1. Effects of six selected antibiotics on plant growth and soil microbial and
enzymatic activities
Feng Liu a
, Guang-Guo Ying a,*, Ran Tao a
, Jian-Liang Zhao a
, Ji-Feng Yang a
, Lan-Feng Zhao b
a
State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, 511 Kehua Street, Tianhe District, Guangzhou 510640, China
b
College of Resource and Environmental Science, South China Agricultural University, Guangzhou 510642, China
Terrestrial ecotoxicological effects of antibiotics are related to their sorption and degradation behavior in soil.
a r t i c l e i n f o
Article history:
Received 20 July 2008
Received in revised form
15 December 2008
Accepted 17 December 2008
Keywords:
Antibiotics
Phytotoxicity
Soil microbial activity
Respiration
Phosphatase
Behavior
a b s t r a c t
The potential impact of six antibiotics (chlortetracycline, tetracycline and tylosin; sulfamethoxazole,
sulfamethazine and trimethoprim) on plant growth and soil quality was studied by using seed germi-
nation test on filter paper and plant growth test in soil, soil respiration and phosphatase activity tests.
The phytotoxic effects varied between the antibiotics and between plant species (sweet oat, rice and
cucumber). Rice was most sensitive to sulfamethoxazole with the EC10 value of 0.1 mg/L. The antibiotics
tested inhibited soil phosphatase activity during the 22 days’ incubation. Significant effects on soil
respiration were found for the two sulfonamides (sulfamethoxazole and sulfamethazine) and trimeth-
oprim, whereas little effects were observed for the two tetracyclines and tylosin. The effective concen-
trations (EC10 values) for soil respiration in the first 2 days were 7 mg/kg for sulfamethoxazole, 13 mg/kg
for sulfamethazine and 20 mg/kg for trimethoprim. Antibiotic residues in manure and soils may affect
soil microbial and enzyme activities.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Tons of pharmacologically active substances are used annually
in human and animal medicines for treatment and prevention of
illness (Dı´az-Cruz et al., 2003; Sarmah et al., 2006). Antibiotics are
specifically designed to control bacteria in human or animals and
help to protect their health. After treatment, most antibiotics are
excreted from the treated body, either unaltered or as metabolites,
some of which are still bioactive (Sarmah et al., 2006). Obviously
this makes them potentially hazardous to bacteria and other
organisms in the environment (Baguer et al., 2000). Different types
of drugs have different anticipated exposure routes to the envi-
ronment (Jørgensen and Halling-Sørensen, 2000). The dominant
pathway for antibiotic release in the terrestrial environment is via
the application of animal manure and biosolids containing excreted
antibiotics to agricultural land as fertilizer (Jørgensen and Halling-
Sørensen, 2000; Dı´az-Cruz et al., 2003, 2006; Golet et al., 2003;
Go¨bel et al., 2005; Kemper, 2008). Antibiotics can also be intro-
duced to agricultural land through irrigation with reclaimed
wastewater, since they have been frequently detected in the raw
and treated sewage wastewaters (Renew and Huang, 2004; Yang
et al., 2005; Gulkowska et al., 2008). Therefore, it is necessary to
understand the environmental impact of antibiotics associated
with application of animal manure, biosolids and wastewater on
agricultural land.
Unlike pesticides used on agricultural land, antibiotics have not
aroused attention as potential pollutants until fairly recently
(Halling-Sørensen et al., 1998; Ku¨mmerer, 2001). Bacterial resis-
tance has been a big issue in terms of human and animal health;
however, antibiotic ecotoxicological relevance is scarcely known
because the potential effects of antibiotics in the environment are
very limited (Pang et al., 1994; Rooklidge, 2004).
When antibiotics get into the arable land, they could possibly
impact vegetation growth and development as well as soil micro-
bial activity (Jjemba, 2002a,b). Phytotoxicity of a chemical can be
assayed using seed germination and plant growth tests. Limited
studies have been conducted to investigate the phytotoxicity of
some antibiotics (e.g. sulphadimethoxine, enrofloxacin and
oxytetracycline) to crop plants (Migliore et al., 1998, 2003; Kong
et al., 2007). The effects of antibiotics on plants in soils were found
different between compounds and between plant species (Batch-
elder, 1982; Jjemba, 2002a; Farkas et al., 2007). Tetracyclines
increased radish yields, but decreased pinto bean yields (Batch-
elder, 1982). When grown in chlortetracycline-treated soil,
a significant increase in the activities of the plant stress proteins
glutathione S-transferases and peroxidases was observed in maize
plants, but not in pinto beans (Farkas et al., 2007).
