Nitrogen and phosphorus removal in the recirculating aquaculture system with water treatment tank containing baked clay beads and chinese

EnvironmentAsia

The international journal published by the Thai Society of Higher Education Institutes on Environment

Available
online
at www.tshe.org/EA
Available
online
at www.tshe.org/EA
EnvironmentAsia 27(1)
(2014)
81-88
EnvironmentAsia
(2009)
50-54

Genotoxicity Assessment of Mercuric Chloride in the Marine Fish Therapon jaruba
Nitrogen and Phosphorus Removal in the Recirculating Aquaculture System
Nagarajan
Arumugam
Kuppusamy
Kumaraguru,
Velmurugan
JanakiCabbage
Devi
with
Water Nagarani,
Treatment
Tank Containing
Baked

Clay Beads
and Chinese
and Chandrasekaran Archana Devi
Aeknarin Thanakitpairin a, b, Wiboonluk Pungrasmi b and Sorawit Powtongsook c, d

Center for Marine and Coastal Studies, School of Energy, Environment and Natural Resources,
a
Madurai
Kamaraj University,
Madurai-625021,
India
Department
of Environmental
Sciences, Faculty
of Science and
Technology,
Rambhai Barni Rajabhat University, Chantaburi, Thailand
b
Department of Environmental Engineering, Faculty of Engineering, Chulalongkorn University, Thailand
c
Abstract
National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand
d
Center of Excellence for Marine Biotechnology, Department of Marine Science, Faculty of Science,
The aim of the present study was to Chulalongkorn
standardize andUniversity,
to assess Thailand
the predictive value of the cytogenetic analysis
by Micronucleus (MN) test in fish erythrocytes as a biomarker for marine environmental contamination. Micronucleus
frequency

Abstract baseline in erythrocytes was evaluated in and genotoxic potential of a common chemical was determined
in fish experimentally exposed in aquarium under controlled conditions. Fish (Therapon jaruba) were exposed for 96
hrs
a single
heavy
metal
(mercuric
Chromosomal
determined Aquaculture
as micronuclei
frequency
toThis
research
aims
to describe
thechloride).
nitrogen and
phosphorus damage
removal was
in Recirculating
System
(RAS)inby
fish
erythrocytes.
Significant
increase
in
MN
frequency
was

observed
in
erythrocytes
of
fish
exposed
to
mercuric
crop plants biomass production. The 3 experiment systems consisted of 1 treatment (fish tank + baked clay beads + Chinese
chloride.
of control-1
0.25 ppm (fish
induced
highest
MN frequency
(2.95
cells/1000
cells compared
cabbage) Concentration
and 2 controls as
tankthe
only)
and control-2
(fish tank
+ micronucleated
baked clay beads),
were performed.
With all
3
to

1
MNcell/1000
cells
in
control
animals).
The
study
revealed
that
micronucleus
test,
as
an
index
of cumulative
experimental RAS, Nile tilapia (Oreochromis niloticus) was cultured at 2 kg/m density. The baked clay beads
(8-16 mm
exposure,
appears
to beasa asensitive
model
to the
evaluate
genotoxictank
compounds
in fish
under
controlled
conditions.

in diameter)
were filled
layer of 10
cm in
water treatment
of control-2.
While
in the
treatment
tank, Chinese
2
cabbage (Brassica pekinensis) was planted at 334 plants/m in baked clay beads layer. During 35 days of experiment, the
Keywords:
mercuric chloride;
micronucleus
average fishgenotoxicity;
wet-weight in control-1,
control-2 and
treatment systems increased from 16.31±1.49, 15.18±1.28 and 11.31±1.49
g to 29.43±7.06, 28.65±3.12 and 27.20±6.56 g, respectively. It was found that the growth rate of 0.45±0.15 g-wet weight/
day in a treatment tank was higher than in those 2 controls, which were rather similar at 0.37±0.16 and 0.38±0.05 g-wet
weight/day, respectively. The fish survival rate of all experimental units was 100%. The average Chinese cabbage wet-weight
fieldallconditions.
2006
Soumendra
1.
Introduction
in treatment
system increased from 0.15±0.02 g to 1.00±0.38 laboratory
g. For waterand

quality,
parameters In
were
within
the acceptet
al.,
made
an
attempt
to
detect
genetic
biomarkers
able range for aquaculture. The assimilation inorganic nitrogen in a treatment tank showed a slower rate and
lower nitrite
accumulation
thosetons
in control
tanks. The
phosphorus
balance Labeo
analysisbata
illustrated
that most of the
in two
fish species,
and Oreochromis
In India,relative
aboutto200
of mercury

andnitrogen
its and
nitrogen and phosphorus
input ininto
all systems
was from feed (82-87%
and 21-87%)
the final
day of experiments,
mossambica,
by while
MN atand
binucleate
(BN)
compounds
are introduced
the environment
nitrogen and
in tilapia
culture revealed
at 15-19% and
4-13%. The in
accumulation
of nitrogen
phosphorus in
erythrocytes
the gill and
kidneyand
erythrocytes
annually

as phosphorus
effluents from
industries
(Saffi, 1981).
the water, up
to 56% has
and 70%,
in control-1as
while
in the tank
with bakedpower
clay beads
had discharge
substantial lower
exposed
to thermal
plant
at
Mercuric
chloride
been was
usedfound
in agriculture
a water
nitrogen and phosphorus concentration. The most important part was unaccounted nitrogen and phosphorus as high as 60%
Titagarh Thermal Power Plant, Kolkata, India.
fungicide, in medicine as a topical antiseptic and
and 17% in treatment and 53% and 10% in control-2 systems. Nitrogen and phosphorus incorporated in plant (treatment)
The present study was conducted to determine
disinfectant,

and in chemistry as an intermediate in
was only 1.31% and 0.11%, respectively. It can be implied from the results that the assimilation in plant was a minor process
the acute
the heavy
metal
compound
the
production
of in
other
mercury
compounds.
for nutrient
removal
this RAS.
On the
other hand, The
the nitrification
andgenotoxicity
denitrificationofoccurred
in the
sediment
layer of
HgCl
in
static
systems.
Mercuric
chloride
is toxic,

contamination
of
aquatic
ecosystems
by
heavy
baked clay beads tank were the major treatment processes to maintain
water quality in the recirculating system.
Without
2
water
it can
theclay
aquatic
metals
andbead,
pesticides
haswaste
gained
attention
baked clay
nitrogen
wasincreasing
accumulated
as nitrate insolvable
the waterinwhile
in hence
treatment
tankpenetrate
with backed

beads,
animals.
Mutagenic
studies
with
native
fish
species
in
recent
exposure
to and
nitrogen
wasdecades.
significantlyChronic
removed by
denitrification
process.

