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The effects of residual tetracycline on soil enzymatic

activities and plant growth
a a b c a
X. Wei , S. C. Wu , X. P. Nie , A. Yediler & M. H. Wong
a
Croucher Institute for Environmental Sciences, Hong Kong Baptist University , Hong Kong,
P.R. China
b
Hydrobiology Research Institute, Jinan University , Guangzhou, GD, P.R. China
c
Helmholtz Zentrum München, German Research Center for Environmental Health, Institute
of Ecological Chemistry , Neuherberg, Germany
Published online: 25 Jun 2009.

To cite this article: X. Wei , S. C. Wu , X. P. Nie , A. Yediler & M. H. Wong (2009) The effects of residual tetracycline on soil
enzymatic activities and plant growth, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants,
and Agricultural Wastes, 44:5, 461-471, DOI: 10.1080/03601230902935139

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 Journal of Environmental Science and Health Part B (2009) 44, 461–471
Copyright
C Taylor & Francis Group, LLC

ISSN: 0360-1234 (Print); 1532-4109 (Online)

DOI: 10.1080/03601230902935139

The effects of residual tetracycline on soil enzymatic

activities and plant growth

X. WEI1 , S. C. WU1 , X. P. NIE2 , A. YEDILER3 and M. H. WONG1

1
Croucher Institute for Environmental Sciences, Hong Kong Baptist University, Hong Kong, P.R. China
2
Hydrobiology Research Institute, Jinan University, Guangzhou, GD, P.R. China
3
Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Ecological Chemistry,
Neuherberg, Germany
Downloaded by [University of North Carolina] at 08:11 12 November 2014

A pot trial was carried out to investigate the adverse effects of tetracycline (TC) on soil microbial communities, microbial activities,
and the growth of ryegrass (Lolium perenne L). The results showed that the presence of TC significantly disturbed the structure of
microbial communities and inhibited soil microbial activities in terms of urease, acid phosphatase and dehydrogenase ( p < 0.05). Plant
biomass was adversely influenced by TC, especially the roots with a reduction of 40% when compared with the control. Furthermore,
TC decreased the assimilation of phosphorus by the plant although the concentration of phosphorus was increased by 20% due to
decreased plant biomass. TC seemed to increase the concentration of dissolved organic carbon (by 20%) in soil. The findings implied
that the agricultural use of animal manure or fishpond sediment containing considerable amounts of antibiotics may give rise to
ecological risks.
Keywords: Soil; tetracycline; bacteria; fungi; Lolium perenne L; plant growth.

