Int. J. Mol. Sci. 2013, 14, 13808-13825; doi:10.3390/ijms140713808 

OPEN ACCESS 
International Journal of 

Molecular Sciences 

ISSN 1422-0067 

www.mdpi.com/journal/ijms 

Article 

Inland Treatment of the Brine Generated from Reverse Osmosis 
Advanced Membrane Wastewater Treatment Plant Using 
Epuvalisation System 

Mohannad Qurie 1,2, Jehad Abbadi 3, Laura Scrano 4, Gennaro Mecca 5, Sabino A. Bufo 1, 
Mustafa Khamis 2,6 and Rafik Karaman 1,7,* 

1 Department of Science, University of Basilicata, Via dell�Ateneo Lucano 10, Potenza 85100, Italy; 
E-Mails: mqurie@ccba.alquds.edu (M.Q.); sabufo@libero.it (S.A.B.) 
2 Department of Chemistry and Chemical Technology, Al-Quds University, Jerusalem, Palestine; 
E-Mail: mukhamis@yahoo.com (M.K.) 
3 Department of Biology, College of Science and Technology, Al-Quds University, Abu Dies, 
P.O. Box 20002, Jerusalem, Palestine; E-Mail: jihadabbadi@yahoo.com 
4 Department of European Cultures (DICEM), University of Basilicata, Via dell�Ateneo Lucano 10, 
Potenza 85100, Italy; E-Mail: laura.scrano@libero.it 
5 Exo Research Organization, Potenza 85100, Italy; E-Mail: g.mecca@exo-ricerca.it 
6 Department of Chemistry, Biology and Environmental Sciences, American University of Sharjah, 
Sharjah, UAE 
7 Department of Bioorganic Chemistry, Faculty of Pharmacy, Al-Quds University, 
Jerusalem, Palestine 

* 
Author to whom correspondence should be addressed; E-Mail: dr_karaman@yahoo.com; 
Tel./Fax: +972-2-279-0413. 
Received: 1 April 2013; in revised form: 28 May 2013 / Accepted: 26 June 2013 / 
Published: 3 July 2013 

Abstract: The reverse osmosis (RO) brine generated from the Al-Quds University 
wastewater treatment plant was treated using an epuvalisation system. The advanced 
integrated wastewater treatment plant included an activated sludge unit, two consecutive 
ultrafiltration (UF) membrane filters (20 kD and 100 kD cutoffs) followed by an activated 
carbon filter and a reverse osmosis membrane. The epuvalisation system consisted of salt 
tolerant plants grown in hydroponic channels under continuous water flowing in a closed loop 
system, and placed in a greenhouse at Al-Quds University. Sweet basil (Ocimum basilicum) 
plants were selected, and underwent two consecutive hydroponic flowing stages using 
different brine-concentrations: an adaptation stage, in which a 1:1 mixture of brine and 


Int. J. Mol. Sci. 2013, 14 13809 

fresh water was used; followed by a functioning stage, with 100% brine. A control 
treatment using fresh water was included as well. The experiment started in April and 
ended in June (2012). At the end of the experiment, analysis of the effluent brine showed a 
remarkable decrease of electroconductivity (EC), PO43-, chemical oxygen demand (COD) 
and K+ with a reduction of 60%, 74%, 70%, and 60%, respectively, as compared to the 
influent. The effluent of the control treatment showed 50%, 63%, 46%, and 90% reduction 
for the same parameters as compared to the influent. Plant growth parameters (plant height, 
fresh and dry weight) showed no significant difference between fresh water and brine 
treatments. Obtained results suggest that the epuvalisation system is a promising technique 
for inland brine treatment with added benefits. The increasing of channel number or closed 
loop time is estimated for enhancing the treatment process and increasing the nutrient 
uptake. Nevertheless, the epuvalisation technique is considered to be simple, efficient and 
low cost for inland RO brine treatment. 