represent an important effort in determining the
accumulation of these chemicals in aquatic biota
Keywords:
Aquaculture
nitrogen potential
removal; phosphorus
nitrification;
effects ofremoval;
toxic agents.
Thisdenitrification;
study was

can
result Recirculating
in tissue burdens
that System;
produceRAS;
adverse
Chinese not
cabbage
carried out to evaluate the use of the micronucleus
effects
only in the directly exposed organisms,
test (MN) for the estimation of aquatic pollution
but also in human beings.
using marine edible fish under lab conditions.
Fish provides a suitable model for monitoring
1. Introduction
Common water treatment processes in the RAS, apart
aquatic
genotoxicity and wastewater quality
from
aeration, and
are sediment
2.
Materials
methodsremoval and nitrification
because of its ability to metabolize xenobiotics and
accumulated
Recently,pollutants.
aquaculture
industry

is
expanding
processes.
In
general,
toxic nitrogen compounds such
A micronucleus assay has
rapidly
duesuccessfully
to an increase
of the world
food
as ammonia
nitrite derived from aquatic animal’s
2.1.
Sample and
Collection
been
used
in several
species
(Dedemand.
Flora,
Environmental
friendly
aquaculture
system
is
therefore
excretion,

feed
residues,
and microbial degradation
et al., 1993, Al-Sabti and Metcalfe, 1995). The
essential
for
sustainable
development.
The
closed(Crab
et
al.,
2007)
must
be
regulated
below
0.5 mg-N/L.
The fish species selected for the
present
study
micronucleus (MN) test has been developed
recirculating
technology
has been
Highcollected
ammoniafrom
and Pudhumadam
nitrite can cause
adverse

health
was
coast
of Gulf
of
together
withaquaculture
DNA-unwinding
assays
as
developing formethods
decades, but
under research.
effects in Southeast
aquatic animals
Mannar,
Coastand
ofcreate
India.environmental
Therapon
perspective
for mostly
mass is
monitoring
of
Recirculating Aquaculture
System
(RAS)
water jarbua
concernsbelongs

if effluent
notorder
properly
treated. Apart
to isthe
Perciformes
of from
the
clastogenicity
and genotoxicity
in fish
anduses
mussels
treatment
technologies
to
treat
wastewater
from
nitrogen
waste
treatment,
phosphorus
accumulation
family Theraponidae. The fish species, Therapon
(Dailianis et al., 2003).
aquaculture
and
reuse
the water

for a long
period.
in the RAS
is also
concerned
but4-4.25
phosphorus
removal
jarbua
(6-6.3
cm in
length and
g in weight)
The MNtank
tests
have
been
successfully
used
as
was selected for the detection of genotoxic effect
a measure of genotoxic stress in fish, under both


Aeknarin Thanakitpairin et al. / EnvironmentAsia 7(1) (2014) 81-88

requires sophisticated sequential anaerobic-aerobic 2.1. Recirculating aquaculture system
process which is not yet commercial available
(Burut-Archanai et al., 2013). In this research, we
The experimental aquaculture system consisted

studied the possibilities of combining wastewater of 38 x 58 x 31 cm3 fish tank (working volume 45 L)
treatment with the production of crop plants biomass connected to the overlay water treatment tank. The
for phosphate removal which could not be treated by water treatment tank (plant tank) was 38 x 58 x 24 cm3
conventional treatment system. The advantage of using plastic tank packed with 10 cm layer of spherical shape
plant is that it is not only assimilating nitrogen waste baked clay beads (8-16 mm in diameter) and Chinese
but it also remove phosphate from the water with the cabbage with approximately 4.83±0.35 cm height
absorption of the root system (Beven, 2010). In an planted at 334 plants/m2. This bead packing performed
aquaponic RAS with substrate support for planting, as suspended solids retainer and biological filtration
the nitrogen assimilation process by plant and the media for inorganic nitrogen treatments (Fig. 1). The
nitrogen degradation process by nitrifying/denitrifying effluent from fish tank was pumped by submersible
bacteria were combined for nitrogen removal from fish pump (Resun SP-6600) through PVC pipes lying over
wastewater. Unlike the aquaponic concept with floating the treatment tank. Water was spray into treatment tank
plants (Rakocy and Hargreaves, 1993; Timmons et al., at 3 L/min for 10 minutes thereafter pump was pause
2002; Wilson, 2005), the proposed system applied for 60 minutes before the next pumping round. Water
baked clay beads layer in a tank, that was not only for from treatment tank was flow back to the fish tank by
supporting plant root but also performed as the media gravity. Continuous aeration in fish tank was provided
for microbial nitrogen removal via nitrification and through diffusive stone aerators in order to maintain
denitrification processes. Moreover, the optimization proper environmental conditions for fish growth and
of nitrogen and phosphorus removal processes was nitrifying process (i.e., well-mixed, DO > 4.0 mg O2/L,
necessary for water quality control in fish tank and pH = 7-8 and alkalinity = 120-160 mg CaCO3/L by
research,
the possibilities
of combining
treatment with the production of
yieldInofthis
plant
(Graberwe
andstudied
Junge, 2009).
In our systems,

