International Journal of Zoology Studies

ISSN: 2455-7269
www.zoologyjournals.com
Volume 1; Issue 2; February 2016; Page No. 01-08

The effects of recirculating aquaculture system effluent water on the growth of Moina macrocopa
(Straus)
*1
Jiun Yan Loh, 2 Han Kiat Alan Ong, 3 Yii Siang Hii, 4 Gideon Khoo
1
Department of Aquatic Sciences, Faculty of Applied Science, UCSI University. No. 1, Jalan Menara Gading, UCSI Heights,
Kuala Lumpur, Malaysia.
2
Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Malaysia.
3
School of Fisheries and Aquaculture Sciences, Universiti Malaysia Terengganu, Malaysia.
4
Faculty of Science, Universiti Tunku Abdul Rahman, Malaysia.

Abstract
In the present study, Moina macrocopa were grew at four different densities (4, 20, 40 and 80 individuals/50 mL), to investigate the
effect of population density on the growth and reproduction using aquaculture effluent throughout 11 days. The organisms in high
population densities i.e. 40 and 80 ind./50 mL of aquaculture effluent showed a lower rate of reproduction and population growth
compared to those at 4 and 20 ind./50 mL. In the lower densities, Moina population increased to the highest densities of 55 (4 ind./50
mL) and 61 individuals (20 ind./50 mL) at the first 5 and 10 days, respectively. In general, two lower densities showed a higher
population increment compared to other two tested densities. These results suggest that high population density suppressed the
growth in M. macrocopa, therefore, lower population densities are recommended for commercial hatchery production using
aquaculture effluent.

