Evaluation of Nitrate Level in Green Lettuce Conventional Grown under Natural Conditions and Aquaponic System

Abstract

Aquaponics integrates growing plants without soil technology with aquaculture, having an important role in recovery of nutrients from effluents. The research aimed to evaluate nitrates level in lettuce (Lactuca sativa) conventional grown under natural conditions and in integrated aquaponic system with a recirculated aquaculture system designed for pikeperch growth (Sander lucioperca). Conventional production (54 plants) has been obtained in the field without fertilizer or pesticide management. Aquaponics productions (54 plants/production) had ponds effluents as a nutritional support from the breeding of pikeperch, tanks were arranged with 255 numbers of pikeperch, each tank of 85 individuals, with a total of fish biomass of 30.76 kg. Fish individual body weight in the experiment was between 66 and 238 grams with an average of 120. 69 g. Chemical analyses were carried out to determine the level of nitrates in 5 plants grown in aquaponic system and respectively, conventional technology. The results have shown that the nitrate level is higher in the salad obtained from the aquaponic system than in conventional technology, however not exceeding the maximum permitted limits.

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244
Evaluation of Nitrate Level in Green Lettuce Conventional
Grown under Natural Conditions and Aquaponic System
Flavius Blidariu1*, Isidora Radulov2, Dacian Lalescu3,
Alexandru Drasovean1, Adrian Grozea1
1 Banat’s University of Agricultural Sciences and Veterinary Medicine from Timişoara,
Faculty of Animal Science and Biotechnologies, 30064 Timisoara, Calea Aradului 119, Romania
2Banat’s University of Agricultural Sciences and Veterinary Medicine from Timişoara,
Faculty of Agriculture, 30064 Timisoara, Calea Aradului 119, Romania
3Banat’s University of Agricultural Sciences and Veterinary Medicine from Timişoara,
Faculty of Food Science and Technology, 30064 Timisoara, Calea Aradului 119, Romania
Abstract
Aquaponics integrates growing plants without soil technology with aquaculture, having an important role in recovery
of nutrients from effluents. The research aimed to evaluate nitrates level in lettuce (Lactuca sativa) conventional
grown under natural conditions and in integrated aquaponic system with a recirculated aquaculture system designed
for pikeperch growth (Sander lucioperca). Conventional production (54 plants) has been obtained in the field without
fertilizer or pesticide management. Aquaponics productions (54 plants/production) had ponds effluents as a
nutritional support from the breeding of pikeperch, tanks were arranged with 255 numbers of pikeperch, each tank of
85 individuals, with a total of fish biomass of 30.76 kg. Fish individual body weight in the experiment was between
66 and 238 grams with an average of 120. 69 g. Chemical analyses were carried out to determine the level of nitrates
in 5 plants grown in aquaponic system and respectively, conventional technology. The results have shown that the
nitrate level is higher in the salad obtained from the aquaponic system than in conventional technology, however not
exceeding the maximum permitted limits.
Keywords: aquaponic, food safety, green lettuce, nitrates, pikeperch
1. Introduction
Current developments of European aquaculture in
sweet water require diversification of its
production through reliable methods for new
species of fish. In the past two decades, in
research it have been made more efforts in order
to develop pikeperch culture (Sander lucioperca),
a commercially valuable fish, with a rate of
increase to the size of the acceptable culture-
intensive marketing [1]. As we continuously add
food in a recirculating system, amounts of nitrates
will be accumulated and it can become dangerous
 * Corresponding author: Blidariu F., Tel 0040-
722.633.123, Email flaviusblidariu@yahoo.com
for the pikeperch. This it can be a good thing for
us because nitrates are the main source of food for
plants [2].
Aquaponics is a vegetable production system,
which integrates the growing technology of plants
without soil and aquaculture. Plants extract
nutrients from wastewater and convert the
metabolic products which could be toxic for fish.
It is a friendly method with the environment due
to the reuse of wastes and nutrients in the resulting
effluents from the fish growth [3].
The food safety for human consumption is
becoming increasingly important worldwide [4].
Terms assigning aquaponics productions as
"natural", "friendly with the environment ", "free
of pesticides", "organic", attract consumers
attention [4].