Introduction residual antibiotics following the application of manure or

Pharmaceuticals, such as antibiotics, have been widely used
during the past decades and are recognized as ubiquitously
occurring persistent contaminants. Antibiotics and their
metabolites can enter the soil environment via several path-
ways, e.g. animal manure as fertilizer. However, limited in-
formation is available related to the impact of antibiotics
on soil ecosystem. Antibiotics have been and still are exten-
sively used in animal farms for disease control. During the
last few years, the abuse and overdose of antibiotics have
brought great impact on environment. Once administered
to an animal, the antibiotics may be present in substantial
amounts in urine and feces as the parent compound and/or
metabolites are adsorbed and partially metabolized. The
use of farm manure (pig or chicken) and fishpond sediment
as organic fertilizer has a fairly long history in agricultural
countries. Nowadays, manure and pond sediment are still
being extensively used as an important component of agri-
culture. Thus, the soil environment may be exposed to these
Address correspondence to M. H. Wong, Croucher Insti-
tute for Environmental Sciences, and Department of Biology,
Hong Kong Baptist University, Hong Kong SAR, P.R. China;
E-mail: mhwong@hkbu.edu.hk
Received August 13, 2008.
pond sediment to agricultural land.
There has been increasing awareness and concern over
the environmental effects of residual antibiotics. They can
provoke resistance in pathogens through long-time ex-
posure, mainly due to genetic variation, and transfer to
plasmids containing an antibiotic resistant gene, either
directly or indirectly from non-pathogenic to pathogenic
microorganisms.[1] Therefore, humans and animals are be-
coming subjected to a rising risk of microbial infections
that cannot be treated with pharmacotherapy. In addition,
residual antibiotics may pose a serious threat to human
health through possible accumulation in food chain.
Tetracycline (TC) is one of the most extensively used an-
tibiotics both on humans and farming animals in China.
China is reported to rank first in the world in terms of an-
nual production of oxytetracycline (OTC) and doxycycline;
two antibiotics from the tetracycline antibiotics group.[2]
For example, Chinese shrimp and prawns have been banned
in Europe and the United States due to the high residual
antibiotics detected.[3] The failure to strictly follow label
directions for the correct usage of TC may pose a more
serious environmental threat than other antibiotics, since
TC belongs to a broad-spectrum antibiotic, which can be
active against both gram positive and negative bacteria.
Compared with most of the persistent organic pollutants
 462
(POPs), the half-lives of most antibiotics are much shorter.
However, it was reported that TC is fairly persistent in the
environment. For example, as reported, TC can persist in
surface water and soil for over one year.[4] It had also been
reported that only a moderate degradation of various TCs
occurred within 180 days in soil.[5] The mean half-life of
oxytetracycline in fish farm sediment was approximately
120 days, thus indicating a moderate persistence of the
antibiotic.[6]
However, unlike other priority pollutants (e.g. heavy met-
als or POPs), the behavior and fate of veterinary medicines
in the environment have not been extensively studied. There
is limited information available on the impacts of an-
tibiotics on soil ecosystem, especially on microorganisms
thriving in soil, which are often several magnitudes more
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sensitive to the toxicity of antibiotics than other higher
organisms.[7] These microbes and plants possess functional
relationship and constitute a holistic system, therefore any
disturbance to the equilibria among microorganisms and
between microorganisms and plants may adversely affect
the stability and productivity of soil ecosystems.[8] This
study aimed at providing a better understanding of the in-
teractions among TC-soil microbes-plants, with emphasis
on the impact of TC to soil microbes and its environmental
fate. More specifically, the objectives of this study were to
investigate (i) the effects of TC on predominant bacterial
and fungal species in soil; (ii) the impacts of antibiotics on
microbial activities in terms of urease, acid phosphatase
