
public scrutiny and legal requirements. The risk of effects
on the environment has led to regulation of new pharma-
ceuticals in the USA,3 and a draft on the environmental risk
assessment (ERA) of new pharmaceuticals is also pro-
requirements concerning the environmental properties
or potential effects of existing pharmaceuticals. Conse-
quently such data are scarce.
An important step in ERA, particularly for drugs, is a
cost–benefit analysis. It is necessary to know the risk to the
environment and the indirect effects on the human body as
a result of contamination of the environment to be able to
decide whether the benefits exceed the costs. If two or
more drugs have the same therapeutic effect, ERA can also
be used to determine which has the lower environmental
impact.
The aim of this paper is to provide data on consumption,
estimated environmental concentration and the effects
on the environment of three antibiotics widely used for
urinary tract infections (mecillinam, trimethoprim and
ciprofloxacin), enabling a preliminary comparison of their
environmental hazard. The structures of these three com-
pounds are shown in the Figure, and relevant physico-
chemical characteristics in Table I. The spread of bacterial
antibiotic resistance genes is another pertinent effect of
antibiotics on the environment. Since this is difficult to
quantify and not included in the ERA assessment of anti-
biotics, it will not be discussed further in this paper.

*Corresponding author. Tel:  45-35-30-64-53; Fax:  45-35-30-60-13; E-mail: bhs@dfh.dk

53
© 2000 The British Society for Antimicrobial Chemotherapy
 B. Halling-Sørensen et al.

Table I. Physicochemical data

Solubility log KOW

Antibiotic Compound group (g/L) Syracusea log KOWb

Mecillinam β-lactam antibiotic 2 not found 0.79  0.38

Trimethoprim dihydrofolate reductase inhibitor 0.3–0.4 0.91 1.33  0.75
Ciprofloxacin quinolone 2 0.28 1.24  0.86
a
From http://esc.syrres.com/interkow/estsoft.htm
b
Estimated with ACD Log P software (Advanced Chemical Development, Toronto, Canada).

(99.8% pure), 2,3-dichlorophenol, sodium acetate and

aniline (analytical grade; Merck, Copenhagen, Denmark)
were used as reference compounds in the toxicity studies
and biodegradation tests.

Figure. Chemical structures of (a) mecillinam, (b) trimethoprim
and (c) ciprofloxacin.
Materials and methods
Chemical substances
Test substances were purchased from their manufacturers
and stored at 2°C for 24 h. Mecillinam [(6R)-6-(per-
hydroazepin-1-ylmethyleneamino)-penicillanic acid, batch
no. V27 (99.3% pure), CAS 32887-01-7] was provided by Leo
Pharmaceutical Products, Ballerup, Denmark; trimetho-
prim [2,4-diamino-5-(3,4,5-trimethoxybenzyl)-pyrimidine;
batch no. 211962, CAS 738-70-5] by Nomeco, Copenhagen,
Denmark; ciprofloxacin–HCl (1-cyclopropyl-6-fluoro-1,4-
dihydro-4-oxo-7-piperazin-1-ylquinoline-3-carboxylic acid
hydrochloride; batch no. 303477A, CAS 85721-33-1) by
Bayer A/G, Wuppertal, Germany. Potassium dichromate
Biodegradation tests
Biodegradation tests were performed in two different test
systems:
(i) Oxytop respirometers (WTW, Weilheim, Germany)
were used to measure biological oxygen demand (BOD)
according to the guidelines published by the Organization
for Economic Cooperation and Development (OECD).5
The Oxytop system is a screening system that only evalu-
ates whether the compound is readily degradable. Sodium
acetate was used as a reference compound. All compounds
were tested in duplicate experiments with added concen-
trations corresponding to theoretical oxygen demands
(TODs) of 30 mg/L. The TOD of 30 mg/L was calculated to
correspond to mecillinam 16.5 mg/L, trimethoprim 19.4
mg/L and ciprofloxacin 17.5 mg/L.
(ii) Biodegradation experiments with activated sludge in
reactors may be used to determine the half-life of the chem-
ical during biodegradation in the activated sludge process
in a waste water system. Primary biodegradation experi-
ments were conducted in aerated batch reactors5 with 1 L
of liquid (sludge). Experiments were conducted at 22°C
and sludge was preconditioned at room temperature.
Sludge was collected from the Lundtofte rensningsanlæg
sewage treatment plant, Lyngby, Denmark and maintained
aerated until use, with no addition of substrate. Most
sludge was used freshly collected (aeration time 1–2 h) or
was preconditioned by aeration for 1 day. Laboratory reac-
tors were fed with sludge (1 L) and mecillinam or trimetho-
prim 500 g/L or ciprofloxacin 250 g/L.
The biodegradation of the chemicals in the reactors was
followed over time by HPLC assay. The half-life (t½) in
days was calculated for the primary degradation of the
three compounds based on first-order kinetics. These half-
lives were applied in the risk assessment described below.
Detailed description and validation of the HPLC methods
used are available from the authors. All three antibiotics
were tested in duplicate reactors. Aniline was used as a
reference compound.

