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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

An investigation into the acute and chronic toxicity of eleven


pharmaceuticals (and their solvents) found in wastewater
effluent on the cnidarian, Hydra attenuata
Brian Quinn, Franois Gagn, Christian Blaise
St-Lawrence Centre, Environment Canada, 105 McGill, Montral, Qubec, Canada H2Y 2E7

AR TIC LE I N FO

ABS TR ACT

Article history:

Summary: Pharmaceuticals previously identified in the effluent from the wastewater

Received 10 July 2007

treatment plant (WWTP) in Montreal discharging into the St. Lawrence river, were tested for

Received in revised form

acute and chronic toxicity using the cnidarian Hydra attenuata. Acute toxicity was based on

16 August 2007

the established technique looking at morphological changes in the Hydra, while recently

Accepted 23 August 2007

developed endpoints of feeding behaviour, attachment and growth (hydranth number) were

Available online 10 October 2007

used to measure chronic effects. The compounds under investigation (ibuprofen, naproxen,
gemfibrozil, bezafibrate, carbamazepine, sulfamethoxazole, sulfapyridine, oxytetracycline,

Keywords:

novobiocin, trimethoprim and caffeine) were tested individually in controlled laboratory

Hydra attenuata

exposures with LC50 and EC50 results calculated. All compounds tested had relatively high

Pharmaceuticals

LC50 values with gemfibrozil, ibuprofen and naproxen having the lowest at 22.36 mg/L and

Solvents

EC50 values based on morphology of 1.18 to 2.62 mg/L (all concentrations are nominal). The

Chronic effects

EC50 values based on feeding were similar to those based on morphology but with increased

Feeding

sensitivity for carbamazepine, bezafibrate and novobiocin. A trend of a reduction in feeding


with deterioration in morphology was observed in the Hydra, with the exception of
novobiocin, where a lower than expected EC50 of 13.53 mg/L was found with no negative
effect on morphology. Significant reductions in attachment and hydranth number were
seen at concentrations of 1 and 5 mg/L for gemfibrozil and ibuprofen respectively. A toxicity
threshold (TT) of 320 g/L was calculated for ibuprofen, only a factor of 102 or 10 higher than
the concentration found in the effluent in the present study (1.19 g/L) and in other
Canadian effluents studied (22 g/L [Brun GL, Bernier M, Losier R, Doe K, Jackman P, Lee HB,
Pharmaceutically active compounds in Atlantic Canadian sewage treatment plant effluents
and receiving waters and potential for environmental effects as measured by acute and
chronic aquatic toxicity. Environ Toxicol Chem 2006; 25(8): 21632176.] respectively. Using
EU directive 93/67/EEC the pharmaceuticals under investigation can be classified as toxic
(gemfibrozil, ibuprofen and naproxen), harmful (carbamazepine, bezafibrate, sulfapyridine,
oxytetracycline and novobiocin) and non-toxic (sulfamethoxazole, trimethoprim and
caffeine) and their potential toxicity for the aquatic environment is discussed.
2007 Elsevier B.V. All rights reserved.

Corresponding author. Birches Lane, Blackrock, Dundalk, Co. Louth. Ireland. Tel.: +353 87 2860874; fax: +353 42 9323063.
E-mail address: quinnbrian@gmail.com (B. Quinn).
0048-9697/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2007.08.038

S CIE N CE OF T H E TOT AL E N V I RO N ME N T 3 8 9 ( 2 00 8 ) 3 0 63 14

1.