and dehydrogenase activities; (iii) the influence of TC on
plant growth; and (iv) the influences of plants on the dissi-
pation of antibiotics.
Materials and Methods
Soil
A loamy soil used for this pot experiment was collected
from an agricultural land at Loi Tung Village, the New Ter-
ritories, Hong Kong (113.52–114.3◦ E, 22.9–22.37◦ N). The
soil was considered clean and free of contamination by an-
tibiotics and suitable for greenhouse trials. The soil samples
were air-dried and passed through a 2-mm mesh sieve be-
fore analyses of physico-chemical properties: pH value (in
a 1:5 mixture of soil:water using a pH electrode), electrical
conductivity (EC) value (in a 1:5 mixture of soil:water), to-
tal organic carbon (TOC) (%) were analyzed using a TOC
analyzer (Shimadzu TOC-5000, Japan) by detecting the ex-
tracts of organic carbon in soil sample (0.5 g) before adding
hydrochloric acid). Total N, total P, total K were measured
−1
using standard methods.[9] NaHCO3 –PO3− 4 –P (mg kg )
was determined in 0.5 M NaHCO3 extracts after shak-
ing for 16 h.[12] Water extractable-K (mg kg−1 ) was ex-
tracted by shaking with water at 1:5 ratio before analysis
by vaporization-inductively coupled plasma-atomic emis-
sion spectrometry.[12] The basic physicochemical properties
of the soil are shown in Table 1.
Wei et al.
Table 1. Basic physicochemical properties of the soil.[27]
Properties Values
pH (soil: water = 1:5) 6.20
EC (µS cm−1 ) 173.8
Texture (%)
Clay 4.6
Silt 19.6
Sand 75.8
Total organic carbon (%) 1.26
Total N (%) 0.11
Total P (%) 0.12
NaHCO3 -PO34 − P (mg kg−1 ) 4.53
Total K (%) 0.95
Water extractable – K (mg kg−1 ) 13.43
Tested bacterial and fungal strains
The bacterial and fungal species in the tested soil were
isolated using Luria-Bertani (LB) medium (United States
Biochemical Corp., Life Science Amersham International
plc., USA) and Rose Bengal medium (KH2 PO4 1.0 g, glu-
cose 10.0 g, MgSO4 ·7H2 O 0.5 g, peptone 5.0 g, agar 20.0 g,
1% Rose Bengal 5.0 mL, distilled water 1.0 L), respectively,
according to the standard suspension dilution and spread-
ing plate technique.[10] The most dominant bacterial and
fungal species grown on agar plates after incubation were
Bacillus megaterium and Penicillium chrysogenum from the
same soil collected from Loi Tung Village (identified by
Prof. Zhengao Li, Department of Soil Microbiology, Insti-
tute of Soil Science, Chinese Academy of Sciences), which
were chosen for the following experiment.
Plant
Lolium perenne L (Perennial ryegrass) was used for this
pot experiment. It is the most predominant forage grass
species in China and has been widely used for stockbreed-
ing, soil stabilization and visual improvement (lawn). The
seeds were purchased from The Ecological Centre of South
China, Zhongshan University, Guangzhou, PR China.
Preparation of bacterial and fungal inocula
Samples (2 mL) of late-exponential-phase starter bac-
teria were inoculated into 300 mL of Luria-Bertani
(LB) broth in 0.5 L conical flasks, under 28 ± 1◦ C and
200 rpm. Bacteria were allowed to grow to the late-
exponential phase before harvest. The bacterial density
in the broth was counted using a haemocytometer. The
bacterial cells were harvested by centrifugation (6000 rpm
and 4◦ C, J25-I Beckman Co., USA) in 250 mL cen-
trifuge tubes and washed three times with 50 mL ster-
ilized 0.9% NaCl solution to remove soluble compo-
nents remaining in the media that might interact with
the antibiotics.[11] Peat moss, purchased from Gartengold
 Effects of residual tetracycline
Company, Germany, was chosen as the carrier for the
microbial inoculum. The mixture served as the microbial
inoculum, in which the final population size of bacteria was
about 1.0 × 109 cfu g−1 inoculum (wet weight). The inocula
were sealed in sterile plastic bags and stored at 4◦ C before
use.
The isolated fungus, P. chrysogenum, was hyphal-tipped
and stored at 4◦ C in slant. The fungal inoculum was pre-
pared according to the standard suspension dilution and
spreading plate technique using Rose Bengal media (Sigma-
Aldrich, Inc., USA).[10] The spores on the fungal plate were
suspended with 10 mL of Milli-Q water and scraped using
a spatula. The spores were then quantified using a hema-
cytometer and a light microscope and the spore suspen-
sion was adjusted to the concentration of 1.2 × 106 spores
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mL−1 . Peat moss, the carrier for the microbial inoculum,
