E3S Web of Conferences 68, 04003 (2018) https://doi.org/10.

1051/e3sconf /20186804003
1st SRICOENV 2018

Phytoremediation Potential of Typha orientalis

and Scirpus littoralis in Removal of Nitrogen
and Phosphorus from Intensive Whiteleg
Shrimp Wastewater
Ngo Thuy Diem Trang1,*,, Lam Thi Nhu Mo1, Vo Chi Linh1 ,and Hans Brix2
1
College of Environment and Natural Resources, Can Tho University, Vietnam
2
Department of Bioscience, Aarhus University, Ole Worms Allé, Building 1135, 8000 Aarhus C,
Denmark (hans.brix@biology.au.dk)
*
Corresponding author’s address: Can Tho University, College of Environment and Natural
Resources, Department of Environmental Sciences, Campus II, 3/2 Street, Ninh Kieu District, Can
Tho City, Vietnam.
Cell: +84 (0) 909 243 703; Fax: +84 (292) 730392

Abstract. The rapid development of whiteleg shrimp farming in the

Mekong delta of Vietnam has an adverse impact on the environment due to
large amount of nitrogen and phosphorus content in wastewater and pond
sludge/sediment. Phytoremediation is a promising technique to use plant
for mitigating environmental impacts from intensively whiteleg shrimp
culture. Growth responses, nitrogen and phosphorus removal of Typha
orientalis and Scirpus littoralis were assessed at three water levels of +15
cm, +30 cm and +45 cm. They were arranged in a completely randomized
design with three replications. The plants were supplied wastewater from
intensive whiteleg shrimp tanks once every two weeks. Waterlogged
assessment was conducted for 71 days. Water levels significantly affected
plant growth rate and nutrient removal capacity. S. littoralis grew well with
a lower mortality rate and had no statistical reduction of biomass compared
to T. orientalis at the highest water level of +45 cm. T. orientalis had the
best perfromance in growth and biomass responses to waterlogged at water
levels of +15 cm and +30 cm. The results indicated that S. littoralis was the
best of choice to grow in waterlogged condition of the shrimp pond for
maintaining water quality.

1 Introduction
Total area and production of shrimp farming in Vietnam in 2013 was 655.156 hectare
and 487.960 tonnes, in which the Mekong Delta (MD) (8°33΄- 10°55΄N, 104°30΄-
106°50΄E), South Vietnam, accounted for over 90% and 60% of Vietnamese shrimp

*Corresponding author: ntdtrang@ctu.edu.vn

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative
Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/).
E3S Web of Conferences 68, 04003 (2018) https://doi.org/10.1051/e3sconf /20186804003
1st SRICOENV 2018

farming area and production [1]. Specifically, whiteleg shrimp (Litopenaeus vannamei) has
increased rapidly in production in recent years, accounting for 38.16% of total shrimp
production from 6.4% of the total farming area of the country. Planning to 2020, the MD
will produce 310.000 tonnes and 60.000 ha of whiteleg shrimp farming. Anh et al. [2]
reported that to produce 1 ton of shrimp 5.345-7.151 m3 wastewater, 259 kg BOD, 769 kg
COD, 1170 kg TSS, 30 kg N, 3.7 kg P and 4.8 kg N-NH3 were discharged into the
environment. Shrimp aquaculture growth in Vietnam has suffered many problems which
similar to Asia’ situation in recent years. The major factors contributing to the problem in
sustaining shrimp aquaculture are disease outbreaks, environmental degradation and poor
management practice [3]. Poor water quality from the river is one of the causes in failure of
3.081 ha whiteleg shrimp [1]. Therefore, there is an urgent need to develop a more
sustainable aquaculture industry that uses less water, and that does not deteriorate the water
quality of the rivers. Phytoremediation is defined as the use of plants and their associated
microbes for environmental clean-up [4]. One of the phytoremediation processes, in which
plants are used to remove contaminants from soils or water into harvestable plant biomass,
is called phytoextraction. Phytoextraction is used mainly for extracting heavy metals from
polluted soils and water [5], but the use of plant uptake and plant-mediated conversions also
has great potential for the removal of nutrients from nutrient enriched waters.
In the present study, Scirpus littoralis Schrab and Typha orientalis C. Presl were chose
to study on waterlogged stress. In fact, T. orientalis and S. littoralis are cultivated in the
paddy field and/or in the shrimp ponds in the MD for human food and providing habitat for
shrimp, respectively. To understand the potential effects of increased water levels on these
aquatic macrophytes, information is needed regarding the duration and water regime
exposure in relation to the response of the plant species in term of growth, biomass and
nutrient removal. Therefore, the present study was carried out to identify waterlogged-
tolerant species through survival, growth, biomass and nitrogen (N) and phosphorus (P)
removal with the hypothesis that S. littoralis had better performance than that of T.
orientalis.