54
 Environmental risk assessment of antibiotics
Ecotoxicology
Toxicity towards the following organisms was tested: activ-
ated sludge bacteria; a green alga, Selenastrum capri-
cornutum (eukaroytic); a cyanobacterium, Microcystis
aeruginosa (prokaryotic); the zooplankton Daphnia magna;
and the zebrafish, Brachydanio rerio. All toxicity tests were
performed in accordance with OECD or International
Organization of Standardization (ISO) guidelines.5–9
Risk assessment
Environmental risk assessment (ERA) was based on the
EU draft guideline document for medicinal products for
human use4 and was conducted in two phases. For most
medicinal products, it is anticipated that only the first phase
of the evaluation will be necessary. Phase II of the ERA,
which involves a comprehensive assessment of ecotoxi-
cology, is considered when the predicted environmental
concentration (PEC) exceeds a threshold value of
0.001 g/L in surface water and 10 g/kg soil. The phase II
assessment involves calculation of a risk quotient between
the predicted (PEC) or measured environmental concentra-
tion and the predicted no-effect concentration (PNEC),
using worst-case assumptions.
For the aquatic compartment, it was assumed that all
antibiotics are used in the year they are purchased; that the
antibiotics are released into the environment without
degradation in man or the sewage system; and that the use
pattern is evenly distributed temporally and spatially. The
concentration in water of the active substance (PECw) was
calculated from the following equation:4
A  (100 R)
PECw  (1)
365  P  V  D  100
where A is the amount used (kg/year), R is the percentage
removal (set to zero when information on biodegradation
in the STP is missing or when worst-case conditions are
assessed), P is the population of Europe (383 million in
1999), V is the volume of waste water per capita per day
(0.2 m3) and D is the dilution factor in the environment (a
default factor of 10 was used). A hydraulic retention time
(HRT) of 10 h was used in the PECw calculations, incor-
porating the degradation.
For compounds sorbing to sludge, the concentration in
sludge can be calculated if the partition coefficient between
sludge and water, Kd, is measured or estimated based on
the octanol–water partition coefficient, Kow, and the frac-
tion of organic carbon in sludge (foc  0.35), and calculated
as follows:
Kd  foc  0.41  Kow (2)
Experimental Kds were obtained in sorption experiments
with sludge (H. C. Holten-Lützhøft and B. Halling-
Sørensen, unpublished results). Kows were from the litera-
ture or calculated with the ACD log P software (Advanced
55
Chemical Development, Toronto, Canada). A Kow cor-
rected for the effect of ionized groups on Kow-dependent
distributions (Dow) can be applied for the Kd calculation
(Equation 3).
Kow
Dow  (3)
1  10pH–pKa
The calculation of the predicted concentration in sludge
(Csludge) is based on no degradation or other loss of the
drugs. The concentration of the antibiotics in sludge can be
calculated as:
Mmedical compound
Csludge  (4)
Vw
/Kd  Msludge
where Kd based on experimental Kd, Dow or Kow, respec-
tively, is inserted for Kd. The total annual volume of waste
water in Denmark (Vw) is 3.8  108 m3. For Europe Vw can
be calculated as P  V  365, i.e. 383 million  0.2 m3  365
 2.8  1010 m3. For the mass of sludge, Msludge, a value of
151 159 tonnes in 1997 was used for Denmark. Mmedicinal
compound, the annual loading of antibiotic to the environmen-
tal compartment, is often assumed to equal consumption
because of lack of information.
Results and discussion
Physicochemical properties of the chemicals
n-Octanol–water partition coefficients were obtained from
literature using the Syracuse database at http://esc.syrres.
com/ and calculated using ACD log P software. No pub-
lished value was found for mecillinam, but agreement
between calculated and literature values for the two other
substances suggests that the range of the calculated value
for mecillinam is appropriate. Based on the available
n-octanol–water partition coefficients, it is considered
appropriate to use static exposure systems in the toxicity
tests.
No values for the vapour pressure of any of the three
substances were found in the literature. However, the
molar weights (200 g/mol) and the presence of several
polar groups in each of the three molecules suggests that
vapour pressure as well as air–water partition coefficients
will be small. Therefore no special precautions were
employed in the test procedure to handle loss of test sub-
stance by evaporation.
Degradation test
None of the compounds was found to sorb strongly to
sludge. The partition coefficients, Kd, between water and
sludge for mecillinam, trimethoprim and ciprofloxacin
were found to be 55, 76 and 417, respectively.
 B. Halling-Sørensen et al.