Introduction

Municipal effluents and environmental pollutants have become more complex and as a result environmental toxicology
is moving away from the obvious acute fish kill type of
pollution, to study more subtle effects reducing the health of
an individual or population. This is aided by the development
of more sensitive analytical techniques, allowing the study of
more subtle effects from a wide array of emerging pollutants.
One such pollutant that has come into focus in recent years is
pharmaceutical compounds, everyday drugs ingested by
western populations that are becoming ubiquitous in the
aquatic environment (Sanderson et al., 2004). Many of the
pharmaceuticals applied in human medical care are not
completely eliminated by the body, often excreted only
slightly transformed or even unchanged (Heberer, 2002).
Once excreted, the majority end up passing through a
wastewater treatment plant (WWTP), from where they are
introduced to surface waters with the treated effluent. As
conventional sewage treatment is generally not effective in
removing many pharmaceuticals and their metabolites, this
effluent is considered an important and continuous source of
drug input into the aquatic environment (Brun et al., 2006).
There are numerous original papers (Ternes, 1998; Jones
et al., 2002; Metcalfe et al., 2003a, 2003b; Roberts and Thomas,
2006; Lishman et al., 2006) and reviews (Heberer, 2002;
Kmmerer, 2004; Brun et al., 2006; Fent et al., 2006; Hernando
et al., 2006) on the occurrence and quantities of pharmaceuticals found in the aquatic environment. Around 80100
pharmaceuticals and their metabolites have been measured
in both effluent and surface waters in numerous countries
(Fent et al., 2006). The most commonly found drugs and their
metabolites included anti-inflammatory drugs (ibuprofen,
naproxen), lipid regulators (bexafibrate, gemfibrozil), anticonvulsion drugs (carbamazepine) and various antibiotics
(trimethoprim, oxytetracycline, sulfamethoxazole) (Fent
et al., 2006). These compounds are mostly detected in the
high ng/L, low g/L range (Heberer, 2002), but may be sufficient
to induce toxic effects (Hernando et al., 2006). Pharmaceuticals
have similar physio-chemical characteristics as harmful
xenobiotics, e.g. they can pass through membranes and are
relatively persistent (Sanderson et al., 2004). Most are specifically designed to affect target organs/systems in the human
body and are designed to be persistent so as to retain their
chemical structure long enough to do their therapeutic work
(Ternes and Hirsch, 2000). Generally little is known about their
affect on non-target species in the environment. The majority
of studies looking at the biological effects of pharmaceuticals
have thus far concentrated on acute studies. As previously
observed by Blaise et al. (2007), there is a notable recurrence of
the use of several well-documented and popular bioassays in
the literature e.g. V. fischeri luminescence inhibition test, P.
subcapitata growth inhibition test and D. magna immobilization and reproduction tests. It has also been observed that due
to their more or less continuous presence at low concentrations in the aquatic environment, pharmaceuticals will most
likely have chronic rather than acute toxic effects (Crane et al.,
2006), e.g. changing behaviour that reduces individual fitness
of an organism (Jones et al., 2002). Therefore standardised
acute tests may not be the most appropriate basis for the

307

ecotoxicological hazard assessment of pharmaceuticals (Ferrari et al., 2004), but currently studies on chronic effects are
lacking for most pharmaceuticals (Carlsson et al., 2006).
In the present study we looked at both the acute and
chronic effects of 11 compounds identified and quantified in
the (primary) treated effluent from the Montreal WWTP in a
series of small scale toxicity tests using the freshwater
cnidarian Hydra attenuata. Hydra are easily cultured and
maintained in the laboratory and being diploblastic, they are
sensitive environmental indicators. For these reasons, Hydra
have been extensively used in toxicity tests to study the
effects of various freshwater pollutants including effluents
(Blaise and Kusui, 1997; Pardos et al., 1999), heavy metals
(Holdway et al., 2001; Karntanut and Pascoe, 2002), estrogenic
compounds (Pascoe et al., 2002; Pachura-Bouchet et al., 2006)
and pharmaceuticals (Pascoe et al., 2003; Blaise et al., 2007).
We used the standardised toxicity test based on morphology
(Wilby, 1988) and the newly developed endpoints looking at
feeding behaviour (Quinn et al., 2007), attachment and growth
(hydranth number). We also discuss the hazard potential of
these compounds by comparing our results to previously
published environmental risk assessments, to classify their
potential toxicity for the aquatic environment.

2.

Materials and methods

2.1.