* Corresponding author. Tel./fax: þ86 20 85290200.
E-mail address: guangguo.ying@gmail.com (G.-G. Ying).
Contents lists available at ScienceDirect
Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol
0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2008.12.021
Environmental Pollution 157 (2009) 1636–1642

2. As antibiotics are designed to be biologically active toward
microorganisms, it would be interesting to understand the poten-
tial effects on soil microbial activity. However, previous reports on
the effects of pharmaceutical antibiotics on soil microorganisms are
scarce and inconsistent (Patten et al., 1980; Thiele-Bruhn and Beck,
2005; Kong et al., 2006; Zielezny et al., 2006; Kotzerke et al., 2008).
A number of soil microbiological parameters, including microbial
biomass carbon and basal respiration, have been suggested as
possible indicators of soil environmental monitoring programs (Yao
et al., 2000; Winding et al., 2005).
In the present study, two classes of antibiotics were chosen to
study the effects on plant growth and soil microbial activity. The
antibiotics used in the study were: tetracyclines (chlortetracycline
and tetracycline, as well as tylosin commonly used in combination
with tetracyclines) and sulfonamides (sulfamethoxazole and sul-
famethazine, as well as trimethoprim commonly used in
combination with sulfonamides). The phytotoxicity was assayed
using seed germination tests on filter paper and plant growth tests
in soil. Soil microbial activity was assessed by measuring soil
microbial respiration and phosphatase activity.
2. Materials and methods
2.1. Chemicals
Chlortetracycline (98% purity), tetracycline (98% purity), tylosin (90% purity), sulfa-
methazine (98% purity), sulfamethoxazole (98% purity), and trimethoprim (96% purity)
were purchased from DeBioChem Reagents & Instruments Co. Ltd. (Nanjing, China). All
the reagents used in the following tests were purchased from Qianhui Reagents &
Instruments Co. Ltd. (Guangzhou, China) and they were of analytical grade.
2.2. Seeds and soil
Seeds of rice (Oryza sativa L.) were obtained as a gift from South China Agri-
cultural University, while seeds of cucumber (Cucumis sativus L.) and sweet oat
(Cichaorium endivia) were purchased from Seeds Collection, Guangdong Academy of
Agricultural Sciences, China. Preliminary incubation showed that all the seeds used
in this study had more than 90% germination rates.
An agricultural soil (0–20 cm deep) was collected from a rice paddy in an
experimental station, Guangdong Academy of Agricultural Sciences, China. The soil
type is classified as Anthrosol based on its properties. The soil was air-dried until
water content reached about 20% of the maximum water-holding capacity (MWHC).
After removal of large pieces of plant materials and soil animals by screening
through a 2 mm sieve, the soil was mixed well and stored at 4 C until use. The soil
had a silt loam texture with a pH value of 5.7 and total carbon content of 18.2 g/kg,
total nitrogen content of 0.959 g/kg, and total phosphate content of 0.215 g/kg.
2.3. Seed germination test
Laboratory tests to evaluate the effects of antibiotics on seed germination of
three plants (rice, cucumber and sweet oat) were carried out using the filter paper
method according to the International Seed Testing Association (ISTA) test protocols
(ISTA,1985). After having been sterilized using 0.1% NaClO and pretreated by soaking
in distilled water for six hours, seeds of cucumber (15), rice (20) and sweet oat (20),
which depended on the size of the seeds, were placed on a filter paper (9 cm
diameter) kept in each Petri dish (10 cm diameter).