Table II shows the results of the biodegradation studies
performed with the Oxytop system and the activated
sludge reactors. Biodegradation in the Oxytop system was
very poor: all three compounds persisted for the entire
28 days of the test period. Using this system, which is con-
sidered an initial screening system, all three compounds
were therefore assessed as being not readily degradable.
The next step was assessment of the primary degrada-
tion of the compounds in sludge reactors. Results per-
formed with an initial concentration of 500 g/L of
mecillinam showed that biodegradation occurred rapidly in
this test system. The estimated t½ (assuming first-order
degradation) was 0.5 days for one sludge column and 0.7
days for the other. β-Lactams such as mecillinam are also
subject to abiotic degradation such as hydrolysis. This may
speed up degradation in the STP.
Biodegradation studies performed with trimethoprim
500 g/L in the reactors showed that degradation had not
reached 50% at day 25. Trimethoprim persisted in the two
reactors with a t½ of 22 days and 41 days, respectively.
Ciprofloxacin 250 g/L was degraded quickly until 50%
had been degraded. Its t½ was 2.5 days in one reactor and
1.6 days in the other. This result is consistent with a similar
study investigating the primary degradation of cipro-
floxacin,12 in which it was found that the t½ for ciprofloxacin
in activated sludge was between 1 and 5 days, depending
on the sludge concentration. Compared with a compound
like pentachlorophenol, which is recognized as barely
degradable under such test circumstances [t½ (first order
model) about 5 days], trimethoprim is extremely persistent,
whereas mecillinam and ciprofloxacin are readily degrad-
able (primary degradation). Because results show that
ciprofloxacin probably was not mineralized under the test
conditions, more studies need to be performed on metabo-
lites. This study and that of Hartmann and co-workers12
indicate that compounds resembling ciprofloxacin persist
in the sludge. The reference compound aniline was degraded
rapidly (t½  0.7 days) in the reference reactor, showing the
ability of the activated sludge to degrade chemical com-
pounds.
Toxicity tests
Table III shows the water concentration giving 50% effect
(EC50) or no-observed-effect concentration (NOEC)
obtained for the reference chemicals and three tested com-
pounds.
Sludge bacteria. Only ciprofloxacin showed high potency
for the sludge bacteria. Mecillinam and trimethoprim were
harmless to the activated sludge bacteria.