addingwastewater
sodium bicarbonate).
plants
biomasscabbage
for phosphate
removal
which could
nottilapia
be treated
conventional
Nilecrop
tilapia
and Chinese
were chosen
for this
Nile
with by
an average
initialtreatment
wet-weight
system.
The
advantage
of
using
plant
is
that
it
is

not
only
assimilating
nitrogen
waste
but
alsowas
study as they are economical important species and fast of 14.27±1.42 g and length of 9.44±0.27it cm
remove
phosphate
from
the
water
with
the
absorption
of
the
root
system
(Beven,
2010).
In
an of
growth rates. The experimental system was carried out in stocked in all fish tanks to obtain the initial density
3
aquaponic
RAS
with
substrate

support
for
planting,
the
nitrogen
assimilation
process
by
plant
and
the
partial controlled condition in which light, temperature, approximately 2 kg/m . Fish was fed twice daily at 8.00
by nitrifying/denitrifying
combined
for nitrogen
removalfeed
DO, nitrogen
moisture,degradation
nutrients andprocess
pests were
regulated to suit am bacteria
and 3.00were
pm with
25% protein
commercial
from
fish
wastewater.
Unlike
the

aquaponic
concept
with
floating
plants
(Rakocy
and
Hargreaves,
1993;was
for both fish and plant living.
pellets at 5% of total fish weight per day (feeding
Timmons et al., 2002; Wilson, 2005), the proposed system
applied
baked
clay
beads
layer
in
a
tank,
adjusted every week following fish biomass). Fish
growth
that was not only for supporting plant root but also performed as the media for microbial nitrogen
2. Materials and Methods
was monitored by length and weight measurement
removal via nitrification and denitrification processes. Moreover, the optimization of nitrogen and
every week and the experimental period was 35 days.
phosphorus removal processes was necessary for water quality control in fish tank and yield of plant

The experiment was conducted at the Center of Growth of Chinese cabbage was measured by weighing

(Graber and Junge, 2009). In our systems, Nile tilapia and Chinese cabbage were chosen for this study
Excellence
Biotechnology,
Department
of growth
at the rates.
initial The
and experimental
the end of 35system
days experiment.
as they for
are Marine
economical
important species
and fast
was carriedLeaf
Marine
Science,
of condition
Science, Chulalongkorn
width, length
canopynutrients
size was
every
out in
partial Faculty
controlled
in which light, temperature,
DO,and
moisture,

andmeasured
pests were
University.
The
treatment
recirculating
aquaculture
week.
regulated to suit for both fish and plant living.
system consisted of fish tank growing Tilapia and
plant2.tank
packed and
withMethods
baked clay beads and Chinese 2.2. Water quality parameters and analytical methods
Materials
cabbage. The fish tank without plant tank and fish
tank + bakedThe
clay experiment
beads tank (no
were assigned

During
the experiment,
water
samples were
wasplant)
conducted
at the Center
of Excellence
for Marine

Biotechnology,
as control-1
and
control-2,
respectively
(Table
1).
All
taken
out
daily
for
ammonia,
nitrite,
nitrate,
alkalinity,
Department of Marine Science, Faculty of Science, Chulalongkorn University. The treatment
experimental
systems
were performed
3 replicates
phosphate,
total
nitrogen
totaltank
phosphorus
analysis
recirculating
aquaculture
system with

consisted
of fish tank
growing
Tilapia
andand
plant
packed with
and placed
in thebeads
greenhouse.
following
method
for tank
water+and
wastewater
baked clay
and Chinese cabbage. The fish tank
withoutstandard
plant tank
and fish
baked
clay
beads tank (no plant) were assigned as control-1 and control-2, respectively (Table 1). All
experimental systems were performed with 3 replicates and placed in the greenhouse.
Table 1. Experimental systems performed in this study

Experimental conditions

Control-1

Control-2

Fish tank only

Fish tank +
Baked Clay Beads

Treatment
Fish tank +
Baked Clay Beads +
Chinese cabbage

2.1. Recirculating aquaculture system
The experimental aquaculture system consisted of 38 x 58 x 31 cm3 fish tank (working
volume 45 L) connected to the overlay water treatment
tank. The water treatment tank (plant tank)
82
was 38 x 58 x 24 cm3 plastic tank packed with 10 cm layer of spherical shape baked clay beads (8-16
mm in diameter) and Chinese cabbage with approximately 4.83±0.35 cm height planted at 334


Aeknarin Thanakitpairin et al. / EnvironmentAsia 7(1) (2014) 81-88

control
and treatment units was 100%. The average Chinese cabbage wet-weight in treatment system
Figure 1. Schematic diagram and photo of the treatment recirculating aquaculture system consisted of fish tank
increased
0.15±0.02
g tobaked
1.00±0.38

g and
increased
and overlayfrom
treatment
tank with
clay beads
andlength
Chinese
cabbage.from 4.83±0.35 cm to 8.04±1.13 cm
(0.11 cm/day). Leaf width increased from 0.77±0.10 cm to 3.26±0.54 cm (0.09 cm/day) and canopy
size expanded
fromwith
1.64±0.18
cm initial
to 11.43±3.22
cm/day).
The
average
growth rate
of
Nile tilapia
an average
wet-weightcmof (0.35
14.27±1.42
g and
length
of 9.44±0.27
cm was
3
Chinese

cabbage
was
equivalent
to
0.02±0.01
g/day.
stocked in all fish tanks to obtain the initial density of approximately 2 kg/m . Fish was fed twice daily
at 8.00 am and 3.00 pm with 25% protein commercial feed pellets at 5% of total fish weight per day
Table 2. Growth characteristics of Tilapia during the experiment (a, b shows was differed significantly)
(feeding was adjusted every week following fish biomass). Fish growth was monitored by length and
weight measurement every week and the experimental period was 35 days. Growth of Chinese
Parameter
Control-1
Control-2
Treatment
cabbage was measured by weighing at the initial and the end of 35 days experiment. Leaf width,
Initial tilapia wet-weight (g)
16.31±1.49
15.18±1.28
11.31±1.49
length
and canopy size was measured every29.43±7.06
week.
Final tilapia wet-weight (g)
28.65±3.12
27.20±6.56
Initial tilapia length (cm)

