Abstract: The aim of this study was to investigate the nitrate concentration on lettuce (Lactuca sativa cv. Px06516006) grown in fish culture water. This study was carried out in Queretaro State, Mexico. Lettuce cultivars were grown in a floating system inside a plastic greenhouse. The cultivation of lettuce was divided into 10 beds; four beds were used as a control group, with a standard nutrient solution and the other six beds were used as treatment group, fish culture water with the addition of missing nutrients. A density of 30 plants m-2 was used. Two trial periods were tested in the summer of 2008, 29 days from April 11 to May 9 for the first experiment and 37 days from May 22 to June 27 for the second experiment. The system was assessed on basis of leaves nitrate content, total fresh weight and total dry weight. In both trials the final nitrate content of leaves was less than 2400 mg kg-1. No significant differences (p<0.05) were found for fresh and dry weights among treatments. Based on these results it is shown that fish culture water is suitable for low nitrate content lettuce production with no detriment to plant quality neither yields during summer.
INTRODUCTION
Nitrate and ammonium are the major sources of nitrogen absorbed by plants. Most of the ammonium has to be incorporated into organic compounds in the roots, whereas nitrate is readily mobile in the xylem and can also be stored in the vacuoles of roots, shoots and storage organs. Nitrate accumulation in vacuoles can be of considerable importance for cation-anion balance, osmoregulation, particularly in so-called nitrophilic species, and in regards to the quality of vegetables and forage plants (Marschner, 1997). Sources which provide humans with nitrate can be of exogenous and endogenous origins. One of the main exogenous sources is the consumption of vegetables which represents as much as 60 to 90% of daily nitrate intakes. The main endogenous source is the L-arginine-NO pathway, which is always active throughout the body and produces NO from the amino acid L- arginine and oxygen (De Graaf, 2006).
Nowadays still there is a controversy whether nitrate has harmful or beneficial effects on human health (L`hirondel et al., 2006; Ward et al., 2006). Harmful effects of nitrate arise when nitrate is reduced to nitrite by bacteria in the gastrointestinal tract. Nitrate and nitrite to smaller extend are involved in metabolisms that can result in formation of N-nitrosamines, which are carcinogenic and can caused gastric cancer and urinary bladder cancer. Another harmful effect is methamoglobinaemia caused by reduction of nitrate to nitrite and nitric oxide that oxidizes hemoglobin in red blood cells to abnormal form known as methaemoglobin. This compound can not transport oxygen causing lack of oxygen in body tissues. This condition generally affects infants up to 12 months old. Beneficial effects consist of relations between nitrate and the killing of pathogens. It may also help to kill ingested pathogens in the stomach and improve gastric mucosal blood flow and mucus secretion. Dental caries, skin infections, urinary tract infections may be inhibited by growth-inhibition or self-destruction of harmful bacteria that are exposed to acidified nitrite (De Graaf, 2006).
The leaves of lettuce plants can accumulate nitrates of up to 6000 mg kg-1 when grown in hydroponics as was done in northern Europe in winter (Gent, 2006). Ioslovich and Seginer (2002) also proposed a control policy to reduced nitrate in lettuce cultivars for fixed spacing systems that consisted to start with the highest permissible temperature and with abundant nitrate supply and then to switch down to the feasible temperature and the lowest permissible nitrate supply, this reduced the final nitrate concentration on lettuce plants. Due to concerns about harmful effects of nitrate on human health, the European Union has tried to minimize accumulation of these chemicals in the environment and food. In lettuce and other leafy vegetables nitrate content has become a quality mark (Abubaker et al., 2007; D`Antuono et al., 2007; Prasad and Chetty, 2007), for greenhouse lettuce cultivations maximum allowable nitrate concentration for lettuce harvested in the winter from October 1 to March 31 is 4500 mg kg-1 and in summer from April 1 to September 30 it is 3500 mg kg-1 (De Graaf, 2006).
The objective of this research was to determine whether fish culture water is suitable for lettuce (Lactuca sativa L.) productions in floating bed system using nitrate plant content as an assessing variable. It is well known that fish can supply plants with nitrogen and phosphorous (Hayashi et al., 2008; Lennard and Leonard, 2006). In this study the phosphorous fish water content was neglected. So, fish culture water was considered as a partial source of nitrogen.
MATERIALS AND METHODS
Experimental Site
The lettuce was grown inside a plastic greenhouse, 9 m wide, 12 m
long with the gutter at 4.2 m and the ridge at 5.9 m high. The lateral
ventilation area was 48 m2 and the roof ventilation area was
7.2 m2, 44 and 6.6% of the total covered area, respectively.