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The aim of this study was to assess the quality and
food safety of lettuce (Lactuca sativa) production
obtained in an integrated aquaponic system with
recirculating aquaculture system of pikeperch
growing, compared to the quality production of
lettuce obtained in conventional technology
2. Materials and methods
Researches were carried out in the Aquaculture
research laboratory of the Banat’s University of
Agricultural Sciences and Veterinary Medicine
from Timisoara, Faculty of Animal Science and
Biotechnologies. Recirculating system used for
this experiment presents a total water volume of 6
m³, consisting of: 7 tanks (1 tank of daily change
water, 1 tank for monitoring and control system of
water physico-chemical parameters, 1 tank for
thermal conditioning water installation, 3 tanks
populated with fisheries biological material and 1
tank was left unpopulated), biological filter,
mechanic filter, sterilization UV filter,
submersible pumps, aerations pumps, feeders,
aquaponic module.
Aquaponic module contains two rectangular tanks
of fiberglass (330 x 40 x 30 cm) placed on a metal
support one over the other.
Plants cultivation supports were made of
polystyrene (2 cm depth). In the polystyrene tiles
it has been made circular holes, representing input
place of seedlings.
Water supply was carried out through a pipe
between the submersible water pump and
sterilizing system with UV lamps, so that the
aquaponic module water supply was made directly
with water pumps from recirculating system. The
total volume of water of a cultivation tank is 317
L, so the total volume of water in the aquaponic
module is 634 L. Water flow has been set equal
for both tanks to a level of 202. 30 L/h, resulting a
daily flow of 4856. 20 L. The total volume of a
cultivation tank is changed once at 1 h and 34
minutes. Water drainage from the aquaponic
module is at the opposite side of the cultivation
tank, to the supply hose, discharge being made
directly into the recirculating aquaculture system
pumps hole.
Fish species used was represented by pikeperch
(Sander lucioperca). In the three tanks were
disposed 255 specimens, 85 in each tank. Total
weight of the 255 specimens was of 30.76 kg. Fish
individual body weight in the experiment was
between 66 and 238 grams with an average of
120. 69 g.
Biological plant material used in the experiments
was represented by lettuce, Lactuca sativa, variety
White Boston. For experiments in the aquaponic
module in Aquaculture Research Laboratory
seedlings were used with an age of 21 days. For
the seedlings production were used as vases,
plastic containers of 10 cm height. Culture vessels
were transparent in order to be able to observe the
level of nutrient solution from the medium culture.
The medium culture was represented by river
sand, placed in a layer of 5 cm. The sand was
previously washed with water from the city's
water network and subsequently sterilized through
boiling. In each culture vessel were conducted 25
sowing nests at 0.5 cm depth, with 1 cm distance
between nests per row and 1 cm between rows.
Seeds were distributed to two in each nest.
Throughout the seedling development, culture
vessels have been kept under direct solar radiation
with slight shading. Irrigation water was
represented by KNOP nutrient solution.
In order not to enter sand in aquaponic module,
plant roots were easily washed with water from
the laboratory supply network. To ensure
seedlings hold in holes plates there were made
cubical support from sponge, median chooped, in
which the seedlings were introduced. The distance
between holes made in the support plates was 15
cm between the holes per row and 15 cm between
rows. This way, the maximum capacity of the
aquaponic module is 108 plants, 54 plants in each
repetition. Each repetition had three rows of 18
plants.
The fish from the recirculating system were fed
with forage type SteCo SUPREME-10 (Coppens
International, Nederlands), 4.5mm grain, with the
following chemical composition: crude protein
(49%), crude fat (10%), crude cellulose (1.8%)
crude ash (9.4%) and 1.4% phosphorous. The
daily amount of feed was 150 g/tank respectively
450g/system, constant until the end of the
experiment.
The quantity mean (X) of water used daily for
daily change was 711 L/day, minimum value
(Min) 442 L/day and the maximum value (Max)
974 L/day.
Water from recirculating system has been
analyzed in terms of physico-chemical
compounds. pH, turbidity, electrical conductivity,

