Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae

Article abstract of DOI:10.1016/j.chemosphere.2004.07.017

The influence of antimicrobial agents approved as veterinary drugs in Japan on the growth of green algae, Selenastrum capricornutum and Chlorella vulgaris, was studied in accordance with the OECD guidelines for testing chemicals. Among the agents tested, growth inhibitory activity was very varied, i.e. erythromycin showed the strongest activity (EC₅₀, 50% effective concentration, =0.037 mg/l), sulfa drugs had activity to some extent (EC ₅₀s of sulfamethoxazole, sulfadiazine, and sulfadimethoxine were 1.5, 2.2, and 2.3 mg/l, respectively), but ampicillin and cefazolin did not inhibit growth (EC₅₀s > 1000 mg/l). We also investigated synergistic effect of combining sulfa drugs with trimethoprim or pyrimethamine, which are commonly used as a combined drug. By adding trimethoprim, the growth inhibitory activity of sulfamethoxazole and sulfadiazine was significantly enhanced. Growth inhibition by sulfa drugs was reduced by the addition of folic acid, indicating that they inhibit folate synthesis in green algae.

Full text of DOI:10.1016/j.chemosphere.2004.07.017

Chemosphere 57 (2004) 1733–1738
www.elsevier.com/locate/chemosphere
Evaluation of antimicrobial agents for veterinary use
in the ecotoxicity test using microalgae
a,
b
a
Kaoru Eguchi *, Hiroyasu Nagase , Manao Ozawa ,
a
Yuuko S. Endoh , Kisako Goto , Kazumasa Hirata ,
a
b
b
Kazuhisa Miyamoto , Haruo Yoshimura
a
a
National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries,
1-15-1 Tokura, Kokubunji-shi, Tokyo 185-8511, Japan
b
Environmental Biotechnology Laboratory, Graduate School of Pharmaceutical Science,
Osaka University, 1-6 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
Received 3 February 2003; received in revised form 27 November 2003; accepted 19 July 2004
Abstract
The influence of antimicrobial agents approved as veterinary drugs in Japan on the growth of green algae, Selena-
strum capricornutum and Chlorella vulgaris, was studied in accordance with the OECD guidelines for testing chemicals.
Among the agents tested, growth inhibitory activity was very varied, i.e. erythromycin showed the strongest activity
(EC50, 50% effective concentration, =0.037mg/l), sulfa drugs had activity to some extent (EC50s of sulfamethoxazole,
sulfadiazine, and sulfadimethoxine were 1.5, 2.2, and 2.3mg/l, respectively), but ampicillin and cefazolin did not inhibit
growth (EC50s > 1000mg/l). We also investigated synergistic effect of combining sulfa drugs with trimethoprim or pyri-
methamine, which are commonly used as a combined drug. By adding trimethoprim, the growth inhibitory activity of
sulfamethoxazole and sulfadiazine was significantly enhanced. Growth inhibition by sulfa drugs was reduced by the
addition of folic acid, indicating that they inhibit folate synthesis in green algae.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Algal toxicity; Combined drug; Synergistic effect; Folate
1. Introduction
metabolized, excreted and thereby released into the
environment. It has been reported that several antimi-
crobial agents are detected in the environment because
they are poorly metabolized in the body and/or de-
graded at ordinary sewage disposal plants (Halling-
Sørensen et al., 1998; Al-Ahmad et al., 1999). Such
antimicrobial agents can have a deleterious effect on
sensitive microorganisms in the field, which may cause
a disorder of the ecosystem. Therefore, the potential im-
pact of antimicrobial agents on the ecosystem must be
evaluated as closely as other hazardous chemicals. Since
Antimicrobial agents are widely used and indispensa-
ble not only for human medication, but also in the vet-
erinary field for livestock and companion animals.
Some antimicrobial agents administered to animals are
*
Corresponding author. Tel.: +81 42 321 1841; fax: +81 42
321 1769.
E-mail address: eguchi@nval.go.jp (K. Eguchi).
0045-6535/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.07.017