For each antibiotic compound, the filter papers in Petri dishes were treated with
5 mL of the antibiotic solution at different concentrations and covered before placing
in an incubator. Seeds were germinated in the incubator under the conditions of 25 C
temperature, 80% humidity and darkness. The seed germination was evaluated using
root length of seedlings as endpoint (primary root ! 5 mm) after 4–5 days (Tiquia
et al., 1996). Except trimethoprim, each antibiotic test had 8 treatments with chlor-
tetracycline concentrations of 0 (CK), 0.1, 1, 10, 50, 100, 200, 500 mg/L, tetracycline
concentrations of 0 (CK), 0.1, 1, 10, 30, 50, 100, 300 mg/L, sulfamethazine or sulfa-
methoxazole concentrations of 0 (CK), 1, 10, 30, 50, 70, 100, 300 mg/L and tylosin
concentrations of 0 (CK),1,10, 30, 50,100, 300, 500 mg/L. The trimethoprim test had 9
treatments with concentrations of 0 (CK), 5% acetone/water solution,1,10, 30, 50,100,
300, 500 mg/L. Each treatment including controls was carried out in three replicates.
2.4. Plant growth test
The effects of the antibiotics on plant growth were assayed in a silt loam soil
using the method modified from the literature (OECD, 1984; Batchelder, 1982;
Baguer et al., 2000). Chlortetracycline and tylosin were directly added in an aqueous
solution to the soil, while the other antibiotics were added by spiking into fine
quartz sand due to their low water solubility. The tests had 8 treatments with
concentrations of chlortetracycline, tetracycline, sulfamethazine, sulfamethoxazole
and trimethoprim at 0 (CK), 1, 10, 30, 50, 70,100 and 300 mg/kg in soil, and tylosin at
0 (CK), 1, 10, 30, 50, 100, 300, 500 mg/kg in soil. Each treatment including controls
was carried out in triplicates. The detailed procedure was described briefly as
follows. The test was conducted in plastic cups (150 mL with a diameter of 7.5 cm).
Each antibiotic was spiked into 100 g of the test soil in each cup in dark, and the soil
was mixed in a shaker for 12 h. Two plant seeds cucumber and rice were chosen in
the phytotoxicity tests. All these seeds were treated in the same way as the seeds
used in the filter paper method. Into each plastic cup 10 seeds of rice or 8 seeds of
cucumber were sown at a depth of 0.5 cm. Then the soil moisture in each plastic cup
was adjusted to 50% of maximum water-holding capacity (MWHC). The treated
plastic cups were placed in a climate chamber at a temperature of 25 C and
humidity of 80% under darkness. After the seeds in the cups were all germinated, the
test conditions of the chamber were changed to a controlled photoperiod (12 h
light:12 h dark). Seven days later following seed germination, plant seedlings were
thinned to five rice plants or four cucumber plants per cup. During the test period,
the soil water moisture was maintained everyday by adding appropriate amount of
water. The plants in the cups were harvested at the 20th day and their shoot and root
lengths were measured.
2.5. Soil respiration and phosphatase activity tests
Soil respiration and phosphatase activity were assayed and used as the indica-
tors of soil microbial activity. The effect of antibiotics on soil microbial respiration
was assayed by the direct absorption method using sodium hydroxide (Wang et al.,
2005; Diao et al., 2006; Yao et al., 2006). The test had 8 treatments with sulfame-
thazine or sulfamethoxazole concentrations of 0 (CK), 1, 10, 40, 70 and 100 mg/kg in
soil and other antibiotics concentrations of 0 (CK), 1, 10, 40, 70, 100 and 300 mg/kg in
soil. Each treatment was conducted in triplicates. The experimental procedure is
described briefly as follows. Each antibiotic was spiked to the test soil (50 g) in each
cup, and 1 mL of 0.1 M glucose solution was also added. Then 10 mL of pure water
was added to obtain soil moisture level at 25% MWHC. The spiked soils were mixed
and left overnight to be acclimatized in the fume hood. After the soil moisture was
adjusted to 60% MWHC, the plastic cups were put into 1 L air-tight plastic jars with
a little cup holding 20 mL of 0.15 N sodium hydroxide in the bottom of each jar and
incubated at 25 C in the darkness. Two blanks without soil but with 20 mL of 0.15 N
sodium hydroxide were also included in the test. The CO2 was determined by
titration of the NaOH solution. At different time intervals (2, 4, 6, 9, 12, 16 and 22
days), the sodium hydroxide in each jar was titrated with 0.1 N hydrochloric acid and
a new 20 mL of 0.15 N sodium hydroxide was placed in the jar. The intensity of soil
respiration was calculated by the following formula: Respiration value (mgCO2 gÀ1
dry soil) ¼ (blank-titer) Â 0.1 Â44/50, where (blank-titer) in the formula is the blank
titration volume of hydrochloric acid in the treatment without soil subtract the
titration volume of hydrochloric acid in the treatments with soil, 0.1 means
concentration of hydrochloric acid, and 50 means weight of dry soil.