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organic compounds, dissolved oxygen, water
temperature were determined with testing water
walls, data recorded by these were stored and
displayed permanently on the HACH LANGE
Gmbh SC 1000 controller, LXG400.99.2B131
type. Determination of total nitrogen of the
samples was carried out using the Kjeldahl
method. For the determination of calcium (Ca) it
was used the complexonometric method.
Determination of sodium (Na) and potassium (K)
out of the water was made with flame emission
method. Determination of magnesium (Mg)
copper (Cu), iron (Fe), manganese (Mn), zinc (Zn)
and phosphorus (P) was performed by atomic
absorption spectrophotometry (Table 1).
Table 1. Values of physico-chemical parameters of the water
from the recirculating aquaculture system
Physico-chemical parameter Min Max Average
pH 6.55 6.80 6.55
Temperature (°C) 26.20 26.20 24.63
Dissolved oxygen (mg/L) 7.07 7.75 7.47
Electrical conductivity (µS/cm) 441.162 569.236 541.160
Turbidity (NFU) 0.524 1.548 0.985
Organic compounds (mg/L) 32.170 45.645 40.469
N (mg/L) 16.15 22.95 20.11
P(mg/L) 3.15 3.60 3.46
K (mg/L) 1.58 2.26 1.86
Ca (mg/L) 8.56 19.92 13.35
Mg (mg/L) 1.14 2.86 2.09
Na (mg/L) 15.07 18.62 17.35
Cu (mg/L) 0.018 0.026 0.02
Zn (mg/L) 0.054 0.067 0.059
Fe (mg/L) - - -
Mn (mg/L) - - -
At the aquaponic module were monitorized some
growth environment parameters (temperature,
humidity and light intensity) on both repetitions
(Table 2).
Table 2. Environment parameter values at the aquaponic module level
Parameter Repetition 1 Repetition 2
Temperature (°C)
- morning
Min. 26.4 25.9
Max. 30.1 29.6
Average 27.9 27.5
- evening
Min. 24.0 24.0
Max. 27.4 27.0
Average 26.4 26.0
Relative humidity (%)
- morning
Min. 70.4 65.5
Max. 85.4 84.8
Average 78.6 77.8
- evening
Min. 51.8 60.0
Max. 72.5 73.6
Average 65.1 66.0
Light intensity (lux)
Min. 10100.00 10133.33
Max. 10400.00 10466.67
Average 10262.00 10265.33

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To determine temperature and relative air
humidity it was used Vaisala HM humidometer
34. Luminous intensity was determined using the
EXTECH luxometer, model 401025.
Lettuce culture, used as Control Version, was
located out in open field. There have NOT been
applied any sort of chemical treatments or
fertilisation on this crop, seedlings used were
produced in the same batch as those that were
transplanted in aquaponic module. The only works
that have been applied to this culture were those of
weeding, weeds removing and irrigation.
In the Control Version seedlings were planted on
2 rows, each with 27 plants, amounting to a total
of 54 plants. Distance between rows was 30 cm,
and 15 cm between plants per row. Chemical soil
analysis showed the following levels of NPK:
0.21% N, 28.09 ppm P, 152 ppm K. Soil pH was
7.57. Determination of soil phosphorus and
potassium was made with Egner-Rhiem-Domingo
method. Determination of total nitrogen content in
the soil was made by Kjeldahl method. The pH
was potentiometry determined.
In the Control Version seedlings were planted on
2 rows, each with 27 plants, amounting to a total
of 54 plants. Distance between rows was 30 cm,
and 15 cm between plants per row. Chemical soil
analysis showed the following levels of NPK:
0.21% N, 28.09 ppm P, 152 ppm K. Soil pH was
7.57. Determination of soil phosphorus and
potassium was made with Egner-Rhiem-Domingo
method. Determination of total nitrogen content in
the soil was made by Kjeldahl method. The pH
was potentiometry determined.
Chemical analyses concerning nitrates level in
plants were carried out over a number of 5 plants
in Repetition 1, 5 plants in Repetition 2 and 5
plants in Control Version, at 25 days since the
introduction of seedlings in culture. At the same
time 3 samples of plant roots were analyzed,
representing the sum of the plants roots from
Repetition 1, Repetition 2 and Control Version.
Determination of nitrate levels in the samples was
carried out using determination of nitric nitrogen
method from the plant in acetic acid extract [5].
Obtained results were statistically processed using
the Statistica program, version 8, Duncan Test.
3. Results and discussion
In Repetition 1 (Figure 1) an average level of
nitrate (NO3-) has been determined in the
consumable part of lettuce plant (Lactuca sativa):
810.69 mg NO3-/kg fresh substance, a minimum of
775.25 mg NO3-/kg fresh substance, and a
maximum of 863.85 mg NO3-/kg fresh substance,
standard deviation being 33.592 mg NO3-/kg fresh
substance.
Figure 1. Basic descriptive statistical indices for NO3- levels of the samples from Repetition 1

