Bulletin USAMV serie……………………70(1-2)/2013

Print ISSN 1843-5262; Electronic ISSN 1843-536X

Vegetable Production in an Integrated Aquaponic System with Rainbow Trout and Spinach

Ștefan Mihai PETREA1), Victor CRISTEA1), Lorena DEDIU1), Maria CONTOMAN1),

Paul LUPOAE2), Mirela (CRETU) MOCANU1), Marian Tiberiu COADA1)
1)
Dunărea de Jos” University of Galati, Faculty of Food Science and Engineering - Aquaculture,
Environmental Science and Cadastre Department - 800008- Galati, Domnească Street, 47, Romania
2)
Natural Sciences Museum Complex Galaţi - Botanical Garden, Street Regiment 11 Siret, no. 6A, 800340,
Romania
*
Corresponding author, e-mail: [email protected], [email protected]
Abstract. Background: In an integrated aquaponic system, the ratio between nitrogen production and
plant assimilation is a key factor for system efficiency. That is why plant density is very important to be
appropriate. Aims: The aim of this study is to expose the performances of growth parameters, both in
terms of quantity and quality, for Nores variety spinach (Spinacia oleracea) produced in an aquaponic
integrated system along with rainbow trout (Oncorhynchus mykiss), under three plant densities (V1 - 59
plants/m2, V2 - 48plants/m2 and V3 - 39plants/m2). Materials and methods: The experimental design
consist in a recirculating aquaculture system with 12 growing units, mechanical and biological water
treatment units and four aquaponic units. Three plants densities were used (59, 48 and 39plants/m2). A fish
stocking density of 136fish/m3 was used. Plants initial and final biomass was taken and a series of periodic
measurements were made. Chlorophyll a, b and carotenoids, ash and dry matter were determined from
spinach leaf. Results: The plant biomass gain registered good values and the quality of final plant
products, given by the chlorophyll a, b, carotenoids and dry matter content were in the optimal variation
interval, comparing to market spinach. Significant differences (p<0.05) between variants were observed.
Conclusion: In the present research work, we demonstrated that significant differences (p<0.05) were
recorded in terms of growth performance between the three experimental variants and that the
quality of spinach growth in an integrated aquaponic system with rainbow trout is similar to that of the
market spinach.

Keywords: aquaponic system, spinach, rainbow trout, chlorophyll, carotenoids, ash, dry matter.

INTRODUCTION

Aquaponics is the combination of closed, recirculating aquaculture and hydroponics

where effluent-rich water that results from raising fish provides a source of nutrients for growing
plants (Beauchamp, 2011). Licamele (2004) categorized it as the most efficient food production
system in terms of amount of product produced per water volume, pointing out that greenhouse
hydroponics production can generate from five to ten times more output compared to
conventional agriculture. One of the facts that contributes to such performance is revealed by
Rakocy et al.(1992), stating that vegetable yields in integrated systems exceed that of field crops
because higher planting densities are possible due to control of nutrients, constant availability of
water and absence of competition from weeds. He also mentioned that over 30 types of
vegetables have been raised in integrated systems. In these circumstances, it can be said that
hydroponic systems have great potential to be profitable, with major annual returns, derived from
both plant and fish production, fact confirmed also by Adler et al. (2000). Rakocy et.al (1992)
highlights the main characteristic of aquaponic systems, categorize them as the only biofilter that
generates income. Nowadays, consumer demand for safer food products is very much on the
increase (Dediu et.al., 2011). Spinach (Spinacia oleracea) is one of the main vegetables cultured
in aquaponics systems because it grows rapidly in response to the high levels of nutrients in
aquaculture water. Causton and Venus (1981), Evans (1972) and Hunt (1990) mentioned that
plant growth analysis is an explanatory, holistic and integrative approach for interpretation of
plant form and function. Growth and yield are functions of a large number of metabolic
processes, which are affected by environmental and genetic factors (Ozalkan et. al. 2010). Studies
of growth pattern and its understanding not only tell us how plant accumulates dry matter, but
also reveals the events which can make a plant more or less productive singly or in population
(Ahad, 1986). From my knowledge, no studies on physiological plasticity and growth
performance of spinach cultured in aquaponic conditions were made.
The aim of this study is to expose the performances of growth parameters, both in terms
of quantity and quality, for Nores variety spinach (Spinacia oleracea) produced in an aquaponic
integrated system along with rainbow trout (Oncorhynchus mykiss), under three plant densities
(V1 - 59 plants/m2, V2 – 48 plants/m2 and V3 – 39 plants/m2).