was mixed with a final population size of 2.0 × 107 cfu g−1
fungal inoculum (wet weight).
Pot experiments
Soil treatment
The collected soil was air-dried for 24 h under room tem-
perature (20 ± 2◦ C), ground and passed through a 4 mm
sieve before sterilization for 2 h at 121◦ C. TC hydrochlo-
ride (SIGMA-Aldrich Co. USA, 97.3%) was dissolved into
deionized water and then spiked into the sterilized soil at
four different concentrations: 0, 1, 10 and 100 mg kg−1 soil,
respectively. The soils were thoroughly mixed and stored
in a dark room at 4◦ C for 7 days until equilibrium. The
soil moisture was adjusted to 60% water holding capacity
(WHC) with addition of autoclaved deionized water. Each
pot (
buttom 10 cm × 17 cm ×
top 15 cm) contained 500 g
soil.
The pot experiment consisted of four groups: bacterial
inoculation (B), fungal inoculation (F), both bacterial and
fungal inoculations (B+F), and control (C) for four dif-
ferent concentrations of TC (0, 1, 10 and 100 µg L−1 , re-
spectively) giving a total of 32 treatments, each with four
replicates. For B and B+F groups, the bacterial inoculum
was added to the sterilized soil with an initial density of
1.0 × 106 cfu g−1 soil before planting while for F and B+F
groups, the fungal initial density was 2.0 × 104 cfu g−1
soil.
The seeds of L. perenne were surface sterilized with
10% hydrogen peroxide (Omega Co. USA) for 30 min and
washed with sufficient deionized water before planting.[12]
Thirty seeds were sown into each pot. All pots were wa-
tered with deionized water daily to maintain the soil mois-
ture content at approximately 60% water-holding capac-
ity (WHC). The pots were arranged under a randomized
block design in a greenhouse with temperature control
(25-32◦ C) and with supplementary illumination (light in-
tensity 250 µmol m−2 s−1 ; under a 14 h/10 h light/dark
cycle).
463
Fertilization
Each pot was amended with Multi-cote plant fertilizer
(Schultz Company MO USA). The initial nutrient con-
centrations were as follows: 270 mg ammonium-N, 90 mg
P (as phosphate) and 180 mg K kg−1 soil, respectively.
Soil analyses
After seed germination, the soil was sampled once ev-
ery five days during the whole experimental period of 60
days. The total bacterial and fungal populations were de-
termined according to the standard suspension dilution
and spreading agar plate method, using LB medium and
Rose Bengal red agar plates, respectively.[10] The micro-
bial activities in terms of urease, dehydrogenase and acid
phosphatase were estimated.[12] After the plants were har-
vested, the soil samples were collected. Extracts of organic
carbon were prepared at a ratio of 1:10 (soil:Milli-Q wa-
ter) and analyzed using a TOC analyzer (Shimadzu TOC-
5000, Japan). The TC concentration in soil was also ana-
lyzed after 60 days via an HPLC system (Hewelett Packard
Series 1100) consisting of a diode array detector (DAD),
autosampler and a reversed-phase octadecylsilyl (ODS)
column (RP-C18, 4.6 mm i.d. x 250 mm; particle size:
5 µm, Agilent).
Plant biomass and nutrient assimilation
At the end of the experiment, plant samples were collected
and divided into above-ground shoots and roots, rinsed
with tap water, washed three times with deionized water,
and oven-dried to constant weight at 60◦ C prior to grind-
ing to 100 µm. The dried plant tissues were then digested
[12]
with concentrated HNO3 for total K determination us-
ing an atomic absorption spectrometer (SpectrAA-20, Var-
ian). Total N and P in plant tissues were measured using
the Berthelot reaction and molybdenum blue methods[12]
after digestion with concentrated H2 SO4 (3 mL) and H2 O2
(1 mL) at 180◦ C. Available soil phosphorus was determined
in 0.5 M NaHCO3 extracts.[12]
Antibiotics analyses
Chemical reagents and apparatus
TC hydrochloride was purchased from SIGMA-Aldrich
Co. USA (97.3%). Acetonitrile and methanol (HPLC
grade, TEDIA, USA) and other chemical reagents were
also obtained from SIGMA-Aldrich Co. USA. The HLB
SPE cartridge (6 mL, 500 mg) was purchased from Waters
Co., USA, and Ultra-Clean SPE SAX (4 mL, 500 mg) from
Alltech Co., USA.
Extraction procedures and clean-up
Samples (1 g) and Na2 EDTA (0.2 g) were placed in glass
homogenizers into which 5 mL citric acid (0.5 M):MeOH
(pH4.7, 90:10) was added, and vortexed for 30 sec. Samples
were exposed to an ultrasonic bath for 30 min before
 464 Wei et al.