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Regarding nitrates level in the plant roots sample
from Repetition 1, NO3- level has been identified,
708.80 mg NO3/kg fresh substance, lower level
than the NO3- average from this repetition.
In Repetition 2 (Figure 2) an average level of
nitrate (NO3-) has also been determined in the
consumable part of lettuce plant (Lactuca sativa):
810.69 mg NO3-/kg fresh substance, a minimum of
730.95 mg NO3-/kg and a maximum of 886.00 mg
NO3-/kg fresh substance, standard deviation being
63.813 mg NO3-/kg fresh substance. In the sample
with plant roots from Repetition 2, NO3- level has
been identified (686.65 mg NO3-/kg) lowest level
than NO3- average from this repetition.
Figure 2. Basic descriptive statistical indices for NO3- levels of the samples from Repetition 2
Figure 3. Basic descriptive statistical indices for NO3 levels
of the samples from Control Version

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The same in Control Version (Figure 3) an
average level of nitrate (NO3-) has been
determined in the consumable part of lettuce plant:
110.75 mg NO3-/kg fresh substance, minimum of
88.60 mg NO3-/kg fresh substance, a maximum of
132.90 mg NO3-/kg fresh substance, standard
deviation being 15.662 mg NO3-/kg fresh
substance. Plant roots sample from this variant has
a NO3- level of 155.05 mg NO3-/kg fresh
substance, higher than average levels of NO3- in
this variant.
The level of nitrates in the sample consisting plant
roots from the Control Version, was lower than
nitrate levels identified in the chemically analyzed
samples in the two repetitions of the aquaponic
system.
To compare nitrate levels (NO3-) in plants grown
in cultivation systems addressed (aquaponic
system for growing lettuce connected to a growth
system of pikeperch and conventional cultivation
system in natural conditions) it was used Duncan
test to multiple comparisons (ANOVA).
Table 3. Nitrate levels (mean±SD) and the
significance of the differences
NO3-
mg/kg fresh substance
Repetition 1 810.69±33.59a
Repetition 2 810.69±63.81a
Control Version 110.75±15.662
Values (mean±SD) in the same column with the same
letters indicate non-significant differences (p>0.05)
The average level of nitrates from consumable
part of lettuce plant of Repetition 1 was 810.69
mg NO3-/kg fresh substance, in Repetition 2 an
average level of nitrate was identified, identical to
that from Repetition 1, the differences being
statistically insignificant (p>0.05).
Analyzing the level of nitrates in chemical
analysed plants identified in Repetition 1 and
Control Version (110.75 mg NO3-/kg fresh
substance), we note that there is a difference of
699.94 mg NO3-/kg fresh substance. The average
level of nitrates from Repetition 1 was
significantly higher (p<0.001), that in the control
variant.
The average level of nitrates in the chemical
analysed plants identified in the Repetition 2 and
Control Version, we observe that there is a
difference of 699.94 mg NO3-/kg fresh substance,
identical to the difference between Repetition 1
and Control Version. The average level of nitrates
from the Repetition 2 was significantly higher
(p<0.001), compared to the average levels of
nitrates in Control Version.
The average of nitrate levels identified in the
aquaponic production do not exceed the normal
upper limit of nitrates (NO3-) from the lettuce
consumable part (400-4800 mg NO3-/kg fresh
substance) [6]. At the same time identified nitrates
levels do not exceed the maximum permissible
limits of 2000 mg NO3 -/kg fresh substance for
growing salad [7].
The average nitrate levels found in the aquaponic
production exceed the limit of nitrate levels
imposed in foods for children consumption, the
limit being maximum 250 mg NO3-/kg fresh
substance [8].
Conclusions
NO3- levels of lettuce aquaponically produced
(mean=810.69) are higher that the NO3- levels
(R1=708.80 R2=686.65) from their roots.
NO3- levels of lettuce conventionally produced in
natural conditions (mean=110.75) are lower than
NO3- levels of their roots (155.05).
The differences between NO3- levels from the
salad aquaponically produced and NO3- levels in
salad conventionally produced in natural
conditions are statistically significant (p<0.001),
those being higher.
Lettuce produced in the aquaponic system, with
the substrate of culture water from recirculating
system in pikeperch grown, shows nitrate levels
which not exceed food safety limits (<2000 mg
NO3-/kg fresh substance).
Acknowledgements
This work was published during the project
“DOCTORAL STUDIES FOR RESEARCH IN
TRAINING", POSDRU /107/1.5/S/80127, co-financed
by the European Social Fund through the Sectorial
Operational Programme for the Human Resources
Development 2007-2013.
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