The fish production tank was inside a plastic greenhouse with almost no
ventilation, to keep the water temperature above 25 °C. These experimental
greenhouses are located in Queretaro State University, campus Amazcala,
Mexico at a longitude of 100° 16`W; latitude, 20° 42`N; altitude,
1920 m.
Lettuce Crop System
Lettuce seeds (Lactuca sativa cv. Px06516006) were germinated
(22 days) using a commercial substrate at about 25 °C and 70% of relative
humidity. Then they were transplanted into the floating system for two
trial periods of 29 days for the first experiment from April 11 to May
9 and 37 days for the second experiment from May 22 to June 27 of 2008.
Lettuce cultivars were grown in a floating bed system divided into 10
beds, 1.2 m wide, 2.5 m long and 0.12 m deep with a total cultivation
area of 30 m2 (Fig. 1). Each bed had a plant
density of 30 plants m-2, 90 plants per bed. Four beds were
used as a control; with a standard nutrient solution, and the other six
beds were used as treatment; with fish culture water with the addition
of missing nutrients. It was assumed that fish only provide a part of
nitrogen to water so nitrogen was complemented and all other nutrients
were added content. The nutrient solution was proposed based on that of
RodrÃguez-Delfin et al. (2001) all quantities were reduced
by 0.75 factor in order to induce a reduction on the final nitrate content
on lettuce plants (Ioslovich and Seginer, 2002; Petropoulos et al.,
2008; Andersen and Nielsen, 1992). The elements quantities and the chemical
compounds used in the nutrient solution are shown in Table
1. The initial nitrogen content was determined as nitrates using an
NO3 meter (Horiba Ltd. Kyoto, Japan). In both, the control
and treatment, all water was recirculated twice a day to provide aeration
for the lettuce root system at 9:00 and 18:00 h.
| Fig. 1: | Schematic distribution of beds |
| Table 1: | Nutrient solutions 75% (ppm) and chemical compounds employed for the nutrient solutions |
| This table was modified from RodrÃguez-Delfin et al. (2001) | |
Aquaculture System
The fish used in the aquaponic system were tilapia (Oreochromis
niloticus). At the beginning of first trial period, the tank was stocked
with 920 Tilapias of an average weight of 15 g at approximately five weeks
old. Fish feeding was calculated as function of fish age and body weight.
Fed times and food rations were determined using the growth table for
tilapia (O. niloticus) production under intensive conditions presented
by Morales-DÃaz (2003). Commercial floating food pellets of 1.5
mm which contains 35% protein, 3% fat and 5% fiber were used to feed the
fish.
The tank was equipped with an aeration system built with stone diffusers, hose and a blower of 2.5 Hp. The aeration system was controlled with an ON-OFF control. Ten minutes each hour from 19:00 to 8:00 h and five min from 9:00 to 18:00 h.
Assessed Variables
Leaves nitrate content; total fresh weight and total dry weight were
the variables, taken nitrate content as a principal variable. At the beginning
of each trial a sample of 10 small plants was taken to determine the variables
initial values. During the experiments a sample of nine plants, chosen
randomly was harvested from each bed, 90 plants in total every week. To
determine nitrate content 30 plants were used (12 for the control and
18 for the treatment) and 60 plants were used to determined total fresh
weight and total dry weight (24 for the control and 36 for the treatment).
All plants were harvested between 8:30 and 9:00 h and then placed in a
dark cold container to minimize plant physiological activity in order
to avoid changes in plant nitrate content (Chandra et al., 2008;
Prasad and Chetty, 2007; Enninghorst and Lippert, 2003).
Nitrate content was determined using an NO3 meter (Horiba Ltd. Kyoto, Japan). The time used to measure nitrate content in plants was determine taking into account that in Mexico lettuce plants are usually harvested in the morning time to avoid quality detriment due to solar heat. On the other hand, it was determined that at morning the content of nitrate in lettuce plants is higher than in afternoon (Fig. 2). To obtain plant sap complete plants were smashed until a homogenous paste was obtained from which the sap samples were taken.
To measure fresh and dry weights a balance (Adventurer, Ohaus Corp. Pine
Brook. NJ. Max. Cap. 210 g, Readability 0.0001 g) was used. All plants
were dried at 75 °C, using a stove (RIOSSA Model HSF-41) until the
constant weight was achieved.
| Fig. 2: | Nitrate content in lettuce plant according time of the day |
Extra Analysis
At the end of each trial of lettuce production a bacteriological analysis
was done checking for Escherichia coli and Salmollella sp.
(NOM-112-SSA1-1994 and NOM-114-SSA1-1994). None of these bacteria were
found. Also, at the end of both lettuce production cycles a fish tissue
analysis was done in order to looking for any excessive metal mineral
concentration taking a sample of 50 g for the control and treatment. These
concentrations were determined by atomic absorption spectrometry (NOM-117-SSA1-1994).