Keywords: Aquaculture effluent, hatchery, Moina macrocopa, population density

1. Introduction It also able to reduce the production cost in Moina cultivation,

Sustainable development in agriculture is a contemporary global simultaneously. Furthermore, Moina grew with fish waste
issue. Bio-conversion of liquid and solid wastes of animal origin materials contained higher nutritional values such as highly
has immense potential for industrial exploitation. These bio- unsaturated fatty acids (HUFAs) [18]. These nutrients could
converted organic products are comparatively safer than subsequently benefit the overall growth performance of
chemical products to the humans and environment [9]. Some fish/shrimp larvae in their early stages. Several studies found
nutritional contents such as nitrogen and phosphorus are present that Moina could not survive in a high population density under
in high amounts in these liquid and solid organic wastes, these extreme environmental conditions such as high ammonia and
waste materials could be recycled as a renewable energy, rather total suspended solids [2, 6, 17]. There is also limited information
than eliminated in an unproductive way [19]. available on the impact of environmental stressors to the growth
Among the zooplankton, Moina is the one of the cladocerans that of M. macrocopa, in particularly the maximum stocking
could be mass propagated using organic wastes [9]. In natural capacity of Moina in a captive environment. Therefore, this
habitats, biotic and abiotic parameters such as water quality, study aimed to investigate the effects of population density on
quantity and quality level of food available, thermal conditions, the growth of Moina macrocopa using aquaculture effluent
inter/intra-competition, predation, and population density are the collected from a Recirculating Aquaculture System (RAS)
most important factors that interact in the population growth of system.
the zooplankton [11]. Among these factors, parameters such as
population density and food availability are the predominant 2. Materials and Methods
factors affecting the growth of M. macrocopa [11, 21]. 2.1 Experimental set-up
Population density determines the production efficiency of M. The cladoceran stock, Moina macrocopa was obtained from a
macrocopa in a captive environment. This could be mainly freshwater hatchery of University Malaysia Terengganu (UMT).
attributed to the high intra-competition of space, food sources, The organisms were subsequently maintained with fresh
and the accumulation of excretory products, which could microalgae in the wet lab of University Tunku Abdul Rahman
eventually lead to a population decline in Moina cultivation. Loh (UTAR) prior to the study. The experiment consisted of 4 culture
et al., [17] proposed that fish faeces could be used as a potential densities in various Falcon tubes (BD FalconTM, USA), labeled
culture medium to propagate M. macrocopa at a hatchery scale. as T1 (4 individuals of M. macrocopa/50 mL effluent medium),
These fish excretion materials might be one of the alternative T2 (20 ind./50 mL), T3 (40 ind./50 mL) and T4 (80 ind./50 mL),
ways to mitigate aquaculture impact to the aquatic environment. and a set of control with the same densities. A total of 48 Falcon
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tubes including controls were prepared for the experiment. The 2.3 Statistical Analysis
experiment was performed in 6 replications for each treatment. All data was analyzed using Minitab version 13 (Minitab Inc.,
Culture media was prepared from non-filter effluent water USA). The data was firstly tested by the Kolmogorov-Smirnov
collected from Nile Tilapia, Oreochromis niloticus recirculating test (Normality) and Levene’s test (homogeneity-of-variance) [5]
aquaculture system (RAS), the RAS consisted of 5 tanks and 20 prior to the one-way ANOVA and Post Hoc Tests (Tukey’s
fishes of each tank. Based on cross examination, the aquaculture tests). Data was tested to compare the differences between
effluent used for this study contained a mixture of 80% fish population density, culture media, and experiment duration.
feaces, approximately 10% uneaten feed and 5–10% protein Statistical significance among the different treatments was
materials such as fish skin and debris. The water quality accepted at p < 0.05 or p < 0.001.
parameters in the RAS system e.g. total hardness, carbonate
hardness, dissolved oxygen (DO), pH, temperature, total 3. Results
ammonia nitrogen (NH3-N), nitrite nitrogen (NO2-N) and nitrate 3.1 RAS system water quality
(NO3-N) were determined using water quality test kits (JBL Dissolved oxygen (DO) levels of the recycled influent water
GmbH & Co., Germany), and Hach colorimeter (DR890, Hach, were recorded at 3.06 to 7.46 mg/L, which were slightly lower
USA) according to the Standard Methods for the Examination than DO (5.78 to 7.84 mg/L) in the fish rearing tanks (Figure 1).
of Water and Wastewater [1]. The non-filter effluent (DO: 4±1 The pH levels of the effluent (waste water) and influent water
mg/L; NH3-N: 0.44±0.15 mg/L; NO2-N: 0.10±0.05 mg/L; NO3- (treated water) were found to be in the range of 7.20 to 7.69,
N: 23.32±11.12 mg/L) contained no algae was used to determine which was relatively constant throughout the 6-month culture
the effect of density in M. macrocopa. For control treatment, de- period (Figure 1). Water temperature did not fluctuate much
chlorinated tap water (DO: 3±1 mg/; NH3-N: 0 mg/L; NO2-N: neither in the culture water nor the treated water. The lowest
0.01±<0.001 mg/L; NO3-N: 1.37±0.05 mg/L) was used as a temperature was recorded at 26.2°C, while the highest was at
comparison. During the study, culture polyether tubes were un- 29.5°C (Figure 1). Total water hardness and alkalinity
capped to allow permeable of oxygen into the culture media. (carbonate hardness) of the culture system were shown in the
Average ambient temperature was recorded at 29±1°C and the Table 1. These two parameters were slightly below the
natural photoperiod at 12 h light: 12 h dark. Culture media in the recommended range, for instance total water hardness for fish
Falcon tubes were replenished twice a week. farming is suggested between 10 to 400 mg/L, while alkalinity
should fall within 10 to 100 mg/L [27]. The NH3-N in the effluent
2.2 Moina macrocopa quantification was ranged from 0.04 to 0.60 mg/L, while the influent water had
For Moina macrocopa quantification, sterile microclothes a significantly lower level at 0.04 mg/L (Figure 1). On the other
(Calbiochem, Merck, Germany) was used to cover the mouth of hand, NO2-N level in the fish rearing tanks was ranged from
the Falcon tubes, and the culture media containing M. 0.006 to 0.071 mg/L (Figure 4.1), which were considerably safe
macrocopa were decanted slowly into Petri dishes. M. for the cultured fishes (suggested concentration <0.1 mg/L)
macrocopa was collected by rinsing the microclothes gently based on US Environmental Protection Agency [27]. However,
with 5 mL culture medium using a glass pipette. This procedure NO3-N level was slightly higher in both effluent and influent
was repeated again until all M. macrocopa were completely water due to intensive stocking density of the fish (averaging
removed from the tubes. M. macrocopa were then transferred 300 g per liter culture water). Nevertheless, no mortality of the
into different Petri dishes for quantification. The counting was Tilapia was found during the culture period. For the effluent
carried out using a Tally counter under a dissecting microscope water, the NO3-N level was ranged from 10.0 to 58.9 mg/L,
(10x to 40x magnification). After quantification, live M. while, for the influent water, it was recorded from 9.4 to 60.9
macrocopa were returned to the culture vessels, and the dead mg/L (Figure 1). This parameter was above the recommended
organisms were discarded. The experiment was carried out for range of the standard water quality (< 3.0 mg/L) suggested by
11 days. EPA [27].