Phosphatase activity was assayed using 0.1 M acetate buffer (pH 5) and 0.5%
disodium phenyl phosphate substrate according to the method described by Guan
(1983). In this acidic phosphatase activity test, the soils were treated in the same
way as in the soil respiration test and incubated at 25 C in the darkness. At different
time intervals (2nd, 5th, 9th, 14th, 19th and 23rd days following treatment), 3 g of
soil was randomly sampled from each container to measure the soil phosphatase
activity. Phosphatase activity was expressed as mg phenol per kg of soil within 1 h
incubation time.
2.6. Extraction and analysis
Antibiotic residues in the soil during the soil respiration and enzymatic tests
were monitored by using high performance liquid chromatography with a diode-
array detector (HPLC-DAD). Sulfamethazine, sulfamethoxazole, trimethoprim and
tylosin in soil samples were extracted with acetonitrile for three times using soni-
cation, while chlortetracycline and tetracycline were extracted with 90% methanol
with 0.8 M oxalic acid and 0.85 M citric acid. After extraction, the extracts were
reconstituted in the initial mobile phase solution. The injection volume was 20 mL
and the column temperature was set at 30 C. Different mobile phases and gradient
programs were applied for the six antibiotics. For the two sulfonamides, acetonitrile
and 0.1% formic acid aqueous solution were used as mobile phase at a flow rate of
0.75 mL/min: 15% at 0 min to 60% of acetonitrile at 10 min, back to 15% at 12 min
which was kept for 3 min. The ultraviolet wavelength (UV) was set at 270 nm. For
trimethoprim, acetonitrile and Milli-Q water were used as mobile phase at a flow
rate of 0.75 mL/min: 30% at 0 min to 60% of acetonitrile at 10 min, back to 30% at
12 min which was kept for 5 min. The UV wavelength for trimethoprim was 230 nm.
For tylosin, acetonitrile and 0.08% acetic acid solution (9 mM ammonium acetate)
were used as mobile phase at a flow rate of 1 mL/min: 35% at 0 min to 60% of
acetonitrile at 15 min, further to 90% at 16 min, back to 35% at 17 min which was
kept for 5 min. The UV wavelength for tylosin was 285 nm. For the two tetracyclines,
acetonitrile and 10 mM oxalic acid solution were used as mobile phase at 0.6 mL/
min: 25% of acetonitrile at 0–8 min, increase to 90% at 10 min, back to 25% at 12 min
F. Liu et al. / Environmental Pollution 157 (2009) 1636–1642 1637

3. which was kept for 5 min. The UV wavelength was set at 370 nm for chlortetracy-
cline and tetracycline.
The instrumental detection limits were 0.30, 0.23, 4.71, 12.6, 14.1 and 66.5 mg/L
for sulfamethazine, sulfamethoxazole, trimethoprim, chlortetracycline, tetracycline
and tylosin, respectively; while their recoveries at the spiking concentration of
10 mg/kg were 99%, 96%, 72%, 70%, 73% and 77%, respectively.
2.7. Data analysis
Unless specified, all reported data were compared by using Duncan’s new
multiple range test at the 5% level. Differences between values at p 0.05 were
considered statistically significant. EC50 values (the concentration causing 50%
effect) as well as EC10 values (the concentration causing 10% effect) of the tested
antibiotics were calculated by plotting logged concentrations versus seed germi-
nation rate or plant growth endpoints (shoot height and root length) by using EC50
calculator program developed by CSIRO, Australia.
After the data had been tested for normality and homogeneity of variance, the
no-observed-effect concentration (NOEC, highest concentration to cause no signif-
icant effect) and the lowest-observed-effect concentration (LOEC, lowest concen-
tration to cause a significant effect) were estimated by SAS 8.2 using Dunnett’s
multiple comparison test to determine which treatments differed significantly from
the controls (1-tailed, p 0.05).