Statistical Analysis
The experiment design adopted was a completely randomized design.
The statistical analysis was done using the SAS, v.9 system software with
one way ANOVA with a significance level of p<0.05.
RESULTS AND DISCUSSION
Nitrate Concentration on Leaves
Figure 3 shows nitrate behavior along the second
trial period. The content of nitrates was higher in the beginning than
at the end in both the control and treatment samples. It can be explained
if we consider that plants need large quantities of nitrogen at the beginning.
In both trial periods the final nitrate content of the leaves was less
than 2400 mg kg-1. According to EU standards for lettuce harvested
between April 1 and September 30 the maximum nitrate content is 3500 and
2500 mg kg-1 for production in greenhouse and on open field,
respectively. In Fig. 3 it can be seen how the nitrate
content of lettuce leaves remained high during most of the time when it
had been expected to decrease from the third week onward. In fact, in
some of the treatment samples nitrate content was higher, in some cases,
more than that of the EU standard. This behavior was observed to be due
to the lack of sun light caused by cloudy days. According to De Graaf
(2006), the nitrate content on lettuce leaves increases when the photosynthesis
plant rate is less than the conversion plant rate and vice versa, Behr
and Wiebe (1992) also found that there exist a close negative correlation
between nitrate content and photosynthesis. In this case the cloudy days
caused low sun radiation and low temperatures so the plant photosynthesis
plant rates decreased causing the nitrate content of the leaves to rise.
This explains why the length of the second trial period was longer than
that of the first. As these investigators were waiting for sunny days
to confirm the decrease of nitrate content in lettuce plants.
| Fig. 3: | Nitrate content development during the second trial period (a) control (b) treatment and (c) average values |
Fresh and Dry Matters
In Fig. 4, it can be seen the behavior of average
values for fresh and dry matter during the second trial period. Figure
5 depicts the behavior for fresh to dry weights ratio. This ratio
decreases rapidly in the first days decreasing from 0.102, in both control
and treatment samples, and reaching values from 0.049 and 0.060 respectively.
Similar values for fresh weight as those found in study have been reported
by Lennard and Leonard (2006). Dry matter was always less than 10% of
fresh weight. In fact this relation tends to decrease from 10 to 5%. Seawright
et al. (1998) carried out an experiment in aquaponic systems with
a complete nutrient solution with different fish biomass. All treatments
showed not statistically differences for plant biomass. However, nitrate
content in plants were not reported.
Fish Analysis
At the end of the first and second trials, a sample of 30 fish were
captured and weighed. The average fish weight was 29.5 and 49.5 g, respectively.
The fish growth corresponds with that on the table by Morales-DÃaz
(2003) in which was based the feeding management. The fish tissue analysis
for the control and the treatment were similar showing no abnormal mineral
concentration. Mohammad and Hossam (2007) found that heavy metals accumulate
in fish organs in different concentrations which trend was liver, gills
and muscle. In this study only muscle samples were taken and this can
explained way not differences were found. From these results it can be
said that the fish may grow in a nutrient solution for lettuce hydroponic
production without any adverse effect on fish growth.
Statistical Analysis
For the first trial in the plant nitrate content there were no significant
differences between the control and the treatment, however in the second
trial period there were significant differences. This is attributed to
the lack of sun light that took place during the second experiment. In
general the plant nitrate content was higher in the treatment than in
the control. In both trial periods no significant differences were observed
between the control samples and treatment samples for fresh matter and
dry matter (Table 2).
| Fig. 4: | Development of average values for (a) fresh weight and (b) dry weight during the second trial period |
| Fig. 5: | Dry to fresh weight ratio development during the second trial period (a) control (b) treatment and (c) average values. DW: Dry weight; FW: Fresh weight |
| Table 2: | Final values for the two trial periods |
| NO3: Nitrate content; TFW: Total fresh weight; TDW: Total dry weight. Different letter mean significant differences (p<0.05). Length of the first trial period 29 days. Length of the second trial period 37 days trial | |
According to the results found in this research it could be concluded that fish culture water can provide nitrogen to the plants and it is suitable for production of lettuce plants with low levels of nitrate on leaves and no detriment on plant quality and yield during summer. It also contributes to the reuse of fish culture water.
ACKNOWLEDGMENTS
This research was partially supported by the Mexican National Council of Science (CONACYT), FIFI 2008 and Proyecto de equipamiento del laboratorio de Biosistemas de la Facultad de IngenierÃa (UAQ), grant No. FIN-2008-07. Authors acknowledge to Adriana MedellÃn and Silvia C. Stroet.