MATERIALS AND METHODS

Integrated aquaponic system description: The present experiment took place between
20th february and 4th april 2013 at the pilot recirculating system station of Aquaculture,
Environmental Science and Engineering Department from Food Science Faculty - ,,Dunarea de
Jos’’ University of Galati. The recirculating system consists in 12 rectangular shape rearing units
with a volume of 0.15m3/unit, 2 rectangular sump units with a volume of 0.29m3/unit, 1
mechanical-quartz sand water conditioning unit with backwash, 1 biological trickling filtration
unit, 1 sterilization UV filter (TETRA POND, Type UV-C 35000 and 36 Watt), recirculating
pumps, oxygenation unit (compressor Resun Air-Pump, Model: ACO-018 A with a flow of
260 l/min) and water quality control sensors. The aquaponic modules consist in 4 rectangular
glass made units (900x600x200mm), placed high above the recirculating system, on a metal
support. A lighting system made of 4 fluorescent lamps, with reddish wavelength and a luminous
power of 1080 lm was placed above the hydroponic units.Regarding the water cycle inside the
integrated system, it must be said that water that flow out from the rearing units pass first through
mechanical filter and after that, by using a recirculating pump, it goes through the biological
filtration unit and then gravitational to aquaponic modules, that flow out the treated water back to
rearing units. The total volume of water from the integrated system is around the value of 2.5-
2.7m3. An equal water flow of 6 L/minute was set for the inlet of all 4 hydroponic units.
Experimental design: Before starting the experiment, the activation of biological trickling
filtration unit was made as described by Dediu et.al. (2012). Daily ammonia, nitrite and nitrate
levels were monitored to determine the degree of ammonia oxidation to nitrate and therefore to
observe when a stable state of bacterial biomass is obtained. For the 44 days experiment, a total
number of 228 rainbow trout (Oncorhynchus mykiss), with an average initial weight of
111.77grams, was used in parallel with spinach (Spinacia oleracea), Nores variety, at an age of
25 days. The total fish biomass from the recirculating aquaculture system, at the beginning of the
experiment, had 25.51kg. Fish were divided in six groups, in duplicate. Three of them were fed
with Clasic Extra 1 P – 41% brute protein and the other three with Nutra PRO-MP-T – 50% brute
protein as in the protocol described by Hayward et.al. (1997). A total amount of 12 363.32 grams
of Clasic Extra 1 P feed and 11 579.54 Nutra PRO-MP-T was administrated during all 44
experimental days. Nores variety spinach was placed in the hydroponic units with the following
stocking densities: (V1 - 59plants/m2, V2 - 48plants/m2 and V3 - 39plants/m2). The seedlings
were obtained at Natural Sciences Museum Complex Galaţi - Botanical Garden. The support
media of spinach, cultivated in the aquaponic system, consisted of polystyrene plates with holes
for plastic special supports. Plants were placed in plastic supports and then, the supports were
filled with a few hydroton balls to ensure their stability. The distance between plants was equal
with 15cm, both for aquaponic and conventional cultured ones. The maximum capacity of an
aquaponic unit is 32 plants. Plants biometric measurements were made both at the initial and at
the end of the experiment, but also among the experiment. A total number of 6 measurements
were made, at almost equal time intervals from one another. The length of both shoot and root,
their final weight and also leaf number and dimension were determined. A set of growth
performance indices were determined by the following formulas (Ozalkan et.al 2010): LAI = L
/ P, (m2/m2); RGR = 1 / Wm * dw / dt, (g/g/day); NAR = 1 / Lm*dw / dt, (g/m 2/day);
Average LAR = ½* (Li / Wi + Lf / Wf), (cm2/g) ; CGR = 1 / P * dw / dt, (g/m2/day); where LAI
= Leaf Area Index, RGR = Relative Growth Rate, NAR = Net Assimilation Rate, Average
LAR = Mean Leaf Area Ratio, CGR = Crop Growth Rate, Wi/f = Initial/Final Dry Weight, dw
= Dry Weight Production in t Days, dt = Number of Days, P = Ground Area, L=leaf area, Li =
Leaf initial area, Lm= arithmetic mean of initial and final leaf area, Wm= arithmetic mean of
initial and final dry weight. The percentage of spinach ash and dry matter content were
determined for all three experimental variants and also for spinach derived from different markets
located in Galati city, so that the comparison between aquaponic derived spinach and market
spinach to be possible. The ash content was made by the procedure described by Akhter et al
(2013). For determining the dry matter content, spinach is oven dried at 80oC for at least for 48
hours, until it reaches constant weight. The chlorophyll a, chlorophyll b and carotenoids from
spinach were calculated using two different methods. The acetone extraction determined the
amount of chlorophyll a, chlorophyll b and carotenoids in mg/dm2 leaf follow the method
described by Wellburn (1994) and the second method, as described by Yasar et. al.(2008) in mg/g
leaf.
Water analysis: Technological water was analyzed in terms of temperature, pH, dissolved
oxygen, electrical conductivity, turbidity, BOD5, NH4+, NO2-, NO3-, PO43-, Fe3+, Mn2+, Mg2+,
Ca2+, K+, Cl-, COD, TOC, total hardness and alkalinity. The temperature and dissolved oxygen
were daily monitored with a portable WTW ProfiLine Oxi 3205 Dissolved Oxygen Meter. The
pH and electrical conductivity were measured with WTW inoLab Multi 720 SET
ph/Cond/Oxygen Meter, the turbidity with VELP portable turbidimeter TB1, BOD5 with VELP
BOD sensor system and NH4+, NO2-, NO3-, PO43-, Fe3+, Mn2+, Mg2+, Ca2+, K+, Cl-, COD, TOC,
total hardness and alkalinity were determined by Spectroquant Nova 400 spectrophotometer, with
Merk compatible kits. The luminous intensity was measured with TESTO 545 light meter.
Statistical methods: Statistical analysis was performed using the IBM SPSS Statistics 20
for Windows. Statistical differences between treatments were tested using T test (α= 0.05) after a
normality test (Kolmogorov-Smirnov). Comparisons between variants were assessed using post-
hoc Duncan test for multiple comparisons (ANOVA).
 RESULTS AND DISCUSSIONS