Test organism

Cultures of H. attenuata have been maintained in the Centre


since the mid-nineteen nineties and used extensively for
various toxicity tests by this and other laboratories (Blaise and
Kusui, 1997; Trottier et al., 1997; Pardos et al., 1999; Quinn et al.,
2007). Hydra were maintained and grown in glass bowels
containing 0.5 L of Hydra medium (110 mg/L TES [N-Tris
(hydroxymethyl) methyl 1-2-aminoethanesulfonic acid],
147 mg/L CaCl22H2O, pH 7) at 20 1 C with a 16 h light and
8 h dark photoperiod, following the procedure adapted from
Trottier et al. (1997).

2.2.

Test solutions and exposure

A previous study (Gagn et al., 2006) identified a number of


pharmaceuticals found in the treated effluent from Montreal
municipal sewage treatment works. These included the
cholesterol lowering agents gemfibrozil and bezafibrate (59
and 72 ng/L respectively), the analgesic ibuprofen and
naproxen (1191 and 217 ng/L respectively), the anti-epileptic
carbamazepine (33 ng/L), the antibiotics sulfapyridine, oxytetracycline, novobiocin, sulfamethoxazole and trimethoprim
(46, 440, 330, 99 and 63 ng/L respectively) and the stimulant
caffeine (22,187 ng/L). These pharmaceuticals were purchased
from Sigma-Aldrich (Canada), dissolved in solution and
investigated for their lethal and sub-lethal effects at various
concentrations between 0.1 mg/L and 50/100 mg/L. Nominal
concentrations were used throughout this experiment as a
previous study by Pascoe et al. (2003) reported the measured
concentrations of all drug solutions (a different suite of drugs,
with only ibuprofen being in common) within 10% of the
nominal concentration, indicating the relative persistence of

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these compounds during exposure. The solvent DMSO was


used for gemfibrozil, naproxen (0.31% final concentration),
sulfapyridine and sulfamethoxazole (0.62% final concentration), while acetone was used for carbamazepine (0.31% final
concentration). Ibuprofen, bezafibrate and trimethoprim were
all dissolved in ethanol (0.31% final concentration) and
oxytetracycline, novobiocin and caffeine were dissolved in
Hydra media. Medium and solvent (where appropriate) controls were used in all exposures. Sub-lethal and lethal toxicity
tests for the 3 solvents (0.16 to 1.25%) were also undertaken in
triplicate to ensure no solvent effects. 4 mL media containing
the relevant chemical in solution were added to 3 wells in a 12
well multi-well plate as previously described (Trottier et al.,
1997; Quinn et al., 2007). Healthy Hydra of similar size having
one bud (2 hydranths) spotted with a binocular microscope
(6), were used in each exposure, with 3 Hydra in each of the 3
wells per exposure concentration (n = 9). The multi-well plates
were covered with parafilm to prevent evaporation and
randomly placed on a work bench at room temperature (20

2 C) for 96 h, after which time they were observed using a


binocular microscope (6 enlargement).

2.3.

Lethal and sub-lethal endpoints

Lethality and sub-lethal effects on Hydra morphology, hydranth number, attachment and ability to ingest prey after
96 h exposure were observed. Hydra were not fed for 24 h
before exposure. Toxicity in Hydra is measured by drastic
changes in morphology, with the contraction of tentacles and
the body, measured and scored from 10 (normal, elongated
tentacles and body) to 0 (disintegrated) on a scale devised by
Wilby (1988). As the amount of toxicant increases Hydra
progressively exhibit signs of toxicity, such as clubbed
tentacles (score 8), shortened tentacles (score 6), a tulip
phase (score 5), loss of osmoregulation (score 2) and disintegration (score 0). Scores 106 are reversible, sub-lethal
indicators while the tulip phase (score 5 and below) is
considered irreversible and used as the endpoint for lethality

Fig. 1 Effects of various concentrations of the three solvents (DMSO, acetone and ethanol) on Hydra morphology (score 106),
attachment and hydranth number. Each point represents the mean score (n = 3) standard error. Significance at * = p 0.05; ** =
p 0.01; *** = 0.001 for attachment and + = 0.05; ++ = 0.01 for hydranth number.