1734
K. Eguchi et al. / Chemosphere 57 (2004) 1733–1738
photoautotrophic microalgae are the primary producers
of essential nutrients in the ecosystem, toxicity against
these organisms is considered to be of particular
importance.
thesized as described below (Section 2.4). Other chemi-
cals (reagent grade) were purchased from Wako Pure
Chemical Industries, Ltd., Sigma Chemical Co., Kanto
Chemicals Co., Inc. (Tokyo, Japan), ICN Pharmaceuti-
cals, Inc. and Nacalai Tesque (Kyoto, Japan).
Environmental toxicity tests defined by the OECD
guidelines for testing chemicals are widely accepted
and performed and the ‘‘Alga, growth inhibition test’’
2.3. Procedure for the growth inhibitory test
(No. 201) is frequently used. For example, Luzhøft
¨
et al. (1999) and Halling-Sørensen (2000) have reported
that several antimicrobial agents used in Europe have
toxic effects on algae.
Growth inhibitory tests were performed following the
OECD guideline for testing chemicals No. 201 with min-
or modifications. In brief, a conical flask (200ml) con-
taining 100ml of distilled water was plugged with a
silicone-foam plug and autoclaved at 121ꢁC for
20min. Condensed medium solution sterilized by filtra-
tion (pore size: 0.2lm) was added and precultivated
algae cells were inoculated. The culture was maintained
at 25ꢁC, at 70% relative humidity, and illuminated at
5000 lx (about 11W/m2) from white fluorescent lamps
in a growth chamber (model ALWG modulated, Hira-
sawa Works, Tokyo, Japan). The flasks were shaken
and changed in position relative to each other 4 times
per day to equalize irradiating light intensity. Cell
growth was measured at 1 day intervals by an automatic
hematology analyzer (Celltac MEK-5133, Nihon Koh-
den Co., Tokyo, Japan).
In most cases, these toxicity tests are performed using
individual chemicals. However, two antimicrobial agents
are sometimes administered to animals simultaneously
as a combined drug. Therefore, it is likely that organ-
isms will be exposed to plural antimicrobial agents in
the ecosystem. For example, ST (a combination of a sul-
fur drug and trimethoprim) or a similar combined drug
(pyrimethamine is often used as a substitute for trimeth-
oprim) is widely used in veterinary medicine. It is ex-
pected that combined drugs would exhibit synergistic
influence beyond their individual influences.
In this study, growth inhibitory tests of several anti-
microbial agents approved as veterinary drugs in Japan
were carried out using algae in accordance with the
OECD guidelines. In addition, we investigated the syn-
ergetic effect of combined drugs consisting of sulfa drugs
and trimethoprim or pyrimethamine in the growth inhi-
bition test.
At first, ‘‘range-finding test’’, which consisted of three
concentrations of test substance in geometric series
(common ratio = 10) and negative control, was carried
out to determine the approximate EC50. Secondary,
the main test was carried out, in which negative control
and five concentrations of test substance in geometric
series (common ratio = 2) determined so that the above
EC50 value located in the center of the concentrationÕs
distribution by the consequence of range-finding test
was used. Each concentrations and negative control of
both tests were designed triplicate.
2. Materials and methods
2.1. Algal species
Selenastrum capricornutum (ATCC22662) and Chlo-
rella vulgaris (ATCC16487) were obtained from the
American Type Culture Collection (ATCC). Both
strains were maintained in recommended media. Before
the test, they were cultivated for at least 6 days in OECD
medium following the OECD-guideline No. 201.
2.4. Synthesis of N4-acethyl sulfa drugs
SAs were dissolved in 0.05mol/l sulfuric acid and ace-
tone (1/1), and chemical equivalent plus more of acetic
anhydride was added in drops with stirring. Occasion-
ally, products and source materials were analyzed by
thin layer chromatography [developing solvent: chloro-
form and methanol (20/1), plate: Silica Gel WF254s/alu-
minium plate (Merck, Darmstadt, Germany)]. After the
spot of source material disappeared under UV irradia-
tion, the reaction solution was neutralized by 1mol/l of
sodium hydroxide and concentrated to half the original
volume under reduced pressure. The precipitated crys-
tals were separated by filtration. The crystals were
recrystallized in methanol twice, and dried at 105ꢁC un-
der reduced pressure. The structure of the product was
confirmed by determining the nuclear magnetic reso-
nance spectrum (1H, 270MHz).
2.2. Chemicals
Sulfadimethoxine (SDM, sodium salt), trimethoprim
(TMP), pyrimethamine (PMT) were purchased from
Sigma Chemical Co. (St. Louis, MO). Sulfamethoxazole
(SMZ), oxytetracycline (OTC, hydrochloride salt),
cefazolin sodium salt (CFZ, sodium salt), ampicillin
(ABPC, sodium salt), thiamphenicol (TP), erythromycin
(EM), tylosin tartate (TST), and norfloxacin (NFLX)
were purchased from Wako Pure Chemical Industries,
Ltd. (Osaka, Japan). Sulfadiazine (SDA), dihydrostrep-
tomycin (DSM) were purchased from ICN Pharmaceu-
ticals, Inc. (Costa Mesa, CA). N4-acethyl sulfa drugs
(AcSAs) derived from each sulfa drugs (SAs) were syn-