Table II. Biodegradation data obtained with the screening test11 (data are expressed as readily degradable or
persistent) and the activated sludge reactors (data are expressed as half-lives (days))

Test Mecillinam Trimethoprim Ciprofloxacin Sodium acetate Aniline

Screening test persistenta persistenta persistenta readily degradable NA

Activated sludge reactor 0.5–0.7 22–41 1.6–2.5 NA 0.7
NA, not assessed.
a
Persisted for 28 day test period.

Table III. Toxicity data: EC50 with corresponding 95% confidence limits or
no-observed-effect concentration (NOEC) (mg/L) for the compounds tested

EC50 (mg/L)

Species mecillinam trimethoprim ciprofloxacin

Bacteria (activated sludge)a 62.1 17.8 0.61 (0.31–1.22)

S. capricornutumb NOEC  300 110 (64–192) 2.97 (2.41–3.66)
M. aeruginosa 0.06 (0.05–0.06) 112 (100–126) 0.005 (0.004–0.006)
D. magna, 48 h test NOEC  300 123 NOEC  60
B. rerio, 72 h test NOEC  100 NOEC  100 NOEC  100
a
3,5-Dichlorophenol was used as a reference compound in the test. EC50 was in accordance with that given in the
guidance protocol, 5.8 (95% CI 3.8–8.8) mg/L.
b
Potassium dichromate was used as reference compound in the test. EC50 was in accordance with that given in the
guidance protocol, 0.59 (95% CI 0.46–0.75) mg/L.