9.96±0.23

9.46±0.27
8.89±0.32
11.92±0.62
11.37±1.02
Initial biomass density (kg/m3)
2.04
2.01
2.14
During
the experiment,
water samples
Final biomass
density
(kg/m3)
3.68were taken out daily
3.81 for ammonia, nitrite,
5.11 nitrate,
Total feed (g)
135.33
176.12
alkalinity,
phosphate, total nitrogen and total
phosphorus analysis
following standard200.50
method for
a
a
b
Average
growth (g/day)

0.37±0.16
0.38±0.05
0.45±0.15
water
anddaily
wastewater
analysis (APHA, 2005).
Suspended solids in
the water was analyzed
every three
Feed conversion
ratio; FCR including DO, pH,
2.06
1.96were measured using
1.69 portable
days.
Physical parameters
temperature and ORP
Survival
rate
(%)
100
100
100in baked
meters. Nitrogen and phosphorus in feed, fish, suspended solids in fish tank, solid retained

2.2.
parameters and analytical
methods
FinalWater

tilapiaquality
length (cm)
11.79±0.73

ORP (mV)

clay beads tank, Chinese cabbage, and baked clay beads were determined at the initial and the end of
It was found
that growth
of Chinese
cabbage
in this
experiment
was
much samples
slower were
than
the experiment
for nitrogen
and phosphorus
budget
analysis.
Nitrogen
content
in dried
conventional
vegetable
planting
in
soil

but
comparable
to
aquaponic
system
by
Graber
and
Junge
analyzed by CHN analysis using dynamic flash combustion, CHNS-O analyzer. Phosphorus was
(2009)
which
the average
2.07plasma
g of wet-weight
and 18spectrometry,
cm in lengthat after
55 days.Equipment
This was
analyzed
usinghad
inductively
coupled
optical emission
the Scientific
3. Results
and Discussion
analysis
(APHA,
2005).

Suspended
solids
in the and
water
probably
due
to
the
limitation
of
nutrients
improper
environmental
condition
in
the
experiment.
Center, Prince of Songkla University, Thailand. Statistical analysis (ANOVA) between the controls
wasLow
analyzed
every three
days. 980-25,410
Physical parameters
light intensity
Lux due
to building
and treatments
was between
calculated
using Microsoft

Excel
2007. shade on the experiment green house in
3.1.
of fish
and Chinese cabbagePotential
including
DO,
pH,
temperature
and
ORP
were
measured
the afternoon was also another factor affecting growth. Growth
Decrease
of Oxidation-Reduction
using
portable
meters.
Nitrogen
and
phosphorus
in
(ORP)
fromand
+290
to +110 mV in baked clay bead layer indicated that accumulation of sediment in
3. Results
Discussion

During
35bead
dayslayer
of experiment,
feed,baked
fish, suspended
tank, solid
retained
clay beads solids
causedinanfish
increase
of oxygen
consumption
in the
(Fig. 2). the average fish
wet-weight
in
control-1,
control-2
and treatment systems
in baked
clay
beads
tank,
Chinese
cabbage,
and
baked
3.1. Growth of fish and Chinese cabbage
increased

from
16.31,
15.18
and
11.31 g to 29.43,
clay beads were determined
at
the
initial
and
the
end
350
28.65
and 27.20
respectively
(Tableand
2).treatment
It was found
of the experiment
for35nitrogen
phosphorus
During
days ofand
experiment,
the budget
average fish
wet-weight
in g,control-1,
control-2

that treatment
withg,baked
clay beads
and2).
Chinese
analysis.
Nitrogen
in dried
were
300
systems
increasedcontent
from 16.31,
15.18samples
and 11.31
g to 29.43,
28.65 andtank
27.20
respectively
(Table
It
cabbage
had
the
highest
growth
of
0.45
g/day
while

analyzed
by
CHN
analysis
using
dynamic
flash
was found that treatment tank with baked clay beads and Chinese cabbage had the highest growth of
250 analyzer.
fish growth
in control-1
control-2
rather
combustion,
Phosphorus
0.45 g/dayCHNS-O
while fish
growth rate
in control-1was
and control-2
wererate
rather
similar atand
0.37
and 0.38were
g/day,
similar
at 0.37
g/day,
respectively.

These
analyzed
using inductively
coupled
plasma
optical
respectively.
These growth
rates were
within
acceptable
range
due toand
the0.38
proper
fish density
between
3
3
200at the fish
2-5 kg/m
while growing
at higher
density e.g.growth
12 kg/m
could
daily
growth
rates
werereduce

within average
acceptable
range
due to the
emission
spectrometry,
Scientific
Equipment
0.16±0.09
as reported
by Azim and
Little (2008).
Statistical
analysis
indicated
that3 while
the fish
in
proper
fish density
between
2-5 kg/m
growing
Center,
Princeg/day
of Songkla
University,
Thailand.
3
150

treatment
tank
had
significantly
higher
growth
rate
than
control-1
and
control-2.
Feed
conversion
ratio
Statistical analysis (ANOVA) between the controls fish at higher density e.g. 12 kg/m could reduce average
(FCR) in treatment
system
wasMicrosoft
1.69 while
FCR daily
in control-1
control-2g/day
wereas2.06
and 1.96,
growth and
to 0.16±0.09
reported
by Azim
and treatments
was calculated

using
Excel
100
theLittle
fish (2008).
in treatment
tanks.
Survival
rate ofthat
all the
and
Statistical
analysis
indicated
2007.respectively. This indicated better feed utilization of
50
0
0

5

10

1583

20

Time (d)