centrifugation (6000 rpm for 10 min and 4◦ C, J25-I Beck-

man Co., USA). After repeating two more times with 5 mL
of buffer, the clear supernatant was carefully transferred to
the Solid Phase Extraction (SPE) cartridges for clean-up
procedure. The clean-up procedure for soil sample was car-
ried out by using HLB-SAX tandem cartridge SPE extrac-
tion, washing with 0.05 M citric acid buffer (pH 4.7) and
eluting the TC from HLB with 0.01 M methanolic oxalic
acid.

Parameters for analysis

The residues of TC were determined by HPLC technique.
The mobile phase consisted of: 10% methanol, 20% ace-
tonitrile and 70% methanolic oxalic acid (0.01 M). The
flow rate was set at 0.8 mL min−1 for soil samples from
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the degradation experiment and detection was preformed

at 280 nm.

Quality administration and quality control

The HPLC retention time of TC under the experimental
conditions was approximately 5.1 min at a flow rate of
0.8 L min−1 (Fig. 1). Calibration curves established for
tetracycline ranged from 0.5 to 200 µg L−1 per injection and
were linear with r 2 > 0.999 for the reversed phase high per-
formance liquid chromatography with diode array detector
(RPHPLC-DAD) procedure. Quantification was obtained
by comparing the peak areas of the sample with the exter-
nal calibration curves, using the autoclaved clean soil (the
same soil used for the pot experiment). The mean percent-
age of recovery (n = 6) of TC for the soil samples subjected
to a series of TC concentration of 1, 5, 10, 20 and 50 µg
kg−1 were 101.0, 90.8, 93.6, 91.8 and 95.2%, respectively,
and the limit of detection was 0.5 µg L−1 .

Data analysis
Analysis of variance was performed on all experimental
data. The differences among four treatments of TC (0, 1,
10 and 100 µg kg−1 ) were compared with Duncan’s Multi-
ple Range Test using SPSS 13.0 software (SPSS, Inc). The
correlations between the bacterial or fungal population size
and the enzymatic activities, the total biomass of plants and
TC levels, the DOC concentrations and the antibiotic levels
were analyzed separately using Pearson’s Correlation Test.
Statistical procedures were carried out with the software
package SPSS 10.0 for Windows.
Results and Discussion
The degradation of TC in soil
Table 2 depicts the average TC degradation rates in differ-
ent initial concentrations throughout the pot trial, which
were 33% at 1 µg kg−1 , 25% at 10 µg kg−1 and 15% at
100 µg kg−1 , respectively. It seemed that all microbes, in-
cluding bacteria and fungi, played an important role in
decomposing TC in soil. Compared with control, the inocu-
Fig. 1. High performance liquid chromatography (HPLC) chro-
matograms of (a) control soil; (b) 50 mg kg−1 standard tetracy-
cline (TC) (TC peak at Rt 5.09 min); and (c) soil sample from the
TC degradation experiment after 30 days (peak at 5.18 min).
lation of bacteria and fungi in soil accelerated TC degrada-
tion, especially when the initial TC concentration was low
(10 µg kg−1 ). However, no obvious combined effect of bac-
teria and fungi on TC degradation was observed. Excluding
the B group at 1 µg kg−1 , a general trend of TC degradation
in soil was observed as follows: F > B > B + F > Control
group. The lower degradation rate in the treatments with
high TC addition (100 µg kg−1 ) could be attributed to the
lower number of microorganisms as a result of the strong
inhibition effect at high concentrations of TC, which subse-
quently lowered the extent of biodegradation of TC in soil.
 Effects of residual tetracycline
Table 2. Residual concentration (µg kg−1 ) of tetracycline (TC) in soil after an experimental period of 60 days.
Bacteria Fungi
Treatment µg kg−1
TC-1 0.73 ± 0.11 0.61 ± 0.08
TC-10 5.57 ± 0.65 6.15 ± 0.41
TC-100 63.2 ± 5.17 68.3 ± 5.80
Control (no TC) ND ND
TC-1, TC-10 and TC-100: 1, 10 and 100 µg kg−1 TC in soil initially.
NA: Not available.
ND: Under the detection limit.
Another possible explanation was the fixation of TC to the
soil surfaces or in the pores of the soil matrix.[13] This may
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have effectively kept TC from biodegradation. This was in
line with the report that TC could remain stable in the soil