Keywords: wastewater treatment; Ocimum Basilicum; reverse osmosis; brine; 

epuvalisation system 

1. Introduction 
The scarcity of freshwater in most countries of arid and semi-arid regions is an escalating problem, 
particularly as their populations continue to grow with constant enhancement of their living standards. 
Water claim is also accelerating due to industrial development and increasing demands of irrigated 
lands [1,2]. One of the alternative solutions for water scarcity is the use of treated wastewater in 
agriculture, which simultaneously avoids the negative impact of wastewater disposal in the 
environment. The reuse of treated domestic wastewater in agriculture has recently expanded and 
forced some governments for its inclusion in their overall water budget [3,4]. Wastewater treatment 
technology ranges from traditional low cost treatment to advanced technologies [5]. Advanced 
wastewater treatment technologies are based on combined processes of biological, chemical and 
mechanical, which include membrane techniques and disinfection. Advanced membrane technologies 
consist of microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) 
processes [6]. These technologies are able to remove particles, turbidity, cysts, bacteria, and even 
viruses. These sophisticated technologies provide treated water of good quality for unrestricted 
irrigation so that water resources can be increased and the environment protected [7,8]. The reverse 
osmosis (RO) process is widely applied after an ultrafiltration process to furnish high quality water 
for indirect potable and direct non-potable use [9]. Together with high quality water, membrane 
technologies, including RO process, generates simultaneously a concentrated by-product called brine. 
Brine is considered as a major problem in treatment plants due to its high salinity and possible content 
of toxic elements. It cannot be discharged without further purification in order to avoid health 
problems, environmental complications [10], and pollution of ground water by salts and harmful 
chemicals [11]. 


Int. J. Mol. Sci. 2013, 14 13810 

There are various options experienced (legally or illegally) for the treatment and disposal of 
generated brine including deep well injection, evaporation ponds, disposal into surface water bodies, 
disposal through pipelines to municipal sewer systems, ion exchange procedures, shrimp breading and 
hydroponic cultivation of salt tolerant plants (halophytic crops) [9�12]. The reuse of RO-brine in 
hydroponic agriculture has been proved to be successful using saline effluent up to 6 g total dissolved 
solids (TDS) per liter with different salt tolerant crops [13,14]. Moreover, this brine was a desirable 
alternative for irrigation of ornamental and landscape plant cropping [15]. The major and important 
factors controlling brine reuse in agriculture are salt concentration and brine chemical composition, 
which can affect soil and ground water quality [16]. Contamination due to toxic elements can pose 
serious hazards for both humans and ecosystems. The presence of toxic elements in wastewater used 
for irrigation can cause their accumulation in soil and plants, and consequently can affect food quality. 
The presence of such elements in the edible part of leafy vegetables should be absolutely avoided 
because of possible health problems for humans and animals [17�19]. Among other factors playing a 
role in brine reuse are: vegetation tolerance, land requirements, hydraulic loading rates, site selection 
and runoff control [20,21]. 

Epuvalisation is a biological wastewater recycling system based on hydroponic cropping 
techniques, which could also be employed as a further purification process of effluent water in tertiary 
sectors of treatment plants. This technique utilizes the roots of plants as bio-filters to remove nitrogen, 
phosphorus and other macronutrients. In addition, toxic elements and salts can be accumulated into the 
plant tissues from wastewater as well as brine [22]. The system mechanism consists of gravitational 
effluent flowing through open channels to keep the water well aerated. The channels host the plant 
roots not only for water absorption purposes but for trickling and biological filter functions as well. 
The roots play a dominant role in taking up the nutrients, thus decreasing the total dissolved solids, 
which includes nitrogen and phosphorus. The technique can be operated in a closed or open loop 
system. The open loop system is less efficient in the removal of nutrients and salinity due to minimal 
contact time, while the closed loop system is more efficient because of a relatively longer retention 
time [23]. The design of channel length, width, depth and slope is very crucial for achieving efficient 
treatment. The recommended channel�s length for an open circuit is 50 m and for a closed circuit is 
10�15 m. The recommended channel�s width in both systems is 50 cm and the depth is 9 cm [23]. The 
selection of the plant is considered an important and critical factor for the achievement of a successful 
epuvalistion system. Several ornamental plants, vegetables and grasses were found to be suitable for 
the purpose, and were selected taking into account the adaptation capacity of each plant species to 
hydroponic growth and their tolerance to salt [23]. The advantages of the epuvalisation technique 
include low cost, easy use and flexibility. On the other hand, frequency of plant replacement, energy 
requirements for water recirculation and cost of the greenhouse (under temperate climate) are 
considered as major disadvantages of this technique [23]. 