309

S CIE N CE OF T H E TOT AL E N V I RO N ME N T 3 8 9 ( 2 00 8 ) 3 0 63 14

Fig. 2 The effect for each of the 3 solvents (DMSO, acetone and ethanol) on the ability of Hydra to feed on Artemia. Each point
represents the mean score (n = 3) standard error. Significance at * = p 0.05;*** = p 0.001.

(Blaise and Kusui, 1997). The feeding test followed the


technique previously described by Quinn et al. (2007). Briefly
after 96 h exposure nine Hydra were placed individually into
each well of a 12 well multi-well plate containing 4 mL of
medium and their morphology noted. At T0, precisely 5 Artemia salina were added to each well and the number of ingested
prey observed every 30 min for 120 min.

2.4.

Statistics

The 96 h LC50 and EC50 values were calculated with their 95%
confidence limits using the Trimmed SpearmanKarber
programme in the ToxTest version 2, US EPA software. The
sub-lethal LOEC (Lowest Observable Effect Concentration) was
reported for 2 Hydra with score 8 or below and the NOEC (No
Observable Effect Concentration) was based on b2 Hydra with
score 8, both calculated based on visual observation following
Trottier et al. (1997). A toxicity threshold (TT) was determined
from the LOEC and NOEC using the following equation: TT =
(NOEC LOEC)1/2 (US EPA, 1989). Variability in feeding between
the exposed and control Hydra was tested by one-way analysis
of variance (ANOVA) using Microsoft Excel. All exposure
effects were compared to solvent controls where appropriate.
Significance was set at p 0.05. Solvent toxicity tests were
undertaken in triplicate with mean scores presented
standard error calculated using Microsoft Excel.

3.

Results

3.1.

Solvent exposure

Three different types of solvent were used to suspend the


pharmaceuticals under study depending on their solubility. In
order to ensure the solvents did not affect the endpoints
measured, they were tested for effects on morphology,
attachment and hydranth number (Fig. 1) and Hydra feeding
(Fig. 2). At 0.31% there was a similar slight shift in morphology
from score 10 to 7 for all three solvents (Fig. 1). This effect is
slightly increased at 0.62% with the emergence of a very low
number of score 6 for both DMSO and ethanol indicating
relatively weak toxicity. This concentration also sees a
significant decrease in attachment for ethanol, but no
significant effects on hydranth number. At 1.25% there is a
large shift to score 6 for both DMSO and ethanol indicating
toxicity, and a significant decrease in attachment and
hydranth growth for both of these solvents. Acetone however
remains relatively non-toxic at this concentration with a small
effect on morphology and no significant decrease in attachment or hydranth growth. Results for the feeding test show no
significant decrease in feeding at 0.31% or 0.62% for all three
solvents (Fig. 2). In fact, at 0.62% acetone was found to
significantly increase feeding.

Table 1 Acute (LC50) and chronic (EC50, LOEC, NOEC) responses (mg/L) with 95% confidence interval (CI) based on
morphology for Hydra attenuata exposed for 96 h to the 11 pharmaceuticals under investigation
Pharmaceutical

96 h LC50

95% CI

96 h EC50

95% CI

Gemfibrozil
Ibuprofen
Naproxen
Carbamazepine
Bezafibrate
Sulfapyridine
Oxytetracycline
Novobiocin
Sulfamethoxazole
Trimethoprim
Caffeine

22.36
22.36
22.36
29.4
70.71
N100
N100
N100
N100
N100
N100

(22.3622.36)
(22.3622.36)
(22.3622.36)
(32.8326.32
(70.7170.71
NC
NC
NC
NC
NC
NC

1.18
1.65
2.62
15.52
25.85
21.61
40.13
NC
NC
NC
NC

(7.150.19)
(2.820.96)
(3.621.9)
(29.028.3)
NC
(34.2313.64)
(46.9534.3)
NC
NC
NC
NC

NC = not calculable, results didn't fit the appropriate test. Toxicity threshold TT = (NOEC LOEC)1/2.

