Effects of six selected antibiotics on plant growth and soil microbial and enzymatic activities

Effects of six selected antibiotics on plant growth and soil microbial and enzymatic activities

Environmental Pollution 157 (2009) 1636–1642 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/lo...

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Environmental Pollution 157 (2009) 1636–1642

Contents lists available at ScienceDirect

Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

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 b

State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, 511 Kehua Street, Tianhe District, Guangzhou 510640, China 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

a b s t r a c t

Article history: Received 20 July 2008 Received in revised form 15 December 2008 Accepted 17 December 2008

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 germination 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 trimethoprim, whereas little effects were observed for the two tetracyclines and tylosin. The effective concentrations (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.

Keywords: Antibiotics Phytotoxicity Soil microbial activity Respiration Phosphatase Behavior

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 environment (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 HallingSørensen, 2000; Dı´az-Cruz et al., 2003, 2006; Golet et al., 2003; Go¨bel et al., 2005; Kemper, 2008). Antibiotics can also be introduced 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 * Corresponding author. Tel./fax: þ86 20 85290200. E-mail address: [email protected] (G.-G. Ying). 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.12.021

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 resistance 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 microbial 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 (Batchelder, 1982; Jjemba, 2002a; Farkas et al., 2007). Tetracyclines increased radish yields, but decreased pinto bean yields (Batchelder, 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).

F. Liu et al. / Environmental Pollution 157 (2009) 1636–1642

As antibiotics are designed to be biologically active toward microorganisms, it would be interesting to understand the potential 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 sulfamethazine, 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), sulfamethazine (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 Agricultural 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 chlortetracycline 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 sulfamethoxazole 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

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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 indicators 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 sulfamethazine 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 g1 dry soil) ¼ (blank-titer)  0.1  44/50, 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 diodearray detector (HPLC-DAD). Sulfamethazine, sulfamethoxazole, trimethoprim and tylosin in soil samples were extracted with acetonitrile for three times using sonication, 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

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F. Liu et al. / Environmental Pollution 157 (2009) 1636–1642

Table 1 Toxicity data from seed germination tests for sweet oat, rice and cucumber (endpoint:root length). Compound

Chlortetracycline Tetracycline Tylosin Sulfamethoxazole Sulfamethazine Trimethoprim a

EC10

EC50

NOEC

LOEC

oat

rice

cucumber

oat

rice

cucumber

oat

rice

cucumber

oat

rice

cucumber

0.2a 14 19 16 2 24

8 16 >500 0.1 6 23

0.7 8 217 >300 6 2

16 57 141 69 37 86

39 69 >500 8 45 118

48 203 >500 >300 >300 >300

<0.1 1 1 1 0.1 <1

1 1 >500 0.1 1 0.1

0.1 1 100 300 1 1

0.1 10 10 10 1 1

10 10 >500 1 10 1

1 10 300 >300 10 10

All concentrations are in mg/L.

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

which was kept for 5 min. The UV wavelength was set at 370 nm for chlortetracycline 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 germination 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 significant effect) and the lowest-observed-effect concentration (LOEC, lowest concentration 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 Table 2 Toxicity data from plant growth tests in soil for rice and cucumber. Compound

Endpoint

EC10

EC50

NOEC

rice

cucumber

rice

cucumber

rice

LOEC cucumber

rice

cucumber

Chlortetracycline

Seedling height Root length

>300a >300

19 300

>300 >300

>300 >300

>300 >300

100 70

300 300

300 100

Tetracycline

Seedling height Root length

>300 >300

300 300

>300 >300

>300 >300

>300 >300

>300 >300

300 300

>300 >300

Tylosin

Seedling height Root length

>500 >500

90 35

>500 >500

343 >500

>500 >500

50 50

500 500

100 100

Sulfamethoxazole

Seedling height Root length

25 2

85 66

38 13

>300 >300

30 1

100 100

50 10

300 300

Sulfamethazine

Seedling height Root length

92 1

249 120

220 43

300 >300

70 1

100 100

100 10

300 300

Trimethoprim

Seedling height Root length

>300 >300

>300 >300

>300 >300

300 300

1 100

>300 >300

10 300

a

All concentrations are in mg/kg dry soil weight.