The objective of this study was to investigate the potential of using an epuvalisation system to treat 
brine generated from an inland RO unit. The system utilizes Basilicum (Ocimum basilicum L.) as the 
salt tolerant plant. Water quality parameters as well as plant growth factors were monitored and the 
objective of reaching zero liquid discharge (ZLD) was achieved. The ZLD consists in the elimination 
of any liquid discharge from the wastewater treatment plant (WWTP) by its purification and recycling [24]. 
This process can be advantageous from both an economical and ecological point of view. 


Int. J. Mol. Sci. 2013, 14 13811 

The novelty in our study is the treatment of RO-brine, characterized by high EC (mS cm-1), using 
Basilicum as a salt tolerant plant. We aimed at prior purification of RO brine water to permit its reuse. 
To our knowledge, no papers were previously published on the purification of RO brine using 
epuvalisation systems. The aim of existing works was normally the direct reuse of brine for plant 
irrigation, avoiding its purification and eventual recycling in the WWTP. In other cases the epuvalisation 
system was used for the direct treatment of wastewater with lower EC compared to RO-brine. 

2. Results and Discussion 
2.1. Results 
2.1.1. Characteristics of RO Brine 
Chemical, physical and biological characteristics of the RO brine used in the epuvalisation system 
are presented in Table 1. Brine was rich in all major ions and its EC value was almost doubled in 
comparison to wastewater (Table 2). This is not surprising, since this water is a concentrate of all 
rejected ions from the RO unit. On the other hand, brine does not contain pathogens, which are 
eliminated in the ultra-filtration sector. In fact, water input to the RO unit came from the permeate of 
the ultra-filtration sector furnished with 20 kD cutoff filters (spiral-wound unit), which has the 
capability to remove bacteria and viruses. 

2.1.2. Brine Quality during Epuvalisation Treatment 
The chemical and physico-chemical characteristics of water flowing out from each sector of the 
treatment plant are presented in Table 2. Careful examination of Tables 1 and 2 reveals that the 
RO brine is two-fold more concentrated than the effluent brine from the activated sludge stage. Our 
aim in this work is to reduce the concentration of RO brine by the epuvalisation technology to a level 
that is equal to or less than the effluent from activated sludge. This is in order to enable the water to be 
recycled within the system for further reuse, thereby achieving ZLD and minimizing its negative 
effects on the environment. 

Table 1. Chemical, physical and biological parameters of brine water generated from 

the RO unit. 

Parameter Mean value . SD (different units) a Ions Mean value . SD (mg L-1) 
pH 7.70 � 0.30 Cl- 
2,560 � 80 
EC 4.50 � 0.50 NO3 
- 
95.0 � 5.0 

TDS 2,250 � 500 PO43- 
2.30 � 0.70 
COD 330 � 55 NH4 
+ 720 � 20 
BOD 120 � 20 Na+ 330 � 25 

K+

FC 0 81.0 � 10 
TC 0 Ca2+ 154 � 15 
Mg2+ 59.0 � 13 

a

 EC, electrical conductivity (mS cm-1); TDS, total dissolved solids (mg L-1); COD, chemical oxygen 

demand (mg L-1); BOD, biological oxygen demand (mg L-1); FC, fecal coliforms (cfu mL-1); TC, total 

coliforms (cfu mL-1); SD, standard deviation of three replicates. 