3. Results and discussion
3.1. Phytotoxicity of antibiotics
In seed germination tests, root length instead of number of
germinated seeds was used as the endpoint in statistical analysis,
which is consistent with the approaches used by previous studies
for metals and organic contaminants (Mishra and Choudhuri, 1999;
Martı´ et al., 2007). Table 1 lists EC10, EC50, NOEC and LOEC values
for the antibiotics tested on three plant seeds (sweet oat, rice and
cucumber). The results (EC50 values) showed that sweet oat and
rice seeds presented more susceptibility to the antibiotics, while
cucumber seeds were less sensitive to all antibiotics. Rice was most
sensitive to sulfamethoxazole with the EC10 value of 0.1 mg/L.
Chlortetracycline and tetracycline inhibited germination of the
three plant seeds with EC50 values less than 300 mg/L. Tylosin was
the least toxic compound, especially toward rice and cucumber
seeds with EC50 values more than 300 mg/L. Sulfamethoxazole and
sulfamethazine also inhibited seed germination of the three plants
with the EC50 values for the two sulfonamides being less than
100 mg/L.
The seed germination tests demonstrated that antibiotics could
negatively affect plant seed germination, but the effects varied
between the plant species and between the antibiotics used in the
tests. Among the three plants, sweet oat was the most sensitive
plant to the six antibiotics although with varying toxicity values.
Tetracyclines and sulfonamides were more toxic to plant seed
germination while tylosin and trimethoprim were less toxic to seed
germination.
In plant growth tests, only sulfonamides (sulfamethoxazole and
sulfamethazine) strongly affected rice growth in soil (Table 2). No
obvious rice growth inhibition was observed when treated with the
other antibiotics. This is in contrast with the results from the seed
germination tests, which showed inhibitory effects by tetracyclines
and sulfonamides. The results from the present study are consistent
with those of previous studies (Batchelder, 1982; Norman, 1955).
Norman (1955) found that root growth of several crops was
inhibited by 5–10 mg/L of oxytetracycline in solution, but the
effects were not observed in soil. The lesser inhibitory effects of
tetracyclines in the soil than in the solution might be attributed to
their strong adsorption onto soil components (clay and organic
matter) (Tolls, 2001; Figueroa et al., 2004; Kulshrestha et al., 2004;
Figueroa and Mackay, 2005; Mackay and Canterbury, 2005; Pils and
Laird, 2007). Sorption coefficients of sulfonamides are very low in
soil (Boxall et al., 2002), which indicates that sulfonamides are
more bioavailable.
As found in the seed germination tests, cucumber was less
sensitive to the antibiotics than rice in terms of plant growth in soil
(Table 2). For sulfonamides, the EC50 values for rice were less than
Table 1
Toxicity data from seed germination tests for sweet oat, rice and cucumber (endpoint:root length).
Compound EC10 EC50 NOEC LOEC
oat rice cucumber oat rice cucumber oat rice cucumber oat rice cucumber
Chlortetracycline 0.2a
8 0.7 16 39 48 0.1 1 0.1 0.1 10 1
Tetracycline 14 16 8 57 69 203 1 1 1 10 10 10
Tylosin 19 500 217 141 500 500 1 500 100 10 500 300
Sulfamethoxazole 16 0.1 300 69 8 300 1 0.1 300 10 1 300
Sulfamethazine 2 6 6 37 45 300 0.1 1 1 1 10 10
Trimethoprim 24 23 2 86 118 300 1 0.1 1 1 1 10
a
All concentrations are in mg/L.
Table 2
Toxicity data from plant growth tests in soil for rice and cucumber.
Compound Endpoint EC10 EC50 NOEC LOEC
rice cucumber rice cucumber rice cucumber rice cucumber
Chlortetracycline Seedling height 300a
19 300 300 300 100 300 300
Root length 300 300 300 300 300 70 300 100
Tetracycline Seedling height 300 300 300 300 300 300 300 300
Root length 300 300 300 300 300 300 300 300
Tylosin Seedling height 500 90 500 343 500 50 500 100
Root length 500 35 500 500 500 50 500 100
Sulfamethoxazole Seedling height 25 85 38 300 30 100 50 300
Root length 2 66 13 300 1 100 10 300
Sulfamethazine Seedling height 92 249 220 300 70 100 100 300
Root length 1 120 43 300 1 100 10 300
Trimethoprim Seedling height 300 0.7 300 300 300 1 300 10
Root length 300 85 300 300 300 100 300 300
a
All concentrations are in mg/kg dry soil weight.