K. Eguchi et al. / Chemosphere 57 (2004) 1733–1738
1735
2.5. Procedure for the inhibitory test of combined
substances and folic acid
0.01mol/l oxalic acid (pH = 3.0), methanol and acetonit-
rile (7/1/1.5) for OTC, 0.01mol/l KH2PO4 and acetonit-
rile (88/12) for ABPC and CFZ, water and acetonitrile
(85/15) for TP, water, methanol and acetic acid (60/40/
0.5) for SAs and AcSAs, 0.3% (v/v) of H3PO4 (adjusted
to pH = 3.0 by triethylamine) and acetonotrile (85/15)
for NFLX. The flow rate was 1.0ml/min. All detections
were performed by UV absorption, in which the wave-
length was 350nm for OTC, 210nm for ABPC and
CFZ, 240nm for TP, 270nm for SAs and AcSAs, and
278nm for NFLX.
The procedure for the growth inhibitory test of ST
combined substances was almost identical to that for sin-
gle substances. The excretion ratio of the test substances
was estimated from their excretion rate after intravenous
administration to pigs according to Shimoda et al. (1981,
1990, 1997), and by Nouws et al. (1991). The ratio was
SMZ:AcSMZ:TMP = 20:105:3, SDM:AcSDM:PMT =
167:8:1 and SDA:AcSDA:TMP = 42:24:1. Folic acid
was added at concentrations of 20 ng/l and 100 ng/l.
2.6. Statistical methods
3. Results and discussion
The Eco-Tox R1.1 program (composed by Yoshioka
et al.), distributed by the Japan Society of Environmen-
tal Toxicology, was used to analyze data. The area un-
der the growth curve at 72h was analyzed by the logit
(planimetry) method. Significance differences between
groups were determined using the StudentÕs T-test for
unpaired samples.
The growth inhibition test for individual antimicro-
bial agents was carried out using freshwater green algae,
S. capricornutum and C. vulgaris, which are used as
standard strains in the OECD test (Table 1). Our results
indicate that S. capricornutum was more sensitive in
general to antimicrobial agents than C. vulgaris. Kasai
and Hatakeyama (1993) and Kasai et al. (1993) also
found the same tendency in the sensitivity of these algae
to several herbicides. EM showed the strongest growth
inhibition (EC50 = 0.037mg/l) against S. capricornutum,
followed by DSM (0.11mg/l), OTC (0.34mg/l) and
TST (0.41mg/l). SAs also exhibited growth inhibition
to some extent (EC50 = 1.53–2.30mg/l) amongst the
agents tested. In contrast, CFZ and ABPC had no influ-
ence on the growth (EC50 > 1000mg/l). In other reports,
several herbicides have shown similar toxicities to
2.7. HPLC conditions
OTC determination was performed using a Chemco-
sorb 3C8 column (4.6 mm i.d. · 125mm L, Chemco Sci-
entific Co., Ltd., Osaka, Japan) and other determinations
using Capcell Pak C18 columns (UG120 S-5lm 4.6mm
i.d. · 250mm L, Shiseido Co., Ltd., Tokyo, Japan).
Column temperature was 40ꢁC. Mobile phase was
Table 1
The results of the growth inhibition test against green algae using antimicrobial agents approved for veterinary use in Japan
Substance
Selenastrum capricornutum
Chlorella vulgaris
EC50
(mg/l)
95% confidence
interval (mg/l)
NOEC
(mg/l)
EC50
(mg/l)
95% confidence
interval (mg/l)
NOEC
(mg/l)
Sulfadimethoxine (SDM)
Sulfamethoxazole (SMZ)
Sulfadiazine (SDA)
2.30
1.53
2.09–2.54
1.40–1.68
1.95–2.42
0.321–0.364
– –
0.529
0.614
<1.00
0.183
>1000
>1000
<0.039
4.06
11.2
–
7.37–15.0
–
<20.3
–
2.19
–
–
6.50–7.55
–
Oxytetracycline (OTC)
Cefazolin Na (CFZ)
Ampicillin (ABPC)
0.342
>1000
>1000
0.107
8.86
7.05
–
<3.58
–
–
– –
– –
>1000
–
>1000
–
Dihydrostreptomycin (DSM)
Thiamphenicol (TP)
Erythromycin (EM)
0.0933–0.121
8.27–9.50
0.0358–0.0399
0.387–0.437
15.5–17.8
74.4–86.7
4.66–5.50
0.150–0.170
–
489–580
31.3–36.4
522
33.8
–
198
12.5
–
0.0366
0.411
16.6
0.0103
0.206
4.01
Tylosin tartate (TST)
Norfloxacin (NFLX)
Trimethoprim (TMP)
Pyrimethamine (PMT)
Pentachlorophenol sodium salta
–
9.72–11.0
10.4
–
4.02
–
80.3
5.06
25.5
0.752
0.105
–
–
8.40
–
7.84–8.97
–
5.02
0.160
–: not tested.
– –: above the limit of detection.
EC50: 50% effective concentration, the concentration of a substance that will reduce cell growth to 50% of the negative control.
NOEC: maximum no-effect concentration.
a
Positive control.

