PHYTOTECHNOLOGY TO TREAT CIPROFLOXACIN CONTAMINATED

WETLANDS: COMPARISION BETWEEN CONTINOUS AND SINGLE

DOSE EXPERIMENTS
Trinh Hong Phuonga, Tu Thi Cam Loanb, Hoang Thi Thanh Thuyc
Hochiminh City University for Natural Resources and Environment
phuong.envigeo@gmail.com; bttcamloan21@gmail.com; chttthuy@hcmunre.edu.vn;

ABSTRACT
Ciprofloxacin is an antibiotic of fluoroquinoloness group that widely applied in shrimp larvae located in Cangio
wetland. Thus, the wetland may be contaminated and could lead to the negative impacts on ecosystem since the
ciprofloxacin residues could modify the microorganism community of receiving water body.
The utility of native wetland plants (Acrostichum aureum and Rhizophora apiculata Blume) for phytoremediation of
ciprofloxacin contaminated wetland was investigated. Two experiments have been set up based on spike
methodology ciprofloxacin: single (10 g/kg and 21 experimental days) and continuous doses (2 mg/kg each time,
for 4 times, at 5 days intervals within 28 experimental days) on planted soils. The results showed that the removal
rate of Acrostichum aureum and Rhizophora apiculata Blume in continuous dose were lower than single dose
experiments. However, phytotechnology still an appropriate and ecological friendly for remediation of antibiotic
contaminated wetland.
Keywords: phytoremediation, ciprofloxacin, wetland, Acrostichum aureum, Rhizophora apiculata Blume

1.

INTRODUCTION

Due to the increased frequency of detection

of
pharmaceutical
compounds
in
wastewater over the past decade, there has
been an increased interest in how to remove
these contaminants. Some advanced
technologies have also been evaluated such
as advanced oxidative processes, activated
carbon adsorption, membrane filtration and
membrane bioreactors (Nakada et al., 2007;
Radjenovic, Petrovic, and Barcel, 2007;
Ternes et al., 2007). However, despite the
sometimes high removal efficiencies
attained, these processes are not widely
used, mainly because of high costs.
Presently, phytotechnology is one of the
environmental friendly on-site treatment
processes
for
removing
emerging
contaminant like pharmaceuticals. This
measure has high efficiencies at reasonable
costs of operation/maintenance and
therefore is appropriate for small-scale
operations such as shrimp farms. Several
examples of successful phytoremediation
have been reported by Gujarathi, Haney and
Linden, 2005; Kotyza et al., 2010.
The study presented here was carried out to
assess the ability of certain naturally
occurring wetland plants to remove

antibiotics. In this study, laboratory tests

were performed to investigate the uptake of
a widely used antibiotic - ciprofloxacin
(CIP) - in pot experiments using
Acrostichum aureum L. and Rhizophora
apiculata Blume Fl. Javae with two kinds
of dose: single and continuous dose.
2.

MATERIAL AND METHOD

2.1 Material
Water and soil used in pot experiments was
obtained from the area adjacent to ponds of
a local shrimp farm in Cangio district,
Hochiminh City, Vietnam. The water and
sediment was determined to be CIP- and
NOR-free prior to use. The pH and Ec of
water were measured in the field. The
nutrient values of sediment together with
sediment
physical
properties
were
determined by standard methods (APHA)
and could be regarded as characteristic of
sediment from former shrimp ponds. All
analyses were performed in duplicate. The
physic-chemical parameters of water and
sediment have been reported in previous
publication (Thuy et al., 2012).
Two plant species with ages of
approximately 4 months old were collected
from wetlands of Cangio district,

Hochiminh City, Vietnam during March

2011 (single dose experiment) and October
2012 (continuous dose experiment).
CIP was purchased from Quality Assured
Laboratory, (Ministry of Health, Vietnam)
with the purity of 98.8%.
2.2 Pot experiment set up
The removal ability of CIP by R. apiculata
and A. aureum was investigated by
preparing plastic planters containing
sediment (2 kg). Water from Cangio area
(1L) was added to make up a 50% water
content to simulate real conditions at the
field. The CIP spiking concentration was 2
mg/kg each time, for 4 times, at 5 days
intervals within 28 experimental days for
planting pots. All planters were then placed
outdoor with natural sunlight. A plastic roof
was made to avoid the impacts of rain. The
planting experiments were performed in
triplicate.
Furthermore, control planters were added
with the same CIPs dose to assess the
antibiotic degradation without plants. In
order to understand the biodegradation of
antibiotics by microorganisms, control
planters containing sterile soil were also set
up. The sediment was sterilized with
autoclave at 121C, 1 atm for 30 min and
then kept in sterile environment. The
control experiments were performed only in
duplicate.
During the experiment, daily sediment
moisture levels were controlled and shrimp
farm water was added as required to keep
the 50 % water content in sediment. The
plant and sediment samples were collected
for CIP analysis on 2, 7 and 28 days after
commencing the experiment for single.
Procedure of single dose experiment has
been reported in previous publication (Thuy
et al., 2012).
2.3 CIPs analysis
The analysis of CIP was carried out at the
Chemical Laboratory of the University of
Natural Sciences, National University of
Hochiminh City. High-performance liquid