LOEC

NOEC

1
1
5
5
1
5
100
100
10
N 100
N 100

0.1
0.1
1
1
0.1
1
50
50
5
N 100
N 100

TT
0.32
0.32
2.24
2.24
0.32
2.24
70.71
70.71
7.07
NC
NC

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Fig. 3 Series of graphs showing the number of Hydra at each morphology state (shift from score 10 to 0) and the mean (n = 9) feeding (ingestion) rate (with standard error) at
various concentrations for each pharmaceutical individually.

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S CIE N CE OF T H E TOT AL E N V I RO N ME N T 3 8 9 ( 2 00 8 ) 3 0 63 14

The only significant reduction in feeding was found after


exposure to 1.25% DMSO, with results for both acetone and
ethanol similar to the control for this concentration.

3.2.

Pharmaceutical exposure

3.2.1.

Acute toxicity effect on morphology

The LC50 and EC50 values with their 95% confidence limits
based on morphology for all eleven compounds are shown in
Table 1. Gemfibrozil, ibuprofen and naproxen all show the
same LC50 value of 22.36 mg/L and similar EC50 values between
1.18 and 2.62 mg/L. Gemfibrozil and ibuprofen are the most
toxic pharmaceuticals tested with a LOEC of 1 mg/L and a
toxicity threshold (TT) value of 0.32 mg/L. Naproxen, carbamazepine and bezafibrate are the next most toxic although
bezafibrate does have a high LC50 value of 70.71 mg/L but a low
TT (0.32 mg/L) due to its low LOEC and NOEC. EC50 values were
also calculated for sulfapyridine and oxytetracycline (21.61
and 40.13 mg/L respectively) but LC50 could not be calculated
as less than 50% of the exposed population were killed, even
after 96 h exposure to 100 mg/L. This was the same for
novobiocin, sulfamethoxazole, trimethoprim and caffeine,
where no 96 h EC50 could be calculated. Sulfamethoxazole
did however show a relatively low TT of 7.07 mg/L due to its
low LOEC and NOEC values.

crease in morphology, feeding remains high. The concentration of each compound having a significant decrease on
feeding is shown in Table 2. As with morphology it is
gemfibrozil, ibuprofen, naproxen, carbamazepine and bezafibrate that show a significant reduction in feeding at 10,10, 50,
50 and 100 mg/L respectively. Novobiocin also shows a
significant reduction in feeding at 100 mg/L despite having
very little effect on morphology (Fig. 3). Table 2 also shows the
EC50 results (with 95% confidence limits) based on feeding.
These results show a similar trend and similar sensitivity as
the EC50 based on morphology with the 5 most toxic
compounds having the lowest scores, but with increased
sensitivity for carbamazepine, bezafibrate and novobiocin.
Novobiocin has a relatively low EC50 of 13.53 mg/L and a
decrease in Hydra attachment at 50 mg/L. A similar trend of
toxicity can also be seen in the attachment results (Fig. 4) with
a significant reduction in attachment at 1, 10, 10 and 25 mg/L
for gemfibrozil, ibuprofen, naproxen and carbamazepine
respectively (Table 2). Interestingly caffeine significantly
decreases attachment at 25 mg/L but significantly increases
it at 50 mg/L. A significant reduction in hydranth number was
only observed at 5, 10 and 100 mg/L for ibuprofen, naproxen
and bezafibrate respectively (Table 2), showing this not to be a
particularly sensitive endpoint (Fig. 4).

3.2.2. Chronic toxicity effect on feeding, attachment and


hydranth growth

4.