Water quality and fish growth performance: The mean values of main physico-chemical
parameters of water, from the integrated recirculating system (Tab. 1) were within optimal range
for both rainbow trout and spinach culture.
Tab. 1
The main physico-chemical parameters of water (mean ± S.E.)
Physico-chemical parameter Average StDEV
NH4+ (mg/L) 0.29 0.12
NO2- (mg/L) 0.15 0.06
NO3- (mg/l) 95.33 6.68
PO43- (mg/L) 3.7 1,35
Fe3+ (mg/L)* 0.37 0.16
Mn2+ (mg/L) 0.48 0.16
Mg2+ (mg/L) 30 8.18
Ca2+ (mg/L) 90.4 16.57
K+ (mg/L) 9.42 3.89
Cl- (mg/L) 27.68 3.5
COD (mg/L) 37.5 18.17
TOC (mg/L) 28.33 10.39
Total Hardness (dGH/°dH) 16.9 3.1
Alkalinity (mg/L) 199.89 71.83
Electrical conductivity(µS/cm) 649.04 66.99
Turbidity (NTU) 1.52 0.56
Temperature oC 16.83 0.77
pH 6.86 0.55
Dissolved oxygen (mg/L) 7.52 0.33
BOD5 (%) 57.1 22.15
*a total of 40g iron chelate was added in the technological water throughout the experimental period.