matrix due to its relatively long half-life in soil of at least
30 days.[14] As reported, TC is susceptible to photodegra-
dation if exposed to direct light.[15] However, it is expected
that photodegradation in the present study was rather
minimal.
As indicated in Figure 2, the concentrations of dissolved
organic carbon (DOC) in soil increased according to in-
creases in TC concentrations. There was a significant cor-
relation between the DOC concentrations and the antibi-
otic levels ( p < 0.05), which implied that the degradation
of TC may have led to the increase of DOC concentra-
tions in the soil. The death and decomposition of other
microbes under high loads of TC in soil may lead to an
increase of DOC concentration. On the other hand, similar
results have reported that the sorption of TC to dissolved
organic matter (DOM) in soil could potentially increase the
water-solubility, therefore increasing the DOC level.[16]
Effects of TC on bacterial and fungal communities and
enzymatic activities in soil
The size of the bacterial populations were significantly af-
fected by the presence of TC within the first 10 days in
both B and B+F groups (Fig. 3) when compared with the
control group. A clear dose-response effect was observed
Fig. 2. Effects of tetracycline (TC) on dissolved organic carbon
(DOC) concentrations in soil. The different letters above the bar
indicate a significant difference between different treatments at
p < 0.05 level.
465
Bacteria + fungi Control
Average degradation rate (%)
0.68 ± 0.13 0.89 ± 0.12 28.3
6.87 ± 0.77 8.11 ± 0.63 33.3
68. 7 ± 6.61 77.1 ± 4.09 30.7
ND ND NA
whereby the bacterial population decreased with increase
in TC concentrations. Thereafter, the number of bacterial
colonies dramatically increased in all the treatments. The
present experiment indicated that for TC on B. megaterium,
the Minimum Inhibitory Concentration (MIC), which is
defined as the lowest concentration of an antimicrobial
that will inhibit the visible growth of a microorganism, was
≤5 µg mL−1 .[17] Compared with other microbial species in
soil, the MIC of TC for B. megaterium in this experiment
seemed to be higher than those for E. coli (≤0.5 µg mL−1 )
and for Salmonella sp (≤2 µg mL−1 ).[18] Our study was
in line with the reported results that the MIC for TC was
≤10 µg mL−1 on B. megaterium.[19] This higher MIC value
also indicated that B. megaterium has a greater resistance
to TC compared to other strains. Nevertheless, the degra-
dation of antibiotics did not seem to result in a decline
in its antibiotic potential in this experiment. The decline
in concentration of TC was not always mirrored by a de-
cline in microbial toxicity.[16] The reason may be that the
metabolites of TC still exhibited bacterial toxicity in soil.[20]
The fungal growth in the soil was also affected by the
presence of TC (Fig. 4). However, no clear dose-response
effect was observed. There was a poor relationship between
the fungal community and concentration of TC under the
different treatments ( p > 0.05). This implied that the fungal
species was fairly sensitive to TC toxicity since the growth
of fungi could be greatly inhibited by TC even under the
lowest concentration. These results were in agreement with
the report which revealed that Penicillium sp. was the most
sensitive species of four fungal strains tested with CMT-
3, a chemically modified tetracycline.[21] According to the
present results, the fungal population seemed to increase
with increasing antibiotic load within 10 days, especially
for the F group. This may be attributed to the degradation
of the antibiotic. The degradation of TC could result in an
increase of dissolved carbon concentration in soil, which
could also lead to fungal growth after ten days.
Effects of TC on the soil enzymatic activities
TC seemed to have obvious inhibition on enzymatic ac-
tivities in soil (Fig. 5 and 6). Even at low concentrations
(1.0 µg kg−1 ), TC significantly inhibited the activities of
urease and dehydrogenase. However, there seemed to be
 466 Wei et al.
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Fig. 3. Effects of tetracycline (TC) on bacterial communities in soil. B-bacteria; F-fungi.