F. Liu et al. / Environmental Pollution 157 (2009) 1636–16421638

4. 300 mg/kg whereas the EC50 values for cucumber were all near or
more than 300 mg/kg. Similar results of sulfonamides’ effects on
cucumber were observed by Migliore et al. (1998). Sulphame-
thoxine at a concentration of 300 mg/kg significantly depressed the
growth of Amaranthus restroflexus, Plantago major, Rumex acetosella,
and Zea mays in vitro, as well as Hordeum disthicum both in vitro
and in soil (Migliore et al., 1998). Species variability was also found
in previous studies (Batchelder, 1982; Farkas et al., 2007). The
growth of radish and wheat was enhanced in the presence of
chlortetracycline and oxytetracycline whereas the growth of corn
was unaffected by these antibiotics (Batchelder, 1982). Chlortetra-
cycline was found to significantly increase the activities of the plant
stress proteins glutathione S-transferases and peroxidases in maize
plants, but not in pinto beans (Farkas et al., 2007).
The concentration of sulfonamides in manure ranged between
10 mg/kg and 91 mg/kg (Pfeifer et al., 2002; Christian et al., 2003;
Jacobsen and Halling-Sørensen, 2006; Martı´nez-Carballo et al., 2007).
In sludge, sulfonamides were also detected with concentrations even
up to 197 mg/kg for sulfapyridine and 73 mg/kg for sulfamethoxazole in
Swiss wastewater treatment plants (Go¨bel et al., 2005; Dı´az-Cruz et al.,
2006). Sukul and Spiteller (2006) proposed that with manure slurry
being applied in the field as fertilizer with a maximum dose rate of
50 m3
/ha,sulfonamideresiduesinsoilcouldreach1 kg/ha,whichisthe
same order of magnitude as the application rate of modern pesticides.
Trimethoprim is used as a synergist to sulfonamides and was detected
with concentrations upto 17 mg/kg in chicken and turkey dung but not
in pig manure (Martı´nez-Carballo et al., 2007). In pig manure, up to
46 mg/kg chlortetracycline, 29 mg/kg oxytetracycline and 23 mg/kg
Chlortetracycline
*
**
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 ˜ 2 2 ˜ 4 4 ˜ 6 6 ˜ 9 9 ˜ 12 12 ˜16 16 ˜ 21
Incubation time (day)
Soilrespiration
(CO2mg/gdrysoil)
0 1 10 40 70 100 300
Tetracycline
*
*
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 ˜ 2 2 ˜ 4 4 ˜ 6 6 ˜ 9 9 ˜ 13 13 ˜ 17 17 ˜ 22
Incubation time (day)
Soilrespiration
(CO2mg/gdrysoil)
Tylosin
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 ˜ 2 2 ˜ 4 4 ˜ 6 6 ˜ 9 9 ˜ 12 12 ˜ 16 16 ˜ 21
Incubation time (day)
Soilrespiration
(CO2mg/gdrysoil)
0 1 10 40 70 100 300
0 1 10 40 70 100 300
Fig. 1. Effects of chlortetracycline, tetracycline and tylosin on soil respiration measured as the cumulative CO2 generated within different incubation periods. The error bars are the
standard deviation (n ¼ 3). The asterisk (*) indicates a significant difference compared to the controls without addition of antibiotics (p 0.05).
F. Liu et al. / Environmental Pollution 157 (2009) 1636–1642 1639

5. tetracycline were reported in Austria (Martı´nez-Carballo et al., 2007).
Tylosin was not detected in swine manure and may have been
degraded during mixing of the manure (Jacobsen and Halling-Sør-
ensen, 2006). Based on the NOEC values in Table 2, only sulfonamides
may affect growth of the plants, especially rice.