Int. J. Mol. Sci. 2013, 14 13812 

Table 2. Physical, chemical and biological characteristics of influent and effluent wastewater 
by different treatment units, which include activated sludge (AS), ultrafiltration-hallow 
fiber (UF-HF), ultrafiltration-spiral wound (UF-SW) and reverse osmosis (RO). Mean 
values . standard deviations (SD) of three replicates. 

Parameter Influent a AS b UF-HF UF-SW RO 
pH 7.50 � 0.30 7.12 � 0.47 7.50 � 0.07 7.45 � 0.08 6.20 � 0.15 
EC (mS cm-1) 1.99 � 0.23 1.90 � 0.22 1.53 � 0.01 1.53 � 0.01 0.03 � 0.01 
TDS (mg L-1) 966 � 105 912 � 92 760 � 10 750 � 16 30 � 12 
COD (mg L-1) 380 � 150 182 � 96 90 � 20 46 � 13 20 � 11 
BOD (mg L-1) 242 � 138 107 � 49 56 � 25 41 � 14 10 � 5 
Cl- (mg L-1) 268 � 62 192 � 100 246 � 20 246 � 10 13.50 � 1.05 
NO3 
- (mg L-1) 15.50 � 10 12.40 � 10 10.02 � 5.02 13.03 � 3.06 2.50 � 0.10 
PO4 
3-(mg L-1) 25.69 � 3.15 14.70 � 2.2 10.16 � 1.50 3.14 � 0.50 0.23 � 0.05 
NH4 
+ (mg L-1) 91 � 64 89 � 56 85 � 11 23.30 � 3.11 5.20 � 0.21 
Na+ (mg L-1) 128 � 45 86 � 50 112 � 14 114 � 10 7.30 � 2.12 
K+ (mg L-1) 41 � 30 36 � 13 38.8 � 10 19.70 � 5.05 1.38 � 1.03 
Ca2+ (mg L-1) 65 � 21 60 � 15 65 � 12 65.70 � 13 1.75 � 0.51 
Mg2+ (mg L-1) 30 � 10 24 � 20 24 � 11 27.30 � 5.01 0.68 � 0.11 
TC (cfu/mL) (6 � 105) � 102 (2 � 104) � 101 120 � 50 0 0 
FC (cfu/mL) (5 � 103) � 102 (2 � 102) � 101 40 � 20 0 0 

a Initial composition of wastewater to be treated; b The effluent from AS is the influent to UF-HF, and so on. 

The influent and effluent quality of brine was monitored during the epuvalisation experiment. 
Water samples were collected and analyzed after 14 days (50% brine) and 28 days (100% brine) from 
plantation. Results are presented in Tables 3 and 4. Examination of both tables reveals that in 50% 
brine treatment (Table 3) the EC, TDS and chemical oxygen demand (COD) values were reduced to 
half of their initial values, while biological oxygen demand (BOD) was reduced to 66%. The reduction 
of ammonium-N was higher than that of nitrate-N. Sodium ion displayed the highest percentage of 
plants� uptake (48%) followed by K+ (25%) and Ca2+ (14%). Chloride and phosphates were reduced up 
to 66% and 22% of the initial concentration, respectively. In the control treatment, the EC and TDS 
values were reduced by 63 and 57% of their concentrations in fresh water, respectively. COD and BOD 
were reduced to 50% of their concentrations. As in the brine treatment, ammonium-N and nitrate-N were 
reduced, but the removal of the former was significantly higher. The plants were found to remove K+ 
in a higher extent than Na+ (opposite to brine treatment) and Mg2+. Chloride was reduced to about 
30%. Although PO43- concentration was low, its removal was quite high (77%). 