The general trend of decreasing morphology from score 10


towards 0 with increasing exposure can be seen for each
compound in Fig. 3. This graph shows that for the five most
toxic compounds (gemfibrozil, ibuprofen, naproxen, carbamazepine and bezafibrate) the majority if not all of the Hydra
have a score of 1 or 0 at the highest exposure concentration. It
is also worth noting that novobiocin and caffeine have a very
small impact on morphology with the majority of exposed
Hydra being unaffected (score 10) up to 100 mg/L exposure.
From this graph a trend in decreasing prey ingestion with
increased toxicity is obvious for the 5 most toxic compounds.
Generally as morphology shifts to score 8 or below there is a
decrease in feeding. However this is not the case for
sulfapyridine or oxytetracycline where despite a small de-

Although there re several routes for pharmaceuticals to enter


the aquatic environment including the application of various
drugs in farming and aquaculture, leachate from landfill and
effluent from hospitals, WWTP effluent is known to be the
most significant of these. Chemical analysis of the primary
treated effluent from Montreal WWTP identified numerous
pharmaceutical compounds, previously reported by Gagn et
al. (2006) and described in depth by Blaise et al. (2007). Briefly,
there were five categories of pharmaceuticals found in the
effluent examined, anti-inflammatory (ibuprofen and
naproxen), lipid regulators (gemfibrozil and bezafibrate),
anti-convulsant (carbamazepine), antibiotics (sulfamethoxazole, sulfapyridine, oxytetracycline, novobiocin and trimethoprim) and a stimulant (caffeine). In general the compounds

Discussion

Table 2 Results for the 96 h chronic toxicity test for Hydra attenuata exposed to each of the 11 pharmaceuticals under
investigation showing the 96 h EC50 (mg/L) based on feeding with 95% confidence interval (CI) and the concentration (mg/L)
where there was a significant (p 0.05) reduction in feeding response, hydranth number and attachment
Pharmaceutical

96 h EC50 (feeding)

95% CI

Feeding response

Hydranth

Attachment

Gemfibrozil
Ibuprofen
Naproxen
Carbamazepine
Bezafibrate
Sulfapyridine
Oxytetracycline
Novobiocin
Sulfamethoxazole
Trimethoprim
Caffeine

1.76
3.85
2.68
3.76
8.59
NC
NC
13.53
NC
NC
NC

(0.93.42)
(0.00.0)
(01.7)
(0.1785.4)
(3.7819.51)
NC
NC
(0.00.0)
NC
NC
NC

10
10
50
50
100
N100
N100
100
N100
N100
N100

N 100
5
10
N 50
100
N 100
N 100
N 100
N 100
N 100
N 50

1
10
10
25
N 100
N 100
N 100
50
N 100
N 100
25 ; 50

NC = not calculable, results didn't fit the appropriate test. indicates increase; indicates decrease.

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Fig. 4 The number of Hydra attached (bars) and the hydranth number (lines) after 96 h exposure to each of the 11 pharmaceuticals
under investigation.

and concentrations found in this effluent were similar to those


reported in other studies (for review see Fent et al., 2006).
Ibuprofen, naproxen, bezafibrate, gemfibrozil and carbamazepine are ubiquitously found in effluents and surface waters in
Germany in the ng/L range and are among the most
commonly found worldwide (Fent et al., 2006). Ibuprofen was
found up to 85 g/L in effluents (Farr et al., 2001), and in
seawater in Norway up to 0.120 g/L (Weigel et al., 2004).
Naproxen had a high concentration in Canadian effluents
with a median level of 12.5 g/L (Metcalfe et al., 2003a).
Concentrations up to 4.6 and 3.1 g/L of bezafibrate were found
in effluent and surface water respectively (Ternes, 1998).
Carbamazepine was found in every Canadian STP effluent at
concentrations up to 2.3 g/L (Metcalfe et al., 2003b) and in
German wastewater up to 6.3 g/L (Ternes, 1998) and has been
consistently found in effluents throughout the world. In a
recent Canadian study bezafibrate, gemfibrozil, ibuprofen and
naproxen were found at concentrations up to 0.8, 1.4, 22 and
14 g/L respectively, in WWTP treated effluent (Brun et al.,
2006).
In the present study all of the compounds identified in the
effluent exceeded the 10 ng/L cut off value in WWTP effluents,
requiring the implementation of the second-tier assessment
based on ecotoxicity data (Hernando et al., 2006). Initially, 96 h
acute toxicity tests based on morphological changes in the
Hydra provided LC50 values ranging from 22.36 mg/L to
N100 mg/L (Table 1). As previously observed by other authors
with such high and environmentally unrealistic LC50 values
the risk of acute toxic effects in the environment are unlikely
(Carlsson et al., 2006; Fent et al., 2006). However due to their
biological activity and continuous release into the environment, environmental classification should be based on
chronic rather than acute toxicity (Carlsson et al., 2006;
Crane et al., 2006). The EC50 values based on morphology
were generally an order of magnitude lower than the LC50
ranging from 1.18 mg/L up to 40.13 mg/L and were roughly
similar to those reported by Blaise et al. (2007), with
differences recorded for ibuprofen, sulfapyridine (the present
study being more sensitive) and bezafibrate (Blaise et al. (2007)
more sensitive). There are several review papers summarizing
the biological effects of pharmaceuticals on numerous species