3.2. Antibiotic effects on soil microbial and enzyme activity
Fig. 1 shows little effects of tetracyclines and tylosin on soil
microbial respiration, with statistically significant variations only
observed at the higher concentration levels. In contrast,
sulfonamides and trimethoprim were found to cause significant
decreases in soil respiration within the first 4 days (Fig. 2). Kotzerke
et al. (2008) also observed reduced microbial activity by antibiotic
sulfadiazine in manure for up to 4 days after manure application.
Soil respiration measured as CO2 decreased significantly with
increasing concentrations of sulfamethoxazole and sulfamethazine
as well as trimethoprim in the soil. The effective concentrations
(EC10 values) in the first 2 days were calculated to be 7 mg/kg for
sulfamethoxazole, 13 mg/kg for sulfamethazine and 20 mg/kg for
trimethoprim. Increased soil respiration activity with antibiotic
concentrations was observed for the two sulfonamides and
Sulfamethazine
*
* *
*
*
* **
*
*
*
*
*
* * *
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Incubation time (day)
Soilrespiration
(CO2mg/gdrysoil)
Sulfamethoxazole
*
*
*
* * *
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Incubation time (day)
Soilrespiration
(CO2mg/gdrysoil)
Trimethoprim
*
*
**
*
*
*
**
*
*
**
*
*
*
*
*
*
*
*
*
* *
*
* *
* *
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
9 ˜ 13 13 ˜ 17 17 ˜ 22
Incubation time (day)
Soilrespiration
(CO2mg/gdrysoil)
300
0 ˜ 2 2 ˜ 4 4 ˜ 6 6 ˜ 8 8 ˜ 12 12 ˜16 16 ˜ 21
0 ˜ 2 2 ˜ 4 4 ˜ 6 6 ˜ 8
0 ˜ 2 2 ˜ 4 4 ˜ 6 6 ˜ 9
8 ˜ 12 12 ˜16 16 ˜ 21
0 1 10 40 70 100
0 1 10 40 70 100
0 1 10 40 70 100
Fig. 2. Effects of sulfamethazine, sulfamethoxazole and trimethoprim on soil respiration measured as the cumulative CO2 generated within different incubation periods. The error
bars are the standard deviation (n ¼ 3). The asterisk (*) indicates a significant difference compared to the controls without addition of antibiotics (p 0.05).
F. Liu et al. / Environmental Pollution 157 (2009) 1636–16421640

6. trimethoprim at certain stages after the first 4 days. In the later
incubation periods, a decreasing respiration activity was followed
in comparison with the activity in the first few days. This indicates
that the effect of these antibiotics (sulfamethoxazole, sulfametha-
zine and trimethoprim) on soil microbial respiration was time
dependent. The increased soil respiratory activity was also reported
in the previous studies (Fru¨ nd et al., 2000; Ingerslev and Halling-
Sørensen, 2000; Halling-Sørensen et al., 2003; Schmitt et al., 2004).
The recovery and increase of soil respiration could be attributed to
a decrease in the bioavailable antibiotic fraction, and an increasing
adaptation and resistance of the microorganisms (Thiele-Bruhn
and Beck, 2005). Chemical monitoring of the soil samples showed
DT50 values (dissipation half-lives) for the three compounds (sul-
famethoxazole, sulfamethazine and trimethoprim) ranged
between 2 and 5 days; therefore, the recovery of soil respiration
after the first 4 days was partially due to the significant loss of these
antibiotics in the soil. Based on the concentrations (up to 91 mg/kg)
detected in manure and soils (Pfeifer et al., 2002; Christian et al.,
2003; Jacobsen and Halling-Sørensen, 2006; Martı´nez-Carballo
et al., 2007) and the EC10 values in the present study, sulfonamides
and trimethoprim have the potential to affect soil respiration in
those lands applied with animal manure and biosolids.
In the present study, no obvious effects of tetracycline, chlortet-
racycline and tylosin on soil respiration could be observed. Sorption
and degradation processes played certain roles in reducing the
effects of these antibiotics. These three compounds exhibited strong
adsorption onto soil, suggesting they are less bioavailable (Sarmah
et al., 2006). Previous studies found that tylosinwas not persistent in
soil and its DT50 was no more than 1 week (Teeter and Meyerhoff,
2003; Hu and Coats,2007). In the presentstudy, tylosinhad a DT50of
8 days in the soil. So tylosin will not accumulate in soil and pose very
little risk to soil microbial respirationprocess (Blackwell et al., 2007).