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K. Eguchi et al. / Chemosphere 57 (2004) 1733–1738
140
120
100
80
S. capricornutum and C. vulgaris as the compounds
tested in this study. For example, the EC50s of symetryn,
pretilachlor, and thiobencarb for S. capricornutum were
10, 2.5, and 38.6lg/l, respectively (Kasai et al., 1993).
Based on the mechanism of action of most herbicides,
it is possible that they will also inhibit the growth of
green algae in the environment. Therefore, the anitimi-
crobial agents showing growth inhibitory activity in this
study, particularly EM, would be expected to cause
damage to green algae in the ecosystem if they were re-
leased into the field at concentrations similar to their
EC50 values. EM inhibits the growth of prokaryotes by
binding to the 50Sribosome, thereby inhibiting trans-
peptidation. However, this mechanism of action will
not inhibit the growth of eukaryotes, so the inhibition
of the growth of green algae by EM cannot be satisfac-
torily explained by the known mechanisms of action of
this compound.
SMZ
TST
OTC
SDA
SDM
NFLX
PMT
TMP
TP
60
40
20
0
0-24
24-48
48-72
Cultivation time (hour)
Fig. 2. Variation of the growth rate. Each test substance was
used at the EC50 concentration. Growth rate was determined as
increase in cell number/h.
Recently, the use of SAs in human medicine has been
reduced because more effective antibiotics are available.
However, SAs are still widely used in veterinary medi-
cine and the livestock industry because of their low cost.
The results presented in this study indicate that excessive
use of SAs in livestock may pose a risk to green algae in
the environment. However, this risk may be reduced as
SAs are metabolized in animals to AcSAs, which were
found to have a much weaker growth inhibitory effect
than that of the parent SAs (Table 3).
(1991) reported that tetracycline and its analogues,
including OTC, are easily oxidized in the presence of
oxygen and light, conditions that accord with those of
the culture medium of the algae used in this study.
Fig. 2 shows the change in the growth rate of S. cap-
ricornutum in the presence of the test agents. Most
agents gave a ‘‘V-shaped’’ curve, indicating that the
growth was inhibited strongly during the middle of the
incubation period (1–2 days). In contrast, OTC and
NFLX inhibited growth early during the incubation per-
iod (0–1 day), allowing growth of the algae to subse-
quently recover. Exceptionally, TST strongly inhibited
growth during the late incubation period (2–3 days).
OTC showed strong growth inhibition for S. capri-
cornutum and C. vulgaris despite of its rapid disappear-
ance. OTC inhibited specifically early in the incubation
period, suggesting that growth inhibition by OTC oc-
curred when its concentration was at a relatively higher
level. Therefore, it is more likely that OTC will cause
damage to algae when it is released continuously into
the environment.
The concentrations of the antimicrobial agents in the
culture medium during incubation with S. capricornutum
were measured. As shown in Fig. 1, a decrease in OTC,
TST, ABPC and CFZ was observed, although other
agents did not decrease significantly. In particular,
OTC decreased most rapidly and was not detectable
within the media within 2 days. Asker and Habib
12
TST
CEZ
10
ABPC
OTC
8
To investigate the synergistic influence of combined
drugs on the growth of green algae, SAs and TMP or
PMT (TMPs) were simultaneously added to S. capri-
cornutum culture. In this experiment, the concentration
of TMPs was fixed at the no observed effect concentra-
tion (NOEC) and the concentration of the SAs were al-
tered. These combined drugs are frequently used in the
veterinary field in Japan. Combination of SMZ and
SDA with TMP rendered the growth inhibitory activity
significantly increased in comparison with their individ-
ual activities (Table 2, Fig. 3A–C). On the other hand,
combination of SDM with PYR did not show such an
effect.
6
4
2
0
0
1
2
3
Cultivation time (day)
Fig. 1. Decrease in concentration of test agents in the growth
inhibition test. Other test substances did not show a significant
decrease during the course of the experiment.