Int. J. Mol. Sci. 2013, 14 13813 

Table 3. Physical and chemical quality of hydroponic recycled water after 14 days of 
epuvalisation treatment with fresh water and 14 days with the 1:1 mixture brine:fresh 
water, compared to treatment with only fresh water during the same period. Mean 
values � standard deviations (SD) of three replicates. 

Brine: Fresh Water 1:1 (v:v) a Fresh Water a 
Parameter Influent Effluent % removal Influent Effluent % removal 
pH 7.30 � 0.1 7.8 � 0.2 7.40 � 0.1 7.5 � 0.10 
EC (mS cm-1) 3.82 � 0.2 1.91 � 0.2 50.0 1.74 � 0.1 1.10 � 0.1 37.0 
TDS (mg L-1) 1910 � 120 930 � 113 51.0 905 � 63.0 520 � 40.0 43.0 
COD (mg L-1) 141 � 15.0 75 � 10.0 47.0 73.0 � 10.0 38 � 10.0 48.0 
BOD (mg L-1) 61 � 10.0 20 � 10.0 67.0 40.0 � 10.0 19 � 10.0 53.0 
Cl- (mg L-1) 1631 � 500 1081 � 200 34.0 420 � 80.0 285 � 60.0 32.0 
NO3 
- 
(mg L-1) 838 � 231 624 � 19.0 26.0 645 � 10.0 332 � 74.0 49.0 
PO4 
3- 
(mg L-1) 2.15 � 0.1 0.50 � 0.1 77.0 2.15 � 0.1 0.50 �0.10 77.0 
NH4 
+ (mg L-1) 292 � 80.0 12 � 4.0 96.0 44.0 � 4.00 10 � 4.0 77.0 
Na+ (mg L-1) 192 � 7.0 100 � 49.0 48.0 54.0 � 2.0 43 � 2.0 20.0 
K+ (mg L-1) 123 � 19.0 93 � 6.0 25.0 110 � 2.0 66 � 26.0 40.0 
Ca2+ (mg L-1) 117 � 2.0 101 � 1.0 14.0 79.0 � 2.0 80 � 1.0 0.0 
Mg2+ (mg L-1) 55 � 1.00 46 � 4.0 17.0 37.0 � 1.0 32 � 1.0 14.0 

a Brine flowing from RO unit. Macro- and micro-nutrients were added as described in �Experimental Section� section. 

Table 4. Physical and chemical quality of hydroponic recycled water after 14 days of 
epuvalisation treatment with fresh water, 14 days with 1:1 mixture brine:fresh water and 
other 21 days with 100% brine, compared to treatment with only fresh water during the 
same period. Mean values � standard deviations (SD) of three replicates. 

100% Brine a Fresh Water a 
Parameter Influent b Effluent c % removal Influent Effluent % removal 
pH 
EC (mS cm-1) 
TDS (mg L-1) 
COD (mg L-1) 
BOD (mg L-1) 
Cl- (mg L-1) 
NO3 
- 
(mg L-1) 
PO4 
3- 
(mg L-1) 
NH4 
+ (mg L-1) 
Na+ (mg L-1) 
K+ (mg L-1) 
Ca2+ (mg L-1) 
Mg2+ (mg L-1) 
7.20 � 0.2 
6.04 � 0.2 
3,000 � 100 
180 � 20.0 
85 � 10.0 
2,463 � 200 
1,017 � 50.0 
3.59 � 0.3 
688 � 10.0 
329 � 10.0 
111 � 25.0 
184 � 10.0 
62 � 5.0 
7.1 � 0.1 
2.51 � 0.1 
1,560 � 50.0 
60 � 20.0 
57 � 10.0 
1,676 � 200 
921 � 15.0 
0.94 � 0.2 
16 � 10.0 
55 � 5.0 
45 � 5.0 
163 � 5.0 
46 � 2.0 
58.0 
48.0 
67.0 
12.0 
32.0 
10.0 
74.0 
98.0 
83.0 
60.0 
11.0 
26.0 
7.02 � 0.1 
2.1 � 0.1 
1,050 � 50.0 
70 � 15.0 
38 � 10.0 
460 � 10.0 
545 �25.0 
2.59 � 0.1 
41 � 10.0 
58 � 5.0 
98 � 1.0 
83 � 2.0 
33 � 5.0 
7.50 � 0.1 
1.05 � 0.1 
510 � 15.0 
38 � 10.0 
12 � 5.00 
337 � 10.0 
44 � 25.0 
0.94 � 0.1 
6 � 4.00 
35 � 5.0 
9 � 5.0 
78 � 5.0 
31 � 2.0 
50.0 
51.0 
46.0 
68.0 
27.0 
92.0 
64.0 
85.0 
40.0 
91.0 
6.0 
6.0 