25

30

35


Aeknarin Thanakitpairin et al. / EnvironmentAsia 7(1) (2014) 81-88

fish in treatment tank had significantly higher growth 3.2. Water quality
rate than control-1 and control-2. Feed conversion
ratio (FCR) in treatment system was 1.69 while FCR in
According the Fig. 3 illustrates inorganic nitrogen
control-1 and control-2 were 2.06 and 1.96, respectively. and phosphate concentrations in control and treatment
This indicated better feed utilization of the fish in systems. High peak of total ammonia nitrogen (TAN)
treatment tanks. Survival rate of all control and treatment up to 1.2±0.3 mg-N/L was found in control-1 during the
units was 100%. The average Chinese cabbage wet- first 5 days, while small TAN peaks at 0.31±0.1 mg-N/L
control
and treatment
was 100%.
The average
cabbage
wet-weight
treatment
system
weight
in treatment
systemunits
increased
from 0.15±0.02
g Chinese

were found
in control-2
and in
treatment
system
which
increased
from
0.15±0.02
g
to
1.00±0.38
g
and
length
increased
from
4.83±0.35
cm
to
8.04±1.13
cm
to 1.00±0.38 g and length increased from 4.83±0.35 cm was within the safety range [below 0.5 mg-N/L (Liao
(0.11 cm/day).
Leafcm/day).
width increased
from
0.77±0.10and
cm Mayo,
to 3.26±0.54

(0.09day
cm/day)
andaccumulation
canopy
to 8.04±1.13
cm (0.11
Leaf width
increased
1972)].cm
During
3-8, the
size
expanded
from
1.64±0.18
cm
to
11.43±3.22
cm
(0.35
cm/day).
The
average
growth
rate ofpeaks
from 0.77±0.10 cm to 3.26±0.54 cm (0.09 cm/day) and of nitrite was found in all tanks after TAN
Chinese
was
equivalent
to 0.02±0.01

g/day. disappearance. This indicated the occurrence of
canopy
size cabbage
expanded
from
1.64±0.18
cm to 11.43±
3.22 cm (0.35 cm/day). The average growth rate of nitrification process via ammonia oxidizing bacteria.
Table 2. Growth characteristics of Tilapia during the experiment (a, b shows was differed significantly)
Chinese
cabbage was equivalent to 0.02±0.01 g/day.
The highest peak of nitrite, up to 3.84±0.8 mg-N/L,

It was found that growth of Chinese cabbage in was also found in control-1, while smaller peaks were
Parameter
Control-1
Control-2
Treatment
this experiment
much (g)
slower than conventional
and treatment11.31±1.49
systems (0.86±0.1
Initial tilapia was
wet-weight
16.31±1.49 found in control-2
15.18±1.28
vegetable
planting
in

soil
but
comparable
to
aquaponic
and
0.38±0.25
mg-N/L,
respectively),
Final tilapia wet-weight (g)
29.43±7.06
28.65±3.12
27.20±6.56and these
system
by
Graber
and
Junge
(2009)
which
had
the
concentrations
were
under
the
safety
range [below 1
Initial tilapia length (cm)
9.96±0.23

9.46±0.27
8.89±0.32
Final
tilapia
length
(cm)
11.79±0.73
11.92±0.62
11.37±1.02
average 2.07 g of wet-weight and 18 cm in length mg-N/L (Hart and O,Sullivan, 1993)]. The nitrification
biomass
(kg/m3) due to the limitation
2.04 was complete within
2.01 10 day when the
2.14accumulation
afterInitial
55 days.
Thisdensity
was probably
3
Final
biomass
density
(kg/m
)
3.68
3.81
5.11
of nutrients and improper environmental condition of nitrate, the end product of nitrification,
occurred

Total feed (g)
135.33
176.12
200.50
in the experiment. Low light intensity between 980- a without nitrite accumulation.
At
the
end
of the
Average daily growth (g/day)
0.37±0.16
0.38±0.05a
0.45±0.15b
25,410
Lux
due to building
of nitrate1.69
was as high as
Feed
conversion
ratio; FCRshade on the experiment
2.06 experiment, concentration
1.96
greenSurvival
house rate
in the
afternoon
was
also
another

factor
100.37±5.6
mg-N/L
in
control-1.
Accumulation
of
(%)
100
100
100
affecting growth. Decrease of Oxidation-Reduction nitrate is generally found in closed aquaculture system in
Potential (ORP)
from
+290that
to +110
mVofin Chinese
baked clay
which
water treatment
process
is nitrification
It was
found
growth
cabbage
in the
thismajor
experiment
was much

slower
than
bead
layer indicated
that accumulation
of sediment
(Nootong
et al., 2011;
Nootong
Powtongsook,
2012).
conventional
vegetable
planting in soil
but comparable
to aquaponic
system
by and
Graber
and Junge
in baked
beads
an increase
was55higher
(2009) clay
which
had caused
the average
2.07 g of oxygen
wet-weightThis

andnitrate
18 cmconcentration
in length after
days. than
Thisthe
wassafety
consumption
in the
layer (Fig.of2).nutrients and improper
concentration
of 50condition
mg-N/L insothe
water
exchange is
probably due
to bead
the limitation
environmental
experiment.
Low light intensity between 980-25,410 Lux due to building shade on the experiment green house in
the afternoon was also another factor affecting growth. Decrease of Oxidation-Reduction Potential
(ORP) from +290 to +110 mV in baked clay bead layer indicated that accumulation of sediment in
baked clay beads caused an increase of oxygen consumption in the bead layer (Fig. 2).
350
300

ORP (mV)

250
200

150
100
50
0
0

5

10

15

20

25

30

35

Time (d)
Figure 2. The variation of Oxidation-Reduction Potential in baked clay bead layer of control-2 (…) and
treatment (‹) system

3.2. Water quality
According the Fig. 3 illustrates inorganic84
nitrogen and phosphate concentrations in control
and treatment systems. High peak of total ammonia nitrogen (TAN) up to 1.2±0.3 mg-N/L was found
in control-1 during the first 5 days, while small TAN peaks at 0.31±0.1 mg-N/L were found in