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 Fig 1: Water quality in the effluent and influent water of the Nile Tilapia (n = 100) RAS system within 6 month culture period.

Table 1: Total water and carbonate hardness of the effluent and influent water
Effluent water Influent water
Month Total hardness Carbonate hardness Total hardness Carbonate hardness
(GH, od) (KH, od) (GH, od) (KH, od)
1 7.0 -8.0 6.0 7.0 - 8.0 6.0
2 7.0 - 14.0 6.0 7.0 - 14.0 6.0
3 >14.0 6.0 14.0 - 21.0 6.0
4 >14.0 6.0 - 10.0 >14.0 3.0 - 6.0
5 7.0 - 8.0 6.0 >14.0 6.0
6 >14.0 10.0 >4.0 6.0

3.2 Effects of effluent water on the population growth of M. 10. Similar growth trend was observed at T2, the M. macrocopa
macrocopa grew slowly for the first 2 days in the effluent culture. The
Figure 2 shows the comparison of population densities of M. numbers dropped slightly on day-3 and day-4. However, the
macrocopa in the control and treatment culture for 11 days. Moina population started to increase after day-4, with an
Population density of M. macrocopa cultured with aquaculture exponential scale (p < 0.05, Table 2) to day-5, and then increased
effluent (T1) was increased significantly (p < 0.05) (Table 2) on again at a much slower rate later today-8 (Figure 3). The
day-3 compared to the control (Figure 2). The population population continued to increase and reached to the peak density
densities of M. macrocopa in the control culture were relatively (61 ind.) at day-8. The population density then dropped (P <
constant with low population density (4 individuals of Moina 0.05; Table 2) at the end of the experiment. On the other hand,
macrocopa/ 50 mL effluent medium). A positive growth trend the number of Moina in control treatment was decreased from
in T1 was observed in the effluent culture (Figure 2), whereby day-1 to day-6. An absolute mortality was noticed at day-10
the population densities increased gradually from day-0 to day- (Figure 3). In T3, the population density of M. macrocopa
4. A total number of 40 individuals were observed at day-4 to showed an increment (4 ind.) at day-1, and started to decrease to
day-5. The population densities reached their peak, recorded at 23 individuals until day-5 (Figure 4). The population started to
55 individuals of M. macrocopa at day-5 (Figure 2). The increase again until day-8 (42 ind.) before it declined to 4
population in T1, however, decreased gradually after the peak individuals at day-11. A drastic effect was observed in the
and slightly increased at day-8, before it declined again at day- control treatment, whereby, no survivors were recorded from

3 
 
day-6 onwards (Figure 4). The population density of M. organism was found in the control started at day-8 (Figure 5).
macrocopa in T4 reached its peak density at the day-1 (after Statistical analysis showed that all population densities were
inoculation), recorded with 108 individuals. However, both found to be significantly influenced (P < 0.05) by the population
population densities in the effluent and control treatments densities and days (Table 3). However, there was no significant
decreased drastically after that. At the end of the experiment, difference (P > 0.05) in the population density among the groups
only 2 organisms were observed in T4 culture medium, no (T1, T2, T3 and T4) on day-8 and day-11 (Table 3).

Fig 2: Population growth of Moina macrocopa at the initial stocking density of 4 ind. per 50 mL culture
media. indicates wastewater effluent (RAS); indicates control (de-chlorinated water).
Error bars show mean ± of the standard deviation.

Fig 3: Population growth of Moina macrocopa at the initial stocking density of 20 ind. per 50 mL culture
medium. indicates wastewater effluent (RAS); indicates control (de-chlorinated water).
Error bars show mean ± of the standard deviation.

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 Fig 4: Population growth of Moina macrocopa at the initial stocking density of 40 ind. per 50 mL culture
medium. indicates wastewater effluent (RAS); indicates control (de-chlorinated water). Error
bars show mean ± of the standard deviation.