a Brine flowing from RO unit. Macro- and micro-nutrients were added as described in �Experimental Section� section; 
b Composition of influent used as hydroponic water in the last 28 days; c Composition of the effluent at the end of the 
whole experimental period (3 cycles). 


Int. J. Mol. Sci. 2013, 14 13814 

Using 100% brine (Table 4), the EC and TDS were reduced to 42% and 52%, respectively. COD 
was reduced to a greater extent than the BOD values. While almost 98% of ammonium-N was 
removed, only 10% of nitrate-N was utilized by plants. Most of the Na+ was removed from brine 
(83%), while only 60% of K+. The concentrations of Mg2+ and Ca2+ were reduced by 26% and 11%, 
respectively, while 32% of chloride was removed and most of PO43- was taken up by the plants. In the 
control treatment using nutrient solution in fresh water, EC and TDS values were halved. BOD was 
reduced more than COD. Most of the ammonium-N and nitrate-N was removed. Potassium was 
reduced 90% of initial concentration, while the Na+ was reduced 40%. Chloride and phosphates were 
reduced 27% and 64%, respectively. 

2.1.3. Real Time Analysis of EC 
Figure 1 illustrates the variation of EC (mS cm-1) vs. the time during the adaptation period in which 
the brine used in the closed loops was mixed at a 50% rate with fresh water. On the 1st and 7th day 
from the beginning of the hydroponic cycle two doses of the same quantity of fertilizers were added 
both to the tank containing the mixture brine/fresh water and the tank containing only fresh water. 
Results for both cases are included in Figure 2 for comparison. Data shows that after the addition of 
fertilizers, there is a gradual decreasing of EC values during the monitoring period. This reduction can 
be attributed to the plant uptake. When the plants were irrigated with the 100% brine water (Figure 2) 
the EC was found to decrease with time as well. Similar results were observed for the control. The 
overall decrease at the end of the experiments was more than 60% in both trials using either 50% brine 
(mixed to 50% fresh water) or 100% brine as irrigation water. This finding indicates that water effluent 
from epuvalisation system can be considered of the same quality as the water effluent from the 
activated sludge unit, as reported in Table 2. This water can be recycled directly into UF and RO 
sectors of the plant for further purification without the need of extra pressure, thus achieving 
ZLD strategy. 

Figure 1. Variation of EC vs. epuvalisation time for two cycles of seven days each using 
two reservoir recharges with the 1:1 mixture brine:fresh water. Fresh water results were 
included as a control. Both treatments contained the same quantity of macro and micro 
nutrients. Mean values � standard deviations (SD) of three replicates. 



Int. J. Mol. Sci. 2013, 14 13815 

Figure 2. Variation of EC vs. epuvalisation time using 100% brine, compared to treatment 
with fresh water as control. Both treatments contained the same quantity of macro and 
micronutrients. Mean values � standard deviations (SD) of three replicates. 