The nitrates concentration was slightly higher, but under the values reported by Licamele
(2009) which recorded concentrations of 50mg/l N-NO3 in a aquaponic integrated system with
lettuce (Lactuca sativa) and Nile tilapia (Oreochromis niloticus). The Fe3+concentration was kept
near to an optimal level for spinach culture by adding iron chelate as mentioned above, the K+
concentration was within the range reported by Villarroel et.al.(2011) that registered K+ values
between 9.52 and 15.9 mg/l in an integrated aquaponic system with Nile tilapia (Oreochromis
niloticus) and strawberry. The alkalinity registered values that are situated within the range
mentioned by Sikawa et.al.(2010) which reported values of alkalinity between 99 – 469 mg/l for
a hydroponic production of lettuce (Lactuca sativa) by using hybrid catfish (Clarias
macrocephalus×C. gariepinus) pond water. The temperature was kept in an optimal range for
both rainbow trout and spinach growth. The Ca2+ and Mg2+ concentration were higher comparing
to other studies as Villarroel et.al.(2011), where concentrations of 36.3mg/l for Ca2+ and 6.11mg/l
for Mg2+ were reported. The turbidity values were near to ones registered by Blidariu et. al.
(2013) who reported a mean value of 0.98 NTU with a maximum recorded of 1.54 NTU in an
aquaponic integrated system with lettuce (Lactuca sativa) and pikeperch (Sander lucioperca).
The percentage value of BOD5 was similar to the one recorded by Endut (2010) who reported
values of BOD5 between 47.3% and 65.5% by growing both african catfish (Clarias gariepinus)
and water spinach (Ipomoea aquatica) in an integrated aquaponic system. Related to rainbow
trout (Oncorhynchus mykiss) growth performance, a total weight gain of 12 440g for the group
fed with Clasic Extra 1 P – 41% brute protein and 14 939g for the ones fed with Nutra PRO-MP-
T – 50% brute protein was recorded. A final biomass of 52 892g (25 208g at the group fed with
Clasic Extra 1 P – 41% brute protein and 27 684g at ones fed with Nutra PRO-MP-T – 50% brute
protein) was registered at the end of experiment. The survival rate was 100%.
Plants growth parameters: At the beginning of the experiment, the total initial weight in
V1 (plant density of 59 plants/m2) was 23.74g, in V2 (48 plants/m2) was 16.52g and in V3 (39
plants/m2) was 15.59g. At the end of the experiment, a total plant weight of 112.88g was recorded
at V1, 101.32g at V2 and 89.47g at V3. A plant yield of 2.78g/plant was registered in case of V1,
3.26 at V2 and 3.88 at V3. By using two multiple comparison test (Tukey and Duncan –
ANOVA), it was observed that differences between V1 - V2 and V2 - V3 were statistically not
significant (p > 0.05), comparing with the differences between V1 and V3 that were statistically
significant (p < 0.05). After applying Kolmogorov-Smirnov normality test on the final shoot
height from all three experimental variants, we found a normal distribution (p>0.05), fact which
allowed us to apply the parametric tests (Fig. 1). Regarding the data series distribution, by
analyzing skewness and kurtosis it can be said that in case of V3 and especially V1, the
mesokurtic distribution has a little leptokurtic tendency, a bit sharper than a normal distribution,
more concentrated around the mean values, a little tilted to the left, with more extreme values to
the right (skewness > 0, kurtosis < 3). In case of V2, the distribution is mesokurtic with slight
platikurtic influence, a bit flatter than a normal distribution, with the tendency of values scatting
over a longer interval around the mean and also a little tilted to the left, with more extreme values
to the right (skewness > 0, kurtosis > 3).