Tetracyclines had DT50 values of more than20 days in the soilused in
the present study. Moreover, tetracyclines have strong adsorption
and complexation with cations such as calcium in soil (Kemper,
2008; Pils and Laird, 2007; Wessels et al.,1998; Zielezny et al., 2006).
This could significantly reduce the bioavailability and effects of
tetracyclines on soil microbial respiration.
Fig. 3 shows inhibition rates of soil phosphatase activity with
addition of antibiotics. The inhibition rates were very variable
during the various incubation periods (22 days). This could be
caused by the heterogeneous nature of soil. The present study
suggests that addition of antibiotics to soil at the concentration
used (1–300 mg/kg) can significantly affect soil phosphatase
activity (p 0.05). The EC10 values calculated for the six antibiotics
ranged from 1 mg/kg for sulfamethazine to 406 mg/kg for tetra-
cycline. Comparing with antibiotic concentrations (up to 91 mg/kg)
in the manure and soils (Pfeifer et al., 2002; Christian et al., 2003;
Jacobsen and Halling-Sørensen, 2006; Martı´nez-Carballo et al.,
2007), inhibition effects may be expected from some antibiotics
such as sulfonamides in real environment. Boleas et al. (2005) also
observed significant effects of oxytetracycline on soil microbial
enzymatic activities (phosphatase and dehydrogenase). However,
Thiele-Bruhn and Beck (2005) found no effects on dehydrogenase
activity even at a concentration of 1000 mg/kg of sulfapyridine and
oxytetracycline. Phosphatase activity was not measured in their
study. The reason behind the inconsistent results on dehydroge-
nase activity remains unclear. Microbial parameters such as enzy-
matic activities could be influenced by various factors and they may
not be specific for antibiotic effects in soil.
4. Conclusion
The different terrestrial toxicological effects of six antibiotics
were observed through using a series of bioassays including plant
seed germination and growth tests in soil, soil respiration and
phosphatase activity tests. Root elongation was found to be
a sensitive endpoint for plant germination and growth tests. The
two sulfonamides (sulfamethoxazole and sulfamethazine) and
trimethoprim were found to be most toxic to plant growth in soil.
Sweet oat and rice were more sensitive to the antibiotic compounds
than cucumber. In comparison with the controls, all antibiotics
tested inhibited soil phosphatase activity at the concentration
range used. Sulfamethoxazole, sulfamethazine and trimethoprim
had temporal effects on soil respiration whereas tetracycline,
chlortetracycline and tylosin had little effects on soil respiration.
The different toxic effects between the two groups of antibiotic
compounds were due to their different behavior in the soil; sorp-
tion, degradation and chelating with metals played important roles
for tetracyclines and tylosin. Considering the environmental levels
and fate of these antibiotics in soil, we would expect low toxic
effects on plant growth and soil microbial activities following
application of wastes with antibiotics such as sulfonamides and
trimethoprim and also a quick recovery from the stress due to the
loss and/or binding of the antibiotics onto soil components.
Acknowledgements
The authors would like to acknowledge the financial support
from the National Natural Science Foundation of China (NSFC
40688001, 40771180 and 40821003) and partial support from
Guangdong Natural Science Foundation. This is contribution No. IS
1026 from GIGCAS.
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
inhibitionrateof
phosphataseactivity(%)
inhibitionrateof
phosphataseactivity(%)
0 10 40 70 100 300
Antibiotic Concentration (mg/kg)
1
0 10 40 70 100 300
Antibiotic Concentration (mg/kg)
1
22 days
CTC
TC
TYL
TMP
SMZ
SMX
2 days
CTC
TC
TYL
TMP
SMZ
SMX
Fig. 3. Inhibition of phosphatase activity by six antibiotics compared to the controls
without addition of antibiotics at different times (2 days and 22 days) during the
incubation. CTC: chlortetracycline, TC: tetracycline, TYL: tylosin, SMZ: sulfamethazine,
SMX: sulfamethoxazole and TMP: trimethoprim.
F. Liu et al. / Environmental Pollution 157 (2009) 1636–1642 1641

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