56
 Environmental risk assessment of antibiotics
Green algae and cyanobacteria. Only ciprofloxacin was
toxic to S. capricornutum (EC50  2.97 mg/L), whereas
mecillinam (NOEC  300 mg/L) and trimethoprim (EC50
 110 mg/L) were toxic only at environmentally irrelevant
concentrations. The highest mecillinam concentration
applied was 300 mg/L, so this concentration is therefore
detected as the lower limit of NOEC. Both mecillinam and
ciprofloxacin were highly toxic to M. aeruginosa (see Table
III), with EC50s in the range 5–60 g/L. M. aeruginosa was
generally much more sensitive than S. capricornutum to
both mecillinam and ciprofloxacin. This is not surprising, as
M. aeruginosa is a prokaryote whereas S. capricornutum
is a eukaryote. Similar results have been reported for
other antibiotics.13 The EC50 of potassium dichromate was
0.59 mg/L (95% CI 0.46–0.75 mg/L), within the limits
indicated in the guidance protocol5 [0.53 mg/L (95% CI
0.20–0.75 mg/L)].
Daphnia magna. Mecillinam was tested in a limit test7 at
two concentrations, 300 and 1000 mg/L. At these concen-
trations it did not kill D. magna. Ciprofloxacin was tested in
a similar limit test at 30 and 60 mg/L (at concentrations of
60 mg/L, ciprofloxacin crystallized and was, therefore, no
longer bioavailable, so it was not tested at higher concen-
trations). At these concentrations, ciprofloxacin did not kill
the organisms studied. Trimethoprim was tested at five
concentrations between 30 and 300 mg/L. Table III shows
the toxicity of the three compounds towards D. magna:
trimethoprim had minor effects on D. magna.
Zebrafish. A limit test was performed in accordance with
OECD guidelines8 at two concentrations (30 and 100
mg/L) for all three compounds. For all three compounds,
an NOEC (72 h) of 100 mg/L was found. Assuming that
toxicity fits a probit-type effect curve, this indicates a 99.9%
confidence level for the true EC50 being 100 mg/L.
Environmental loading
Because data on environmental loading were not available
for the antibiotics tested, the environmental loading was
500)
assumed to be the greatest possible, i.e. equal to the
consumption of the compounds. Data for Europe (Spain,
Portugal, Italy, Greece, Germany, Switzerland, France,
Table IV. Estimated PECs, PNEC (mg/L) and PEC/PNEC ratios based on European consumption in 1999
Compounds PEC1 (mg/L)a PEC2 (mg/L)b
Mecillinam 7.0  10–6 5.9  10–8
Trimethoprim 1.7  10–4 1.7  10–4
Ciprofloxacin 6.7  10–4 6.3  10–4
a
Calculated without including biodegradation (R  0 in Equation 1); simulates a worst-case assessment.
b
Calculated including biodegradation (R  (percentage degraded in the STP during the 8 h HRT) in Equation 1) and simulates a best-
case assessment.
Austria, Denmark, Belgium, The Netherlands, UK, Ire-
land, Norway, Sweden and Finland; total population 383
million) was obtained from IMS14 for the first 3 months of
1999 and extrapolated to give a value for the whole of 1999.
These IMS data exclude consumption at hospital level in
Spain, Portugal, Greece, Switzerland and Ireland. The total
environmental loading in 1999 for mecillinam, cipro-
floxacin and trimethoprim was estimated as 2.0, 186.2 and
47.8 tonnes/year, respectively.
Preliminary risk characterization
Risk characterization is the estimation of the incidence of
the adverse effect occurring in an environmental compart-
ment as a result of actual or predicted exposure to a sub-
stance. It generally involves the integration of an effect
assessment (calculating the PNEC) and an exposure assess-
ment (calculating the PEC). We calculated PEC in two
ways: (i) not including degradation data (worst-case
scenario) (we call this PEC1) and (ii) including degradation
data (PEC2) (Table IV).
Many international regulatory frameworks often
express risk as a PEC/PNEC ratio. It should be noted that
these ratios are not absolute measures of risk.
The worst-case PEC estimations, PEC1 (R  0 in
Equation 1), for the three compounds show that (based on
European consumption data) none of the three compounds
exceeds the threshold values in the EU (1994) draft guide-
line4 of 0.001 g/L for surface water. With today’s pattern
of use, the compounds would not, therefore, require phase
II assessment. However, changes in the consumption
pattern of the three compounds throughout Europe or in
certain member countries could enhance the PEC and
therefore trigger a demand for more information.
The aim of this study was to compare the environmental
fate and effects of the three compounds, as part of the
phase II assessment, on the aquatic environment using
bacteria (activated sludge), green algae and cyanobacteria,
daphnia and fish as target organisms. The terrestrial com-
partment was not assessed because none of the compounds
was found to sorb strongly to activated sludge (Kd
and are not used in veterinary therapy. An assessment fac-
tor of 100 was used in accordance with OECD guidelines,5
as acute toxicity data on algae, crustaceans and fish were
PNEC (mg/L) PEC1/PNEC ratio PEC2/PNEC ratio
0.0006 0.01 9.8  10–3
0.18 9.5  10–4 9.4  10–4
0.00005 13.3 12.7
57
 B. Halling-Sørensen et al.
available. The lowest EC50 (see Table III) obtained for the
three compounds were therefore recalculated to a PNEC
of 0.0006, 0.18 and 0.00005 mg/L for mecillinam, trimetho-
prim and ciprofloxacin, respectively. Based on these
PNECs, the PEC/PNEC ratio was calculated.
Table IV shows PEC/PNEC calculations for the three
antibiotics at European level, excluding the degradation
data (PEC1/PNEC; worst-case scenario) and including
these data (PEC2/PNEC). A PEC1/PNEC ratio of 1 was
found for ciprofloxacin; the ratios for mecillinam and
trimethoprim were both
1. This is a result of the lower
consumption of trimethoprim, even though it is assessed as
the most persistent of the three compounds. The picture
did not change much after taking the biodegradation of the
compounds into consideration because mecillinam and
trimethoprim still had PEC2/PNEC ratios
1, suggesting tion Test. Environment Guideline no. 202, OECD, Paris.
that no environmental hazard would occur with today’s
pattern of use. The PEC2/PNEC ratio was still 1 for
ciprofloxacin, identifying a possible environmental hazard
with today’s pattern of use. This study would suggest that
more information on the fate and effects of ciprofloxacin in
environmental matrices should be provided.
Acknowledgement
The authors gratefully acknowledge the technical assis-
tance of Susanne Hermansen, The Royal Danish School of
Pharmacy.
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