Aeknarin Thanakitpairin et al. / EnvironmentAsia 7(1) (2014) 81-88

therefore needed (Hart and O,Sullivan,1993).On generally known that plant can take up inorganic
the other hand, nitrate in control-2 and treatment nitrogen and phosphorus compounds as nutrients for
systems were 47.24±4.1 mg-N/L and 26.66±3.7 growth, however, phosphate concentration in treatment
mg-N/L respectively. The lower nitrate accumulation tank containing Chinese cabbage was slightly higher
in control-2 and treatment systems indicated that than control-2 which had only baked clay beads. Hence,
baked clay beads played a significant role in nitrate the role of phosphorus uptake by plant in this experiment
removal.
was still unclear and further detailed study is therefore

It could be summarized that nitrification was the recommended.
major process for water quality control in fish tanks
It was found that the baked clay beads tank was
control-2
which
was within not
the only
safetyremove
range inorganic
[below 0.5nitrogen
mg-N/Lbut(Liao
and
without
bakedand
claytreatment
beads andsystem
plant. This
nitrification

it also
retain
Mayo,
1972)].
During
day
3-8,
the
accumulation
of
nitrite
was
found
in
all
tanks
after
TAN
peaks
activity occurred with the natural biofloc (suspended suspended solids (Fig. 4). Water in the fish tank without
disappearance.
This indicated
theculture
occurrence
of nitrification
process (control-1)
via ammonia
bacteria.
solids)
that accumulated

during fish
(Nootong
beads filtration
hadoxidizing
the suspended
solids
The highest peak of nitrite, up to 3.84±0.8 mg-N/L, was also found in control-1, while smaller peaks
et al., 2011). In contrast, when baked clay beads tank concentration as high as 352.22±56.01 mg/L below
were found in control-2 and treatment systems (0.86±0.1 and 0.38±0.25 mg-N/L, respectively), and
was applied to the fish culture system, nitrate was the safety concentration of ,80 mg/L (Timmons et al.,
these concentrations were under the safety range [below 1 mg-N/L (Hart and O Sullivan, 1993)]. The
significantly removed by denitrification process in the 2002) throughout the experimental period. In general,
nitrification was complete within 10 day when the accumulation of nitrate, the end product of
anaerobic
layer ofoccurred
the bakedwithout
clay bead
tank.accumulation.
Moreover, suspended
higher
than 500 mg/L
must be avoided
nitrification,
nitrite
At the endsolids
of the
experiment,
concentration
of
phosphate

concentration
was
also
low
in
control-2
due
to
it
obstruct
visibility
while
it
was
only
46.11±8.55
nitrate was as high as 100.37±5.6 mg-N/L in control-1. Accumulation of nitrate is generally found
in
and closed
treatment
system. At
the end
of thethe
experiment,
mg/L and 55.00±22.55
mg/L in control-2
andettreatment
aquaculture
system
in which

major water treatment
process is nitrification
(Nootong
al.,
accumulation
of phosphate
(8.84±0.4 mg-P/L)
was nitrate
system,
respectively.was
Hence,
baked
claythe
beads
tanks in
2011; Nootong
and Powtongsook,
2012). This
concentration
higher
than
safety
,
found
in
control-1.
Lower
phosphate
concentrations
this

experiment
were
successfully
maintain
suspended
concentration of 50 mg-N/L so water exchange is therefore needed (Hart and O Sullivan, 1993). On
werethefound
control-2
and
treatment
solidswere
in fish
tank. Other
water quality
parameters
other in
hand,
nitrate in
control-2
andsystems
treatmentatsystems
47.24±4.1
mg-N/L
and 26.66±3.7
mg-were
5.72±0.1
mg-P/L
and
3.38±0.5
mg-P/L,

respectively.
It
is
within
the
acceptable
range
for
aquaculture
(i.e.,
N/L respectively. The lower nitrate accumulation in control-2 and treatment systems indicated thatpH =
8.23-8.55; alkalinity = 100-163.33 mg CaCO3/L; DO
baked clay beads played a significant role in nitrate removal.

Figure 3. The water quality analysis in fish tanks from control-1 (S), control-2 (…) and treatment (‹) systems.
(The horizontal dot lines indicate safety concentration for aquaculture)

It could be summarized that nitrification was the major process for water quality control in
fish tanks without baked clay beads and plant. This nitrification activity occurred with the natural
85 fish culture (Nootong et al., 2011). In contrast,
biofloc (suspended solids) that accumulated during
when baked clay beads tank was applied to the fish culture system, nitrate was significantly removed


It is generally known that plant can take up inorganic nitrogen and phosphorus compounds as
nutrients for growth, however, phosphate concentration in treatment tank containing Chinese cabbage
was slightly higher than control-2 which had only baked clay beads. Hence, the role of phosphorus
uptake by plant in this experiment was still unclear and further detailed study is therefore
recommended.
Aeknarin Thanakitpairin et al. / EnvironmentAsia 7(1) (2014) 81-88

Total suspended solids (mg/L)

450
400
350
300
250
200
150
100
50
0
0

5

10

15

20

25

30

35

Time (d)

Figure 4. The suspended solids concentration in control-1 (S), control-2 (…) and treatment (‹) systems

It was found that the baked clay beads tank was not only remove inorganic nitrogen but it also
retain suspended solids (Fig. 4). Water in the fish tank without beads filtration (control-1) had the
suspended solids concentration Control-l
as high as 352.22±56.01 mg/L
below the safety concentration
Control-2
Treatment of 80
mg/L
(Timmons
et
al.,
2002)
throughout
the
experimental
period.
In
general,
suspended
higher
Parameter
Nitrogen per tank (g)*
Nitrogen per tank (g)*
Nitrogen solids
per tank
(g)*
than 500 mg/L must be avoided
due

to
it
obstruct
visibility
while
it
was
only
46.11±8.55
mg/L
and
In put
at final day
In put
at final day
In put
at final day
55.00±22.55 mg/L in control-2 and treatment system, respectively. Hence, baked clay beads tanks in
Feed
7.15 (82.00%)
9.31solids
(85.73%)
10.59 (86.45%)
this experiment were successfully
maintain -suspended
in fish tank.- Other water
quality parameters
Fish
1.53 (17.54%)
(17.89%) 1.51