chromatography (HPLC), coupled with UV

detection, was used for determining the
concentrations of CIP. The determination of
FQs in soils was based on published
methods with slight modifications (Mostafa,
S., El-Sadek, M., and Alla, E.A. 2002;
Ferdig, M., Kaleta, A., and Buchberger, W.
2005; Christodoulou et al. 2007). The
concentrations of CIP in soil samples were
determined by adding 5 g of soil to 5 mg/kg
of internal standard and 10 mL ammonium
formiate buffer in an ultrasonic bath for 30
min. The extraction was carried out twice
and the combined extract filtered through a
0.45m glass fiber filter. The filtrate was
then cleaned up by through a conditioned
solid phase extraction cartridge (SPE C18)
and eluted with 5 mL of 25%
methanol-ammonia solution (20:80) and
then was evaporated off under a gentle
stream of nitrogen (30 40 minutes). The
concentrated
aqueous
extract
was
reconstituted with mobile phase, and then
the CIP concentrations were detected by
their UV absorbance at 278 nm and
compared with internal standards. Limit of
detection (LOD) and limit of quantification
(LOQ) were 0.1 mg/kg and 0.5 mg/kg.
3.

RESULTS AND DISCUSSIONS

3.1 Effect on plant growth

As shown in table 1, no significant negative
effect on plant growth was measured as
both R. apiculata and A. aureum remained
healthy and continued to grow.
For continuous spiking pots, the increased
biomass has been observed with both plants
in comparison with no CIP spiking plants.
The plant biomasses (dry weight) of
different are shown in Table 1.
This results were consistent with the last
results of single dose experiment, in which
R. apiculatas biomass increased by 11%.
In particular, the A. aureums development
was illustrated by the development of new
roots; the biomass increased by 24% (Thuy
et al., 2012).

100

Table 1. The results of single and continuous

doses experiments

28

compartment

Single dose

Continuous
dose

Dry biomass (g)

A. aureum - P

73 1.0

A. aureum - C

Removal rate (%)

80

Parameter/

85 43

60

40

20

44 17

0
NS-C

R. apiculata - P

17 2.0

A. aureum - C

308 17
278 51

76 4.1

A. aureum

99 0.0

80 3.0

R. apiculata

97 0.1

68 6.9

SC

42 0.4

70 2.5

CIP (mg/kg)

80 0.1

R. apiculata

SC

10

CIP in soils
(mg/kg)
NS-C

A. aureum

Figure 1. Time variation of CIP removal rate

in single dose experiments

NS-C

A. aureum

R. apiculata

0
7

14

21

Days

Note: aNS-C: Non-sterile control and SC: Sterile

control

3.2 CIPs degradation

The data after 2 days were not reliable since
some pots have higher levels of CIP as
compared to CIPs added amount. In
general, the CIP concentrations in planted
soils were lower than sterile control pots. At
day 7, a clearly decreasing tendency of CIP
was observed in all of unplanted non-sterile
and sterile as well as planted pots. However,
it is interesting to note that the removal rate
of R. apiculata was stable till day 28 (end
of experiment). In contrast, the increased
removal ate up to 80% has been reported
for A. aureum. The reason for that may be
due to the development of A. aureums root
system.
As compared to single dose experiment, the
CIPs removal rate in continuous dose
experiment was lower for both R. apiculata
and A. aureum. During the first 7 days of
the experiments, the CIP concentrations
soils rapidly decreased (Figure 1).

Figure 2. Time variation of CIP

concentrations in single dose experiments

Then, the CIP levels further decreased. Till

the end of experiment, only 2 % of the
initial CIP was recovered from planted pots,
compared to more than 19% and 50% from
the unplanted non-sterile and sterile pots
(Table 1).
Therefore, if this contaminant is frequently
released into receiving water body, the side
effect related with antibacterial resistant
bacteria will be occurred.
4.

CONCLUSION

The present study confirmed that CIP a

commonly used in shrimp farming - can be
actively taken up by various species of
wetland plants. After a maximum 28-day
exposure to the CIP in wetlands soils, no
effect on growth rate was noted for both
Rhizophora apiculata and Acrostichum
aureum. Hence, the removal rate of
continuous dose could simulate better the
removal rate of both plants as compared to
single dose since this contaminant is

discharge frequently to the wetland

environment. In addition, the removal rate
of wetlands plants is depending on the
loading of CIP.
However, the study results have also
indicated that phytotechnology using native
wetland plants like Rhizophora apiculata
and Acrostichum aureum could be effective
measure to the minimize the side effect of
CIP accumulated in wetlands soils.

The single dose experiments were

supported
by
Vietnam's
National
Foundation for Science and Technology
Development
(NAFOSTED),
Project
105.09.30.09. The continuous dose
experiments were supported by Ministry
of Natural Resources and Environment,
Project TNMT.04.30. The authors would
like to thank MSc. Truong Lam Son Hai
and MSc. Dao Phu Quoc for their
laboratory assistance.

ACKNOWLEDGEMENTS

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