2 Materials and Methods

2.1 Experimental Setup

A completely randomized design consisting of two plant species (Scirpus littoralis
Schrab and Typha orientalis C. Presl) and three water levels (+15, +30 and +45 cm) was set
up in triplicates. The corresponding unplanted containers were filled with three water levels
of +15, +30 and +45 cm which were considered as control treatments. The experiment was
conducted in the greenhouse at the College of Environment and Natural Resources of Can
Tho University, Vietnam (10.03° N latitude and 105.76° E longitude).

2.2 Plant Materials and Growth Condition

Rhizomes and young plants of the two studied species were collected from the fields.
The plastic containers with the size of 50-L, 45-L and 120-L were filled with a 25-L, 30-L
and 80-L of wastewater that made up water levels at +15, +30 and +45 cm above sediment,
respectively. Sixteen kg of sediment (76.2% dry weight) from the intensive whiteleg shrimp
pond was filled in each container to mimic the bottom pond condition. The individual
young and similar size plants of each species were placed in the container for acclimation
two months before flooding. The plants were thinned out to keep a density of 15 plants/m2

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[6]. Wastewater from the intensive whiteleg shrimp culture tanks with stocking density of
400 shrimps/m3 water that were used as growth solution for the plants. A valve connected
with Ø 34 mm PVC tube was fitted through a hole in the side of the containers at the
middle of water level to sample water (Figure. 1).

Fig.1. An overview of the treatment plots which represented one plant species for theree water levels
of +15, +30 and +45 cm above sediment

2.3 Plant Growth and Biomass Measurement

At harvest, plants were carefully removed from the sediment and the roots were
carefully washed to remove particle and sediment. The fractions were weighed for fresh
biomass and dried at 60°C until constant weight to determine for dry weight. Relative
growth rate of biomass was calculated from the difference of natural logarithm of harvested
biomass and beginning biomass divided by time.

2.4 Water Sampling and Analysis

Water samples were taken every two weeks at 6:00 - 7:00 AM. Temperature (°C),
dissolved oxygen (DO, mg/L), redox potential (Eh, mV) of the water in the containers were
measured at the experimental site using respective portable meters. Water samples were
collected and transferred immediately to the laboratory for analysis of COD, NH4-N, NO2-
N, NO3-N, TKN, PO4-P and TP. All the analytical measurements were carried out
according to Standard Methods [7].

2.5 Statistical Analysis

Data were tested for normal distribution, variance homogeneity (Levene’s test) and
logarithmically transformed if necessary. Differences in water quality were identified by
two-way repeated measures ANOVA (General Linear Models) using Type III sum of
squares. Differences in plant growth and biomass were identified by one-way ANOVA.
Tukey Honestly Significant Differences (HSD) was used to compare significant differences
between treatments at the 5% probability level. The software Statgraphics Centurion XV
(StatPoint, Inc., USA) was used for all statistical analyses.

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3 Results and Discussions

3.1 Plant Growth and Biomass Allocation

Water levels significantly affected plant survival rate, number of new shoot and root
length (p<0.05; Figs. 2A, 2B, 2C), but did not affect plant height and growth rate of shoot
and root (p>0.05; Fig. 2). The same findings in Typha domingensis when the plants were
flooded at 40-137 cm depth for six weeks [8]. T. orientalis in the water level of +45 cm
showed stress symptom of leaf rolling and wilting at the 6th week of waterlogged, and the
major part of shoots eventually dried out at the 10th week of waterlogged. Therefore, at the
harvest we counted the number of survival plant for T. orientalis in the water level of +45
cm which was zero due to no green leaves, but number of new shoot and biomass of those
plants was collected and measured. S. littoralis had an average of 21% mortality at the
water level of +45 cm (Figure. 2A). Although two tested species have been known as
wetlands plants and they were grown in the commercial extensive shrimp pond in the MD,
they both showed stress of waterlogged at the water level of +45 cm. It can be explained
that the growth condition with sallow sediment layer with low nutrient concentration in the
sediment and in the wastewater, which might cause unfavorable condition for plant growth
[9].