1.59 (14.64%)
1.60 (13.06%)
2.28 (18.61%)
were within the acceptable
range for1.56
aquaculture
(i.e.,(13.90%)
pH = 8.23-8.55;
alkalinity
= 100-163.33
mg
Ñ
CaCO
/L;
DO
=
7.07-9.07
mg/L;
temperature
=
26.50-30.07
C).
3
TN in water
0.04 (0.46%) 4.83 (55.39%) 0.04 (0.37%) 2.18 (20.07%) 0.04 (0.33%)
1.21 (9.88%)
Table 3. The nitrogen balance in the recirculating aquaculture systems

Suspended solid in fish tank

-

0.56 (6.42%)

-

0.07 (0.65%)

-

0.08 (0.65%)

Solid retained in baked clay beads tank

-

-

-

1.28 (11.79%)

-

1.19 (9.71%)

3.3. Nitrogen and phosphorus mass balance

The nitrogen balance- analysis in Table
3 shows -that nitrogen -input in 0.02

all systems
Chinese cabbage
(0.16%) was
0.16mostly
(1.31%)
from feed (82-87%) and fish (13-18%) while at the end of experiments; nitrogen in fish was between
Baked clay beads
15-19%. These results were comparable to the report of Avnimelech and Rityo (2003), which
Unaccountedexplained that the input nitrogen
(20.30%) was accumulate
5.74
7.33 (59.84%)
and1.77
phosphorus
in(52.85%)
fish 22% and -16% respectively.
Moreover,
in
many
research
reports
notified
the
proportion
of
ammonia
nitrogen
in
RAS
that

39.29%
Total
8.72 (100%)
8.72 (100%) 10.86 (100%) 10.86 (100%) 12.25 (100%) 12.25
(100%)
was from feed, 26-28% was from fish excretion while the final portion of 24% was accumulated in
* CHNS-O Analyzer,
CE Instruments
Flash EA
1112
Series,
Thermo
Quest,
Italy
sludge suspended
solids
(Lin
and
Nash,
1996;
Funge-Smith
and Briggs, 1998).Accumulation of
nitrogen in the water, up to 56%, was found in control-1 while water in the tank with baked clay beads
had substantial lower nitrogen concentration. The most important part was unaccounted nitrogen as
= 7.07-9.07
temperature
= 26.50-30.07ºC).
was This
accumulated
in sludge

highmg/L;
as 53%
in control-2
and 60% in treatment24%
system.
was assumed
as the suspended
nitrogen gassolids
loss
3.3. Nitrogen
anddenitrification
phosphorus mass
balance
(Lin2005;
and Nash,
1996; Funge-Smith
Briggs,
1998).
through
process
(Rafiee and Saad,
Funge-Smith
and Briggs,and
1998).
Nitrogen
Accumulation
nitrogen
in theresults
water,from
up tothis

56%,
was
incorporated in Chinese cabbage (treatment system)
was only of
1.31%.
Hence,
study
that nitrogen
removal
in 3our
RAS was
mainly
by nitrification-denitrification
processes

Theillustrated
nitrogen balance
analysis
in Table
shows
found
in control-1
while water in the tank with
baked
while nitrogen
by plant
the minor
role.substantial lower nitrogen concentration.
that nitrogen
input in uptake

all systems
wasincorporated
mostly fromwithclay
beads had
feed (82-87%) and fish (13-18%) while at the end of The most important part was unaccounted nitrogen as
experiments; nitrogen in fish was between 15-19%. high as 53% in control-2 and 60% in treatment system.
These results were comparable to the report of This was assumed as the nitrogen gas loss through
Avnimelech and Rityo (2003), which explained that denitrification process (Rafiee and Saad, 2005; Fungethe input nitrogen and phosphorus was accumulate in Smith and Briggs, 1998). Nitrogen incorporated in
fish 22% and 16% respectively. Moreover, in many Chinese cabbage (treatment system) was only 1.31%.
research reports notified the proportion of ammonia Hence, results from this study illustrated that nitrogen
nitrogen in RAS that 39.29% was from feed, 26-28% removal in our RAS was mainly by nitrificationwas from fish excretion while the final portion of denitrification processes while nitrogen uptake by plant

86


Aeknarin Thanakitpairin et al. / EnvironmentAsia 7(1) (2014) 81-88

Table 4. The phosphorus balance in recirculating aquaculture systems
Control-l
Parameter

Phosphorus
per
tank
(g)*
Nitrogen per
tank
(g)*

Control-2

Phosphorus
tank
Nitrogen perper
tank
(g)*(g)*

Treatment
Phosphorus
tank
(g)*
Nitrogen perper
tank
(g)*

In put

at final day

In put

at final day

In put

at final day

Feed

0.55 (87.02%)

-

0.50 (21.35%)

-

0.56 (20.47%)

-

Fish

0.08 (12.66%)

0.08 (12.66%)

0.08 (3.42%)

0.10 (4.27%)

0.09 (3.29%)

0.10 (3.65%)

TP in water
TN

0.002 (0.32%) 0.439 (69.46%) 0.002 (0.08%) 0.298 (12.72%) 0.004
0.04 (0.15%)

0.186 (6.80%)

Suspended solid in fish tank

-

0.11 (17.41%)

-

0.02 (0.85%)

-

0.03 (1.10%)

Solid retained in baked clay beads tank

-

-

-

0.28 (11.96%)

-

0.21 (7.68%)

Chinese cabbage

-

-

-

-

0.001 (0.04%)

0.003 (0.11%)

Baked clay beads

-

-

1.76 (75.15%)

1.41 (60.21%)

2.08 (76.05%)

1.76 (64.35%)

Unaccounted

-

0.003 (0.47%)

-

0.234 (9.99%)

-

0.446 (16.31%)

0.632 (100%)

0.632 (100%)

2.342 (100%)

2.342 (100%)

2.735 (100%)

2.735 (100%)

Total

* Optical Emission Spectrometer, Optima 4300 DV, Perkin Elmer Instruments, USA

incorporated with the minor role.