2.1.4. Plant Growth Parameters 
The plants grew very well in the hydroponic system. Plant growth parameters (plant height, fresh 
weight and dry weight) of Basilicum cultivated in brine and fresh water hydroponic system is 
summarized in Figures 3 and 4. The plant height was found to increase normally with time. There was 
no significant difference between plant height in the case of brine and fresh water treatments. This 
finding is not surprising since Basilicum plants can tolerate saline water application up to 80% salt 
without any significant reduction of plant height [25]. No significant change of fresh and dry weight of 
Basilicum plants was observed between both treatments. 

Figure 3. Variation of Basilicum height after 14 days of epuvalisation experiment using 
fresh water, 14 days using the 1:1 mixture brine:fresh water, and other 26 days using 100% 
brine, compared to treatment with fresh water as control. Mean values � standard 
deviations (SD) of three replicates. 



Int. J. Mol. Sci. 2013, 14 13816 

Figure 4. Basilicum fresh and dry weight after 14 days of epuvalisation experiment using 
fresh water, 14 days using the 1:1 mixture brine:fresh water, and other 26 days using 100% 
brine, compared to treatment with fresh water as control. Mean values � standard 
deviations (SD) of three replicates. 


2.1.5. Chemical Composition of Plant Tissues 
Basilicum plants grown in brine accumulated a higher Na+, K+, and Cl- amount in their leaves 
compared to plants irrigated by using only fresh water (Figure 5). But both brine and fresh water 
treated plants accumulated the same amount of N and P. Basilicum grown in brine water was found to 
accumulate much more Na+ in the stems than plants grown in fresh water (Figure 6). 

On the other hand, the same amounts of K+, Cl-, N, and P were found in the plant stems in both 
cases. Similarly, roots of Basilicum grown in both brine and fresh water accumulated independently 
the same amount of the following elements: Na+, K+, Cl-, N, and P (Figure 7). 

Figure 5. The total nutrients content of Basilicum leaves after 14 days of epuvalisation 
experiment using fresh water, 14 days using the 1:1 mixture brine:fresh water, and other 
26 days using 100% brine, compared to treatment with fresh water as control. Mean 
values � standard deviations (SD) of three replicates. 



Int. J. Mol. Sci. 2013, 14 13817 

Figure 6. The total nutrients content of Basilicum stems after 14 days of epuvalisation 
experiment using fresh water, 14 days using the 1:1 mixture brine:fresh water, and other 
26 days using 100% brine, compared to treatment with fresh water as control. Mean 
values � standard deviations (SD) of three replicates. 


Figure 7. The total nutrients content of Basilicum roots after 14 days of epuvalisation 
experiment using fresh water, 14 days using the 1:1 mixture brine:fresh water, and other 
26 days using 100% brine, compared to treatment with fresh water as control. Mean 
values � standard deviations (SD) of three replicates. 


The macronutrients N, P and K+ in leaves, stems and roots of Basilicum grown in brine and fresh 
water did not show any significant difference between both waters. Potassium was accumulated in 
higher amounts compared to the other macronutrients. The high content of K+ was recorded during 
vegetative stage for all plants. Sodium content in roots was found not to differ in plants treated with 
both waters. No particular accumulation of chloride was observed in the roots, stems and leaves of the 
Basilicum plants. 

2.2. Discussion 
Techniques involving the recirculation of the wastewater after biological treatment and/or 
filtration promote hydroponic systems as methods with a high potential for the treatment and reuse of 
wastewater [26]. Substantial research has been done on the use of plants in wastewater treatment using 
different techniques such as algae culture, floating emerged or submerged plant culture, raft or 
suspended culture, nutrient film technique (NFT), aeroponics and static culture (combination of inert 


Int. J. Mol. Sci. 2013, 14 13818 

medium and water culture) which represent different water culture designs [27]. Although some of 
these systems are effective, the production of large quantities of aquatic plants with low economic 
value poses a big challenge [28]. Thus, crops can be cultured hydroponically in recirculating systems 
to produce a valuable by-product, while improving the water quality to a highly desired level. 
High-value vegetable crops, such as tomatoes, lettuce, cucumbers and sweet basil, have been cultured 
in a variety of hydroponic media [29]. 