from various taxa (Carlsson et al., 2006; Crane et al., 2006; Fent
et al., 2006, Blaise et al., 2007) where it was reported that fish
are less sensitive than algae and invertebrates, with LC50
values at the mg/L level.
Little is known about the chronic effects of these compounds on non-target species, particularly invertebrates,
where the relevant pathways (e.g. COX enzymes, effects on
the CNS) have yet to be studied. This is further complicated
since pharmaceuticals form a heterogeneous group consisting
of compounds with diverse chemical properties and biological
effects, with little known about their chronic effects. For this
reason we developed a toxicity test based on chronic endpoints. Animal feeding behaviour has already been identified
as a potential endpoint for the study of the more subtle effects
of pharmaceuticals (Fent et al., 2006). The results for the
feeding test were similar to those for the EC50 based on
morphology but showed increased sensitivity (lower EC50) for
carbamazepine, bezafibrate and novobiocin (Table 2). The
feeding results for novobiocin were particularly interesting,
showing an EC50 (based on feeding) of 13.53 mg/L with little or
no effect on morphology, indicating that exposure to this
antibiotic affects feeding behaviour unrelated to morphological effects. A previous study by Pascoe et al. (2003) also looked
at the effect of feeding using a different suite of pharmaceuticals (except ibuprofen) at lower concentrations (0.0110 mg/
L) for an extended period (7 days) and reported a significant
reduction in feeding for ibuprofen and acetylsalicylic acid. In
the present study the lowest concentration showing a
significant reduction in feeding was 10 mg/L (gemfibrozil
and ibuprofen). However gemfibrozil was found to significantly reduce attachment at 1 mg/L and gemfibrozil, ibuprofen and
bezafibrate had a NOEC based on morphology of 0.1 mg/L.
In numerous studies, environmental risk assessment has
been used to estimate the risk of pharmaceuticals most
frequently found in wastewater effluents and surface waters,
based on the comparison between the predicted environmental concentrations (PEC) and the worst-case predicted no effect
concentrations (PNEC) estimated from toxicity assays (Jones
et al., 2002; Sanderson et al., 2004; Hernando et al., 2006). A key
piece of information needed in risk assessment is the
concentration at which a chemical produces adverse effects

S CIE N CE OF T H E TOT AL E N V I RO N ME N T 3 8 9 ( 2 00 8 ) 3 0 63 14

313

Table33- -Level
Table
Level
of of
toxicity
toxicity
for for
each
each
of the
of 11
thepharmaceuticals
11 pharmaceuticals
underunder
investigation,
investigation,
based on
based
the chronic
on theEC
chronic
ECfrom
Hydrafrom
50 results
50 results
attenuata
Hydra attenuata
in the current
in the study
current
using
study
theusing
classification
the classification
from EU directive
from EU
93/67/EEC
directive 93/67/EEC