Fig.1. Distribution histograms of final shoot height, for all three experimental variants (V1-HU1F; V2-
HU2F; V3-HU3F)

From the evolution of shoot height (Fig. 2) we can state that in case of V3 the evolution
was more symmetric comparing with V1 and V2 that was less symmetric. The distribution of
values in case of V3 had tended to the maximum value recorded at the last two measurements. It
can be also concluded that plants from V3 had a more constant growth rate comparing to those
from other two variants. It can be observed that after 18 days of experiment, plants from V2
registered the highest growth and that plants from V3 had increased their growth rate in the last
10 days of experiment. At the end of the experiment, at V1 a mean individual plant height of
14.46cm was registered, at V2 the value was 17.28cm and at V3 was 28.97cm. The differences of
plant height between the three experimental variants, at the end of experiment, were significant
(p<0.05).
 Fig. 2. The evolution of shoot height among the experimental period
(V1-HU1; V2-HU2; V3-HU3)

Plants growth indices: Growth, survival and reproduction are the three imperatives of any
organism. In plants, growth is particularly important because both survival and reproduction
depend on plant size and therefore on growth rate (Shipley, 2006). The following plant growth
indices were calculated: CGR, RGR, NAR, LAR, LAI and root-shoot ratio. The RGR evolution is
influenced by NAR and LAR. Analyzing spinach RGR makes possible to compare growth
differences that arise between the experimental variants. This method of growth analysis is
popular with many forest researchers and it is believed by some to be one of the most
ecologically significant and useful indices of plant growth (Pearcy et al.1989). The
photosynthetic efficiency of plants is indicated by NAR. We can observe (Fig. 3) that individual
NAR average values are greater in V1 (mean value 0.32 g/m2/day), comparing to V2 (mean value
0.23 g/m2/day) and V3 (mean value 0.18 g/m2/day). The differences between the variants are
statistically significant (p<0.05). Slight NAR increase and the decrease in the values of the LAR
indicate dry matter accumulation in leaves and stems. LAR is characterized as a measure of
payments balance between income and expenditure because it deals with the potentially
photosynthesizing and potentially respiring components of the plant (Hunt, 1990). The mean
individual average LAR values were as follows: 556.6cm2/g at V1, 771.59cm2/g at V2 and
1012.46cm2/g at V3. The differences between the variants are statistically significant (p<0.05).
The final mean leaf area at V1 is 253.08cm2, at V2 is 438.51cm2 and for V3 we registered a value
of 569.15cm2. From both final mean leaf area and LAR values, it can be concluded that spinach
leaves are thinner in case of V2, comparing to V1 and V3 were leaves are thicker. This particular
fact can be given on account of the plant adaptability to new growth conditions. Mean individual
value for RGR registered at V1 was 0.027 g/g/day, at V2 was 0.029 g/g/day and in case of V3,
the RGR value was 0.03 g/g/day.

Fig 3. Plant RGR, NAR and LAR for each experimental variant (V1-HU1; V2-HU2; V3-HU3)
A plant with optimum LAI and NAR may produce higher biological yield as well as seed yield
(Dutta and Mondal 1998). In our case, mean individual CGR registered the following values:
0.009 g/m2/day at V1, for V2 the value was 0.011 g/m2/day and for V3 was 0.01 g/m2/day (Fig.
4). The differences between the variants are statistically not significant (p > 0.05). Also, LAI
evolution in time shows that 9 days after the start of the experiment the values in case of V3
detach from one of V1 and V2 by getting higher while after another 9 days the same thing
happens with V2 (Fig.4).

Fig. 4. Plant CGR, LAI and root-shoot ratio for each experimental variant
(V1-HU1; V2-HU2; V3-HU3)