0.7 85

F. Liu et al. / Environmental Pollution 157 (2009) 1636–1642

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3.5

Soil respiration (CO2 mg/g dry soil)

Chlortetracycline 3.0

**

2.5 2.0 1.5

*

1.0

*

0.5 0.0

0˜2

2˜4

4˜6

6˜9

9 ˜ 12

12 ˜16

16 ˜ 21

Incubation time (day) 0

1

10

40

70

100

300

Soil respiration (CO2 mg/g dry soil)

3.0 2.5

Tetracycline

*

2.0

*

1.5

* *

1.0 0.5 0.0

0˜2

2˜4

4˜6

6˜9

9 ˜ 13

13 ˜ 17

17 ˜ 22

Incubation time (day) 1

0

10

70

40

100

300

3.5

Soil respiration (CO2 mg/g dry soil)

3.0

Tylosin

2.5

*

2.0 1.5

*

1.0 0.5 0.0

0˜2

2˜4

4˜6

6˜9

9 ˜ 12

12 ˜ 16

16 ˜ 21

Incubation time (day) 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).

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). Sulphamethoxine 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). Chlortetracycline 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, sulfonamide residues in soil could reach 1 kg/ha, which is the same order of magnitude as the application rate of modern pesticides. Trimethoprim is used as a synergist to sulfonamides and was detected with concentrations up to 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

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F. Liu et al. / Environmental Pollution 157 (2009) 1636–1642

4.0 Sulfamethazine

Soil respiration (CO2 mg/g dry soil)

3.5

** *

3.0 2.5 2.0

*** *

1.5

*

*

*

1.0

** *

* **

0.5 0.0

0˜2

2˜4

4˜6

6˜8

8 ˜ 12

12 ˜16

16 ˜ 21

Incubation time (day) 0

1

10

40

70

100

4.0

**

Soil respiration (CO2 mg/g dry soil)

3.5

*

3.0 2.5 2.0

*

*

*

**

Sulfamethoxazole

** *

*

** *

*

1.5

*** *

* * *

1.0 0.5 0.0

0˜2

2˜4

4˜6

6˜8

8 ˜ 12

12 ˜16

16 ˜ 21

Incubation time (day) 0

Soil respiration (CO2 mg/g dry soil)

3.0 2.5 2.0

** **

* **

1

10

40

*

*

Trimethoprim

* *

**

100

*

** *

*

70

1.5

*

*

* **

*

*

* *

1.0

*

* **

0.5 0.0

0˜2

2˜4

4˜6

9 ˜ 13

6˜9

13 ˜ 17

17 ˜ 22

Incubation time (day) 0

1

10

40

70

100

300

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).

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-Sørensen, 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

F. Liu et al. / Environmental Pollution 157 (2009) 1636–1642

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, sulfamethazine 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 HallingSørensen, 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 (sulfamethoxazole, 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, chlortetracycline 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 tylosin was not persistent in soil and its DT50 was no more than 1 week (Teeter and Meyerhoff,

inhibition rate of phosphatase activity (%)

35 2 days

30

CTC

TYL

SMZ

TC

TMP

SMX

20 15 10 5 0 1

10

40

70

100

300

Antibiotic Concentration (mg/kg)

inhibition rate of phosphatase activity (%)

35 30

22 days

CTC

TYL

SMZ

TC

TMP

SMX

2003; Hu and Coats, 2007). In the present study, tylosin had a DT50 of 8 days in the soil. So tylosin will not accumulate in soil and pose very little risk to soil microbial respiration process (Blackwell et al., 2007). Tetracyclines had DT50 values of more than 20 days in the soil used 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 tetracycline. 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 dehydrogenase activity remains unclear. Microbial parameters such as enzymatic activities could be influenced by various factors and they may not be specific for antibiotic effects in soil. 4. Conclusion

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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; sorption, 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.

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Antibiotic Concentration (mg/kg) 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.

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.

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