Fig 5: Population growth of Moina macrocopa at the initial stocking density of 80 ind.
Per 50 mL culture medium. Indicates wastewater effluent (RAS); indicates control
(De-chlorinated water). Error bars show mean ± of the standard deviation
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Table 2: The F and P-value from one-way ANOVA analysis used in the comparison of population density between the effluent cultures (T1, T2,
T3 and T4) along the culture period.
Number of Moina in culture treatments
Day no. T1 T2 T3 T4
F-value P-value F-value P-value F-value P-value F-value P-value
1 0.063 0.806 0.356 0.564 0.186 0.068 1.245 0.291
2 0.022 0.886 0.148 0.708 0.209 0.657 1.268 0.286
3 8.368 0.016* 0.481 0.504 0.094 0.765 0.826 0.385
4 18.060 0.002** 4.946 0.050 3.637 0.086 0.910 0.363
5 49.574 0.000*** 29.325 0.000*** 80.369 0.000*** 18.857 0.001**
6 21.076 0.001** 36.019 0.000*** 25.490 0.001** 25.900 0.000***
7 28.212 0.000*** 18.386 0.002** 24.690 0.001** 33.380 0.000***
8 10.485 0.031* 6.825 0.059 33.707 0.004** 24.103 0.008**
9 48.332 0.000*** 55.944 0.000*** 52.137 0.000*** 128.273 0.000***
10 19.692 0.011* 108.000 0.000*** 43.750 0.003** 36.750 0.004**
11 49.000 0.002** 24.143 0.008** 48.000 0.002** 36.577 0.004**
P-values with asterisk denote the significant level of study (*p < 0.05; **p < 0.01; ***p < 0.001). T1 denotes the density of 4 ind.; T2: 20 ind.;
T3: 40 ind. and T4: 80 ind. /culture.

Table 3: The significance values of all population densities in experimental period (in days) (A), and the interaction of
density versus days in various population densities in the study (B).
A) B)
Population densities Interaction F-value P-value
Day no. T1, T2, T3 and T4 Population density:

F-value P-value T1
1 15.790 0.000*** Density × Days 14.464 0.000***
2 6.561 0.003**
3 5.341 0.007** T2
4 4.926 0.010* Density × Days 7.388 0.000***
5 8.735 0.001**
6 4.505 0.014* T3
7 3.635 0.031* Density × Days 2.208 0.028*
8 1.640 0.256
9 9.500 0.000*** T4
10 12.367 0.002** Density × Days 7.743 0.000***
11 3.690 0.062
P-values with asterisk denote the significant level of study (*p < 0.05; **p < 0.01; ***p < 0.001). T1 denotes the density
of 4 ind.; T2: 20 ind.; T3: 40 ind. and T4: 80 ind. /culture.

4. Discussion the density T1 is preferable with regarding on the cost and time
The present study evaluated the growth performance of Moina consuming. This also means that M. macrocopa could be
macrocopa at different stocking densities using effluent water as harvested within a shorter period of time, thus allowing more
a culture medium from a Nile Tilapia RAS system. There are number of cultivation batches per cycle. A short production time
several factors that could affect the life history of M. macrocopa is also important for commercial livefeed producers as it could
in a culture environment, such as population density, trophic greatly reduce operational time and cost in the hatchery [7].
conditions, water quality, and water-borne chemical cues [23]. Generally, M. macrocopa has a higher density adaptation in a
Among these, population density is presumably the major captive culture environment compared to other cladoceran
limitation to the overall growth performance of M. macrocopa, species e.g. M. micrura. According to Jana and Pal [15], the
this is mainly because of the competition of space, food and growth performance of M. micrura was limited at the population
bioaccumulation of excretion materials. density of 75 ind./ L. Nevertheless, M. macrocopa showed a
The population density of M. macrocopa at T1 (4 ind./ 50 mL, better adaptation at the density of 100 to 500 ind./ L, which is
or equivalent to 100 ind./ L) showed a better growth equivalent to 4 ind. and 20 ind./ 50 mL shown in the study. Our
performance compared to T3 and T4. M. macrocopa grew at T2 study suggested that M. macrocopa should not be exceeded 500
shared the similar growth trend as in the density of T1. In terms ind./ L in an enclosed culture environment, because high
of time and efficiency, M. macrocopa at T1 reached its peak stocking density may possibly lead to a population collapse. This
population on day-5, which was 3 days earlier than the density could be caused by insufficient of space, food availability,
of T2 (highest production at day-8). Furthermore, M. macrocopa sexual transformation, and/or allelopathic effects [4, 8, 10, 12, 13, 14,
16, 22, 25, 26]
cultivated at T2 required higher starting density as compared to . The food supply of Moina embryos are closely related
T1. Therefore, instead of using a higher culture density (≥ T2), to the energy reserves of the females where the embryos are fed
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