on the organism. This toxicity data can be interpreted using


EU directive 93/67/EEC, which classifies substances according
to the measured effective concentrations (EC50 value) (CEC,
1996). When this scheme was applied to the data obtained in
the current study gemfibrozil, ibuprofen and naproxen were
classified as toxic (Table 3), having an EC50 between 1 and
10 mg/L. Carbamazepine, bezafibrate, sulfapyridine, oxytetracycline and novobiocin were all classified as harmful (EC50
between 10 and 100 mg/L) and sulfamethoxazole, trimethoprim and caffeine were considered non-toxic (EC50 N 100 mg/L)
(Table 3). These results are similar to other studies that
considered ibuprofen, naproxen, gemfibrozil and carbamazepine as high risk pollutants in WWTP effluents (Hernando et
al., 2006; Blaise et al., 2007). In the present study the toxicity
threshold value for ibuprofen was 320 g/L, only a factor of 102
or 10 higher than the concentration found in this effluent
(1.19 g/L) and in other Canadian effluents (22 g/L (Brun et al.,
2006)) respectively. In a previous study, exposure of Hydra to
carbamazepine for 48 h induced oxidative metabolism with an
increase in lipid peroxidation at a threshold concentration of
7.1 g/L (Quinn et al., 2004), similar to maximum concentration of 6.3 g/L reported in WWTPs (Ternes, 1998). These
results add to a growing volume being published indicating
that pharmaceuticals may be present in effluents entering the
environment at concentrations high enough to have chronic
effects. As these compounds enter the environment as a
mixture or cocktail of drugs it is possible that they have a
cumulative effect, with each compound behaving in an
additive fashion, contributing to the total effect of the mixture
and further increasing its toxicity. The toxicity of these
pharmaceutical mixtures is currently being investigated. As
Hydra occupy one of the lower trophic levels within freshwater
foodwebs, changes in their population could have an indirect
but significant effect on the rest of the freshwater community.
As mentioned above, the pharmaceuticals assessed in this
study comprise of 11 different compounds with different
chemical structures. This array of compounds produced
practical complications as each one had to be suspended in
solution up to relatively high concentrations (50/100 mg/L),
before exposure to Hydra. To achieve this we used 3 different
solvents (DMSO, acetone and ethanol) and tested each one on
our acute and chronic endpoints. All toxicity results for the
pharmaceuticals were compared against the solvent controls

and separate toxicity tests undertaken to ensure no solvent


effects. It is surprising to note that numerous publications
make no reference to what solvent was used for the dissolving
the pharmaceuticals. Pascoe et al. (2003) used up to 10%
ethanol which resulted in Hydra mortality. Other solvents
used include dimethyl sulfoxide for the suspension of
gemfibrozil (Zurita et al., 2007). Little difference in toxicity
was observed between the three solvents tested with no
significant effects observed at the concentrations used.

5.

Conclusion

It is generally thought that the aquatic environment is


particularly at risk to pollution by pharmaceuticals due to
their continued introduction into surface waters from
WWTPs. Although it is unlikely that these pollutants will be
found at concentrations high enough to illicit an acute effect,
evidence is mounting to suggest that they may be present in
sufficient concentrations to cause chronic effects. In the
present study the five most common pharmaceuticals (ibuprofen, naproxen, bezafibrate, gemfibrozil and carbamazepine) were found to be the most toxic to H. attenuata with EC50
results in the low mg/L level and toxicity threshold as low as
320 g/L. Based on environmental risk assessment gemfibrozil, ibuprofen and naproxen may be categorized as toxic
compounds, with their toxicity threshold only one or two
orders of magnitude above concentrations found in the
environment and may therefore be considered high risk
compounds in WWTP effluents, particularly as they may act
cumulatively in a mixture found in effluents. However the
current lack of chronic toxicity data for the majority of
pharmaceuticals is a major hindrance to their adequate risk
appraisal and needs to be addressed by the use of chronic
toxicity tests and the development of new chronic endpoints.

Acknowledgements
The authors are grateful to Jasmine Nahrgang and Pavleta
Pavlota for their help in conducting these experiments. This
research project was undertaken under the Saint-Lawrence
Centre's Municipal Effluent Research Program and partly

314

SC IE N CE OF T H E TOT AL E N V I RO N ME N T 3 8 9 ( 2 00 8 ) 3 0 63 14

funded by an NSERC visiting Fellowship in Canadian Government Laboratories.

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