Regarding mean individual root-shoot ratio, at the end of experiment, the following results were
obtained: 0.24 at V1, 0.22 at V2 and 0.25 at V3. The root-shoot ratio helps to assess the overall
health of plants. The root - shoot ratio high values from V1 and V3 could be as well an indication
of a healthier plant, given that the increase came from a greater root weight and not from a
decrease in shoot weight.
Plant quality parameters: Spinach ash and dry weight were determined from all three
variants and also from market spinach. Ash content is a measure of the total amount of minerals
present within spinach while dry matter content reflects the amount of fats, proteins, vitamins,
minerals, and antioxidants. The dry matter of spinach was determined from both shoot and root
(Fig. 5). As a conclusion it can be said that shoot dry matter percentage of V2 and V3 variants
(7.57% and 7.82%) were both over the shoot dry matter content of market spinach (7.31%). The
first variant registered a shoot dry matter percentage of 7.1%. The same thing happened in case of
root dry matter where percentages as 8.99% and 9.57% were registered at V2 and respectively
V3, compared with 7.8% in market spinach. Lower root dry matter content was recorded in case
of V1 (5.43%). The values were higher than those of Roe et.al (2013) that reported 6.5% shoot
dry matter for spinach growth in field and lower than the values reported by Hannatu et.al.(2011)
who registered a shoot dry matter content of 18.25 for spinach growth also in field. Ash content
of spinach growth in aquaponic conditions registered lower percentages (V1 – 1.24%, V2 –
1.42% and V3 – 1.63%), compared with market spinach ash percentage with was 2.15% (Fig. 5).
The ash content was near the one mentioned by Akhter et.al. (2013) 1.26% and Hannatu
et.al.(2011) 1.62%, higher than the one mentioned by Umar et.al (2007) 1.08% and lower than
the ash content reported by Roe et.al (2013) 2% for spinach growth in field. Both ash and dry
matter content differences between experimental variants were statistically significant (p<0.05).
 Fig. 5. Spinach root and shoot dry matter content and ash content g% for each experimental variant
(V1-HU1; V2-HU2; V3-HU3) and also for market spinach.

Chlorophyll and carotenoid content are also important indicators of plant quality. The values of
chlorophyll a and b from aquaponic cultured spinach were measured both as mg/dm2 and also
mg/g and compared with one of market spinach. Also carotenoid content was determined for
aquaponic cultured spinach. The final value of chlorophyll a at V1 was 0.2 mg/dm2 and 0.35
mg/g, at V2 was 0.18 mg/dm2 and 1.04 mg/g and at V3 was 0.19mg/dm2 and 1.24 mg/g. The
chlorophyll b final values were 0.12 mg/dm2 and 0.17mg/g at V1, 0.13 mg/dm2 and 0.5 mg/g at
V2 and 0.21 mg/dm2 and 0.54 mg/g at V3. The carotenoid values at the end of experiment were
0.11 mg/dm2 at V1, 0.09 mg/dm2 at V2 and 0.14 mg/dm2 at V3. Both chlorophyll a and b values
for market spinach (0.98mg/g and 0.39mg/g) were lower than values registered at V2 and V3 and
higher than those recorded at V1 (Fig. 6).

Fig. 6. Chlorophyll a, b and carotenoid evolution for each experimental variant

(V1-HU1; V2-HU2; V3-HU3).

The evolution of chlorophyll a, b shows an increase after 20 experimental days for V2 and V3
variants, followed by a decrease till day 44 (Fig. 6). The differences between experimental
variants were significant (p<0.05).

CONCLUSIONS

From present study we can conclude that significant differences were recorded in terms
of growth performance between the three experimental variants. Both final height of plants and
leaf area were influenced by plant stocking density and therefore by water nutrients availability.
It can be also concluded that signs of stress related to high stocking density and maybe also to
angle – light accessibility were observed at V1 and V2, manifested by leaf shape and thinness.
 Also, it is obvious, from the evolution of LAI, that plants from V3 had an accelerated leaf
area growth rate. The root-shoot ratio, generated by a greater root weight, show off a better
health condition for plants from V3.
Regarding plants quality, ash and dry matter percentage emphasizes the fact that the
content of fats, proteins, vitamins, minerals and antioxidants from V2 and V3 aquaponic cultured
spinach is similar and even higher than that of the market spinach. The same thing must be
mentioned also for chlorophyll a and b content. Lower values are registered in case of V1
experimental variant.
Therefore, it can be concluded that plants growth in aquaponic condition are similar in
terms of quality to those growth in field and also a certain range for plant stocking density must
be found, related to both technical parameters of integrated aquaponic system and water physico-
chemical parameters, so that the final products will meet the marketable conditions. In case of
our integrated aquaponic system, the plant stocking density of 39plants/m2 is recommended to be
applied because it generated an increased stem length, overall plant mass, chlorophyll and
carotenoid content and better leaves quality.
The plant density of 59plants/m2 is too high for maintaining an optimal plant
physiological status, fact revealed also from final average individual plant biomass values (V1-
3.52g and V2-3.89g, compare with V3-4.27g).

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