Fig. 2. Survival rate (%, A), number of new shoot (B), shoot height and root length (C) and growth
rate of shoot and root (D) of T. orientalis and S. littoralis grown at different water levels. Values are
the means of 3 replicates ± S.D. Different letter a,b,x,y indicates significant difference based on a Tukey
HSD test (p<0.05).
Number of new shoots of S. littoralis was higher than that of T. orientalis in all water
levels and it was also reduced at the highest water level of +45 cm (p<0.05; Figure. 2B).
The root length of both species tended to be shortened at the highest water level of +45 cm
(p<0.05; Figure. 2C). Between two tested species S. littoralis showed more tolerant to
waterlogged condition in term of survival. Grace [10] and Newman et al. [9] reported that

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nutrient enrichment and shallow water depths (i.e., 20 - 30 cm) were usually favor the
dominance of Typha species in wetlands. While T. latifolia and T. domingensis can grow at
a wide range of water depths (0 - 115 cm), these species have high shoot density and
flowering incidence only within a narrow range of water depths [10]. As water depth
increases, T. domingensis increases its shoot height, produces fewer but larger ramets, and
decreases the incidence of flowering. Increasing water depth reduces the anchorage
capacity of plants by decreasing biomass allocation to rhizomes and roots [10]. Water
levels significantly affected shoot and root dry and fresh weight at the harvest of the two
species (p<0.05; Figure. 3) but did not affect relative growth rate (RGR) of their biomass
(p>0.05). The species factor also influenced fresh weight and dry weight of the shoots and
the roots. Although the biomass was reduced in both species in response to water levels
(p<0.05), it nevertheless was significantly greater in T. orientalis than S. littoralis under
+30 cm water levels (p<0.05) S. littoralis showed no statistical reduction in fresh weight
and dry weight of the shoot and the root fractions across water levels while T. orientalis had
a reduction of these parameters at the highest water level of +45 cm.

Fig. 3. Dry weight (A), fresh weight (B), relative growth rate (RGR) of dry weight (C) and fresh
weight (D) of T. orientalis and S. littoralis grown at different water levels. Values are the means of 3
replicates ± S.D. Different letter a,b indicates significant difference between treatment in the shoot
fractions based on a Tukey HSD test (p<0.05). Different letter x,y indicates significant difference
between treatment in the root fractions based on a Tukey HSD test (p<0.05).

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3.2 Water Quality in the Growth Containers

3.2.1 Dissolved Oxygen, Redox Potential, Temperature and Chemical Oxygen Demand
Values in the Water
There were significant differences for dissolved oxygen (DO), redox potential (E h),
temperature and chemical oxygen demand (COD) in the water between treatments and
between sampling times (p<0.05; Figure. 4). Dissolved oxygen is the most critical water
quality for shrimp growth. The DO concentrations tended to increase over time (p<0.05;
Figure. 4A) and was at the lowest value in the containers planted T. orientalis at the water
level of +45 cm (p<0.05; Fig. 4A). Although the water DO concentration was the lowest in
the treatment of T. orientalis at +45 cm water level but it was higher than that of the
permitted limit (≥3.5) for requirement on shrimp rearing water quality regarding to
Appendix 1 of the Vietnamese standards No. 02-19/2014/BNNPTNT (issued on July 29,
2014) [11]. In the case of submerged sediment in our study, penetration of atmospheric
oxygen into the sediment was limited due to low rates of oxygen diffusion and, hence, low
redox potential, which inhibits plant growth through inhibition of respiration and
production of toxins in reducing conditions. Therefore, the reported water redox potential
values had similar trend to DO concentration, it increased over time and had the highest
values at the end of the study (p<0.05; Figure. 4B). The average values of Eh in the water of
the treatments were in the range of -101 to -75 mV and were classified as reduced
environment [12].