K. Eguchi et al. / Chemosphere 57 (2004) 1733–1738
1737
Table 2
Synergistic growth inhibition between SA and TMP or PMT
binations above mixed with the corresponding AcSAs at
a ratio according to the concentrations detected in the
urine of pigs fed with SAs. A similar synergistic effect
to that described above (Table 2 and Fig. 3) was ob-
served with combinations of SMZ, TMP, and AcSMZ
(Table 3). On the other hand, combination of SDM or
SDA with their acetylate and PMT or TMP did not
show a synergistic effect on growth in excretion ratio.
A reason must be that the concentration of TMP used
was not enough to express synergistic influence in com-
bination with SDM or SDA. These results indicate that
several combined drugs that show a synergistic effect in
vitro may have an actual synergistic effect on algae in
ecosystem although excretion ratio can vary in animal
condition or other factors. The synergistic effect ob-
served by the combination of SAs and TMPs in this
study indicates that the simultaneous release of several
antimicrobial agents may result in greater toxicity to
microorganisms in the environment than the release of
the same agents individually.
Substances
EC50
(mg/l)
95% confidence
interval (mg/l)
NOEC
(mg/l)
SMZ
SDA
SDM
TMP
PMT
1.53
2.19
2.30
80.3
5.06
1.40–1.68
1.95–2.42
2.09–2.54
74.4–86.7
4.66–5.50
0.614
<1.00
0.529
25.5
0.752
SMZ + TMPa
SDA + TMPa
SDM + PMTa
0.275b
0.465b
2.36b
0.239–0.309b
0.419–0.517b
2.17–2.57b
0.2b
<0.125b
1.00b
TMP and PMT were used at their minimum no-effect observed
concentrations (NOEC) and the concentration of SA was
varied.
a
TMP and PMT were used at their NOECs.
Concentration of SA in each combination.
b
The growth inhibitory activity of the combination of
SDA and TMP was significantly reduced by the addition
of 20 ng/l of folic acid to the medium. Significantly, folic
acid exhibited a similar effect when SDA was tested
alone, but not when TMP was tested alone (Fig. 4). Both
SAs and TMPs inhibit the folate synthesis pathway in
bacteria, but their inhibition sites are different. SAs inhi-
bit dihydropterinic acid synthetase (DHPS), thereby
inhibiting the synthesis of folic acid. On the other hand,
TMPs inhibits dihydrofolic acid reductase (DHFR),
which converts folic acid to 7,8-dihydrofolic acid (7,8-
DHF) and 5,6,7,8-tetrahydrofolic acid (5,6,7,8-THF),
both active forms of folic acid suitable for utilization.
Therefore, the synergistic effect of the combination of
SAs and TMPs is likely to be due to the cumulative effect
of their actions on two different sites in the folate biosyn-
thesis pathway. Since SAs block the synthesis of folate,
the growth inhibitory effect of this compound can be re-
versed by the addition of folate. In contrast, TMP
blocks enzymes downstream of folate in the synthesis
pathway, thus addition of folate will not reverse the
growth-inhibiting effect of this compound. Since algae
Fig. 3. The dose–response curve of sulfur drugs combined with
TMP or PMT (A: SMZ, B: SDM, C: SDA).
SAs are thought to be partly metabolized to AcSAs
in the bodies of animals (Vree et al., 1985). Therefore,
the test of combined drugs was done by using the com-
Table 3
Synergistic growth inhibition by the ST combined drug estimated to be excreted by pigs
Substance and mixing ratio
(SA:AcSA:TMPs)
EC50
(mg/l)
95% confidence
interval (mg/l)
NOEC
(mg/l)
SMZ:AcSMZ:TMP (20:105:3)
SDM:AcSDM:PMT (167:8:1)
SDA:AcSDA:TMP (42:24:1)
0.784a
2.17a
2.08a
0.717–0.856a
1.95–2.40a
1.88–2.33a
0.2a
0.5a
0.5a
AcSMZ
AcSDM
AcSDA
>100
>100
>100
–
–
–
80
>100
>100
–: above the limit of detection.
a
Concentration of SA in each combination.

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K. Eguchi et al. / Chemosphere 57 (2004) 1733–1738
Acknowledgments
We are grateful to Dr. Fumie Kasai, National Insti-
tute for Environmental Studies, and Dr. Hotaka Saito,
Mitsubishi Chemical Safety Institute Ltd. for their ad-
vice, and Dr. Hatsuo Maeda, Graduate school of Phar-
maceutical Sciences, Osaka University for synthesizing
the AcSAs.
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