 Effects of residual tetracycline 467
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Fig. 4. Effects of tetracycline (TC) on fungal communities in soil. B-bacteria; F-fungi.

 468 Wei et al.
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Fig. 5. Effect of tetracycline (TC) on urease activity.

Fig. 6. Effects of tetracycline (TC) on dehydrogenase activity.

 Effects of residual tetracycline
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Fig. 7. Effects of tetracycline (TC) on plant growth (a) total biomass of plants; (b) potassium uptake in plants; (c) phosphorous
uptake in plants. The different letters above the bar indicate a significant difference between different treatments at p < 0.05 level.
no effect on acid phosphatase activity. The degradation
of the antibiotic in soil probably caused the gradual in-
crease of urease activity. On the other hand, urease activity
seemed to increase gradually (Fig. 5). A significant correla-
tion (r = 0.009, p < 0.01) was found between bacterial pop-
ulation size and urease activity for all the treatments since
soil microbes, including bacteria and fungi, contributed
greatly to soil enzymatic activities.[22] Bacteria in soil are
responsible for a number of enzymatic reactions, e.g. ni-
trogen fixation and nitrification are of importance to the
growth of plants.[23] The same trend was also observed in
dehydrogenase activity, especially when the TC concentra-
tion was low (1 µg kg−1 ) (r = 0.012, p < 0.05). The total
activity of microorganisms could be estimated by measur-
ing the activity of a living cell-associated enzyme such as
dehydrogenase, which plays an important role in the res-
469
piratory pathway of microorganisms.[22] The inhibitory ef-
fects of TC on soil dehydrogenase activity (DHA) (Fig. 6)
led to an inhibition of the breakdown of organic matter in
soil, and hence adversely affected the growth of plants. The
great inhibition of enzymatic activity by TC also explained
the low concentration of available phosphorus in soil, espe-
cially when under high concentrations of TC (100 µg kg−1 )
(r = 0.019, p < 0.05).
Effect of TC on the plant growth parameters
Plant biomass production
TC seemed to have an adverse effect on the total biomass of
plants, however the biomass of shoots was not significantly
affected by the antibiotics (Fig. 7). There was a significant
negative correlation ( p < 0.05) between the concentrations
 470
of antibiotics and plant biomass, which proved that the
presence of antibiotics had a significant adverse effect on
the plant growth. This was probably due to the effect of TC
on the microbial communities in soil, whereby enzymatic
activity decreased causing a negative impact on the phy-
toavailability of nitrogen and phosphorous, which are both
essential for plant growth. Efficient recycling of reduced
nitrogen (in the form of urea) is important for plant growth
since urea contains a significant amount of this element.[24]
Accordingly, the plant growth was adversely affected espe-
cially during the early stage (t = 5 days and t = 10 days) of
the experiment, which indicated that young seedlings were
more susceptible to TC than mature ones.
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Potassium, phosphorous and nitrogen uptake in plants
Figure 7 depicts the uptake of K, P and N in plants. TC did
not seem to exert an obvious effect on the uptake of K and
N in plants. Since soil microorganisms control the cycling
of several essential elements such as N in soil,[25] the in-
hibitive effect of TC on bacteria and fungi would affect the
supply of N and therefore the N uptake of plants. TC could
also reduce the urease activity as a urease inhibitor, which
reduces the growth of microbes and blocks the formation
of urease, thus decreasing the amount of urease in the soil.
Subsequently, the rate of hydrolysis of urea is decreased.
This could, furthermore, decrease the ineffective degrada-
tion of urea and thereby affect the nutrients absorption
in plants. TC seemed to increase the uptake percentage of
phosphorus in plants due to the lower biomass imposed
by the presence of TC, but it actually reduced the total
quantity of phosphorus. On the other hand, saprophytic
fungi contribute to nutrient cycling in soils, as they cap-
ture, store, release and recycle carbon and other nutrients
(e.g. phosphorous, nitrogen, etc). Arbuscular mycorrhizas
fungi (AMF) can contribute to plant growth and nutrient
uptake, especially phosphorous.[26] Hence, the decrease in
fungal growth with increase of TC load seemed to increase
the quantity of phosphorous in plant.
Conclusions
All the concentrations of TC investigated had obvious in-
hibition effects on both bacteria and fungal communities,
and consequently affected enzymatic activities in soils in
the pot experiment. TC seemed to affect plant growth by
significantly reducing the biomass of plants especially in
roots even when exposed to low levels of TC (1.0 µg kg−1 ).
The fate of the antibiotic in soil was also investigated in
this study. Approximately 30% of TC had been degraded
after 60 days. The residual concentrations of tetracycline
in soil proved that the degradation of the antibiotic in soil
could cause an increase in urease activity. The presence of
bacteria and fungi in soil could accelerate the degradation
of TC in soil at 1.0 or 10 µg kg−1 . Plants, Lolium perenne L,
also played an important role, such as providing root exu-
Wei et al.
dates to maintain a considerable population size of bacteria
and fungi which could accelerate the biodegradation of TC
in the soil.
In order to reduce the risk of environmental contami-
nation, caution should be exercised regarding the use of
antibiotics. Guidelines should be strictly followed when us-
ing antibiotics as veterinary drugs in animal farming and
aquaculture.
Acknowledgments
The authors would like to thank Dr. Jinshu Zheng for help-
ful suggestions and critical comments on the clean-up pro-
cedure and the RPHPLC-DAD analytical method for TC
detection. Financial support from the Academic Link of
Deutscher Akademischer Austausch Dienst (DAAD) and
Research Grants Council of Hong Kong (GER/JRS/05-
06/01) is gratefully acknowledged.
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