Fig. 4. Dissolved oxygen (A), redox potential (B), temperature (C) and chemical oxygen demand (D)
in the water over time of T. orientalis (BB), S. littoralis (NT) and unplanted (KC) at +15, +30 and
+45 cm water levels. Values are the means of 3 replicates. Asterisk ( *) indicates significant difference
between treatments within sampling time. Different letter a,b,c indicates significant difference between
sampling times based on a Tukey HSD test (p<0.05).
The average water temparature of the treatments were in the range of 30.3 - 31.4°C
(Figure. 4C) which was in the permitted limit (18.0 - 33.0°C) for requirement on shrimp

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rearing water quality (Vietnamese standards No. 02-19/2014/BNNPTNT). COD was not
listed in the former Circular No. 45/2010/TT-BNNPTNT [13] for permitted limit in water
quality for shrimp growth or in discharged water; however, in the updated Vietnamese
standards [11] COD parameter was placed in the permitted limit for discharge effluent after
treatment of wastewater (≤150 mg/L). In our study, COD concentrations in all the
treatments fluctuated and tended to increase at the end of the study, but they were 6-times
lower than that of the permitted limit. In sum, DO, temperature and COD concentrations in
the water across the treatments meet the Vietnamese standards requirement on shrimp
rearing water quality. The water in the treatments was qualified and can be reused for
rearing shrimp.

3.2.2 Nitrogen and Phosphorus Concentrations in the Water

Nitrite nitrogen (NO2-N) is toxic to shrimp and exposure to high concentrations may
cause retarded growth and mortalities [14]. The average NO2-N concentrations in the
treatments tended to reduce over time and increase again at the last sampling time (p<0.05;
Figure. 5A). Similar trend was observed in NO3-N, NH4-N and TKN concentrations
(p<0.05; Figure. 5B, 5C & 5D) that reflected debris of plants and microorganism body
degraded causing water re-pollution at the end of the study. In general, water NO2-N
concentrations in the treatments were in the range of 0.03 - 0.3 mg/L which was within the
permitted range for normal shrimp growth (Circular No. 45/2010/TT-BNNPTNT, <0.35
mg/L) [13]. However, NO2-N concentration the in the unplanted treatments was slightly
higher which was the same finding reported by Doan et al. [6] who using constructed
wetlands planted Typha orientalis to purify intensive white leg shrimp waste water. Gross
et al. [14] suggested a safe concentration for whiteleg shrimp production in the ponds to be
less than 0.45 mg/L NO2-N. The NO3-N concentration is not harmful for shrimp growth;
however, higher accumulation of NO3-N might be toxic to shrimp [15]. In this study, NO3-
N concentration likely reduced overtime for all treatments, except for the last sampling, and
were negligible ranging from 0.14 to 0.37 mg/L. The NH4-N concentrations in the
treatments were in the range of 0.3-0.8 mg/L in which the planted treatments had the lower
NH4-N concentrations compared to in the unplanted treatments (p<0.05; Fig. 5C). The
planted treatments with +15 and +30 cm water levels had lower NH4-N concentrations
because the plants grew well in these treatments resulting in a better NH4-N uptake rate by
the plants leading to the lower TKN concentrations.

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Fig. 5. Nitrite nitrogen (A), nitrate nitrogen (B), ammonium nitrogen (C) and total kjeldahl nitrogen
(D) in the water over time of T. orientalis (BB), S. littoralis (NT) and unplanted (KC) at +15, +30 and
+45 cm water levels. Values are the means of 3 replicates. Asterisk ( *) indicates significant difference
between treatments within sampling time. Different letter a,b,c indicates significant difference between
sampling times based on a Tukey HSD test (p<0.05).

Fig. 6. Orthophosphate (A) and total phosphorus (B) in the water over time of T. orientalis (BB), S.
littoralis (NT) and unplanted (KC) at +15, +30 and +45 cm water levels. Values are the means of 3
replicates. Asterisk (*) indicates significant difference between treatments within sampling time.
Different letter a,b,c indicates significant difference between sampling times based on a Tukey HSD
test (p<0.05).
The concentrations of PO4-P and TP of all the treatments reduced over time but
increased at the last sampling point (p<0.05; Figure. 6A & 6B). There were significant
differences between the treatments within sampling time. Although P is not harmful to
aquatic animals, P supports algae and aquatic plants growth [16]. In sum, water quality
parameters across the treatments meet the Vietnamese standards requirement on shrimp
rearing water quality. The treatments with plants helped to maintain better quality than that
of the unplanted treatments.

4 Conclusion
The treatment with presence of plants helped to maintain a better water quality than that
in the unplanted treatments. The results indicated that S. littoralis was the best of choice to
grow in waterlogged condition of the shrimp pond for maintaining water quality. However,
further studies must be conducted using a commercial shrimp pond or a settlement pond to
grow S. littoralis together and/or separate to further assess the feasibility of using this
species.

Acknowledgments
This work was financially supported by the project grant B2016-TCT-10ĐT from Ministry of
Education and Training of Vietnam.

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