The phosphorus balance in all systems is show in
Table 4. It was found that phosphorus input was mostly
from feed (21-87%) and fish (3-13%). At the end of
experiments, phosphorus in fish was between 4-13%
which was slightly lower than previous report (15.98%)
by Rafiee and Saad (2005), phosphorus accumulation
in the water was up to 70% in control-1, while tanks
with baked clay beads had substantial lower phosphorus
concentration. Unaccounted phosphorus was as high as
17% in treatment, but control-1 and control-2 system
were lower at 1.0% and 10%, respectively. Phosphorus
in suspended solids ranged between 1-18% while
phosphorus incorporated in plant (treatment) was only
0.11%. Moreover, it was assumed that most of the
nutrients were accumulated in suspended solids and
solid deposited in baked clay beads tank.

Promotion and National Research University Project of
Thailand, the Office of the Higher Education Commission
(FW1017A). Additional support was also obtained from the
Ratchadaphiseksomphot Endowment Fund of Chulalongkorn
University (RES560530068). Moreover, this research also
received an extra funding from a master thesis grant of the
National Research Council of Thailand for the year 2011 and
received a partially financial support by the graduate thesis
grant, Chulalongkorn University. Equipments and facilities
in this research were provided by the Center of Excellence
for Marine Biotechnology, Department of Marine Science,
Faculty of Science and Department of Environmental
Engineering, Faculty of Engineering, Chulalongkorn

University, Thailand. Some equipment (microplate
spectrophotometer) is provided for by the Thai Government
Stimulus Package2 (TKK2555) under the Project for
Establishment of Comprehensive Center for Innovative
Food, Health Products and Agriculture, Chulalongkorn
University.

4. Conclusion

References


With the proposed RAS, toxic nitrogenous
compounds such as ammonia and nitrite were
maintained within the safety level for fish. Significant
amount of nitrogen compounds were removed mostly by
degradation especially nitrification and denitrification
processes while nutrients (nitrogen and phosphorus)
assimilation in plant was the minor process. This RAS
concept has high potential for further development.

APHA. Standard Methods for the examination of water and

wastewater. 21st ed. APHA, AWWA, WPCF, Washington

DC, USA. 2005.
Avnimelech Y, Rityo G. Shrimp and fish pond soils processes

and management. Aquaculture 2003; 264: 140- 47.
Azim ME, Little DC. The biofloc technology (BFT) in indoor

tanks: Water quality, biofloc composition, and growth

and welfare of Nile tilapia (Oreochromis niloticus).

Aquaculture 2008; 283: 29-35.
Beven S. The Aquaponics Guidebook. 3rd ed. Aquaponic

Europe, London, UK. 2010.
Burut-Archanai S, Eaton-Rye JJ, Incharoensakdi A,

Powtongsook S. Phosphorus removal in a closed

recirculating aquaculture system using the cyanobacterium

Synechocystis sp. PCC 6803 strain lacking the SphU

regulator of the Pho regulon. Biochemical Engineering

Journal 2013; 74: 69-75.

Acknowledgements

This research was financially supported by the Integrated
Innovation Academic Center Chulalongkorn University
Centenary Academic Development Project (CU56-FW14),
with the partial supports from the Higher Education Research

87

Aeknarin Thanakitpairin et al. / EnvironmentAsia 7(1) (2014) 81-88

Crab R, Avnimelech Y, Defoirdt T, Bossier P, Verstraete W.

Nitrogen removal techniques in aquaculture for a

sustainable production. Aquaculture 2007; 270: 1-14.
Funge-Smith SJ, Briggs MRP. Nutrient budgets in intensive

shrimp ponds: implications for sustainability. Aquaculture

1998; 164: 117-33.
Graber A, Junge R. Aquaponic systems: Nutrient recycling

from fish wastewater by vegetable production. Desalination

2009; 246: 147-56.
Hart P, O’sullivan D. Recirculating system: Design, construction

and management. Turtle Press Pty, Australia. 1993.
Liao PB, Mayo RD. Salmonid hatchery water reuse system.

Aquaculture 1972; 1: 317-35.
Lin CK, Nash GL. Asian Shrimp News, Collected Columns.

Asian Shrimp Culture Council (1989-1995) 1996; 125
36.
Nootong K, Pavasant P, Powtongsook S. Effects of organic

carbon addition in controlling inorganic nitrogen

concentrations in a biofloc system. Journal of the World

Aquaculture Society 2011; 42(3): 339-46.
Nootong K, Powtongsook S. Performance evaluation of the

compact aquaculture system integrating submerged

fibrous nitrifying biofilters. Songklanakarin Journal of

Science & Technology 2012; 34(1): 53-59.
Rakocy J, Hargreaves J. Integration of vegetable Hydroponics

with fish culture: A review. In: Techniques for modern

aquaculture, proceedings of aquaculture engineering

conference, Washington on June. 21-23, 1993.
Rafiee GR, Saad CR. Nutrient cycle and sludge production

during different stages of red tilapia (Oreochromis sp.)

growth in a recirculating aquaculture system. Aquaculture

2005; 244: 109-18.
Timmons MB, Ebeling JM, Wheaton FW, Summerfelt ST,

Vinci BJ. Recirculating aquaculture systems. 2nd ed.

Cayuga Aqua Ventures, Ithaca, New York, USA. 2002
Wilson G. Australian barramundi farm goes aquaponic.

Aquaponics Journal 2005; 37: 12-16.

Received 16 November 2013
Accepted 2 December 2013
Correspondence to
Mr. Aeknarin Thanakitpairin
Department of Environmental Sciences,
Faculty of Science and Technology,
Rambhai Barni Rajabhat University,
Chantaburi, Thailand
Tel: +668 4347 3739
E-mail:

88