Results of this study revealed the plants under study were found to grow very well in the 
hydroponic system. The concentration of dissolved solids in brine declined during the plants growth 
period. This is might be attributed to the ability of plant roots, as they develop, to act as filters thus 
reducing the dissolved solids values in the effluent. The root system was also capable of absorbing 
dissolved solids as plant nutrients. The reduction of TDS from brine water as an effect of plant uptake 
was 50%. 

In this study, the percentage of COD reduction by plant activity in both diluted and not diluted brine 
was 47% and 67%, respectively. The large decrease in COD could be related to their fully developed 
plant roots, which effectively act as filter for suspended solids and absorb dissolved nutrients, thus 
leading to this significant reduction. Jiang and Xinyuan [30] documented that 44% reduction of COD 
was achieved in zoo wastewater using floating (water hyacinth and mosquito fern), submerged 
(curly pondweed, eelgrass and parrot feather), floating leaf (hindulotus) and emerged (swamp 
morning-glory and alternanthera alligator) plants. 

The nitrate-N concentration in the final effluent was reduced by plant absorption. The rate of this 
reduction was found to increase as plants grow and develop. The plants had a high content of nitrogen 
in the first growth stages while the nitrogen content decreased in the seed-setting stage [31]. Although 
the reduction of NO3 
- nitrogen was only 26% and 10% of the influent concentration containing either 
50% or 100% brine, respectively, the total NO3 
- removal was 107 and 96 g in the respective 
treatments. On the other hand, the total reduction of ammonium-N was between 96% and 98%. This 
percentage corresponds to a total removal of 140 and 336 g in 50% and 100% brine, respectively. The 
high removal of ammonium-N during the epuvalisation system could be attributed to both direct plant 
uptake by the plants and/or nitrification. Gloger et al. [31] reported that lettuce was responsible for the 
removal of 9% of nitrogen applied as feed, while Jiang and Xinyuan [30] documented a 73% removal 
of total nitrogen of zoo wastewater (110 kg total nitrogen/year) using different plant types. Furthermore, 
Naegel [32] reported a nitrate level reduction in aquaculture wastewater of about 78% (from more than 
450 to about 100 mg/L) in 8 weeks when using tomatoes and about 89% (from more than 450 to 
about 50 mg/L) in 4 weeks when using lettuce. On the other hand, Mantet et al. [33] reported 58% 
nitrogen removal using Salix viminalis grown in gravel hydroponic system to treat primary settled 
sewage wastewater. 

Phosphorous is considered to be a major growth-limiting nutrient in aquatic systems [34]. 
Wastewater application was therefore a beneficial source of phosphorous required by plants. Under 
normal conditions, phosphorous occurs either as orthophosphate (HPO42- or H2PO4 
-) ions or organic 
compounds dissolved in water. Phosphorous is converted from inorganic to organic and vice versa, by 
microorganism activity. Biologically available phosphorous in aquatic system includes soluble reactive 
and unreactive phosphorous. The latter is available as a result of enzymatic hydrolysis [35]. Our results 
indicate that about 75% of the applied P was removed in both 50% and 100% brine treatments. The 


Int. J. Mol. Sci. 2013, 14 13819 

total amount of P removed by plants as PO43- was low (0.83 g and 1.33 g in both 50% and 100% brine 
treatment, respectively). Nevertheless, the plants did not show any P deficiency symptom [36]. Jiang 
and Xinyuan [30], achieved 62% removal of total phosphorous from zoo wastewater using different 
plant types. Other investigators [37] reported a removal efficiency of 99% and 97% of total and 
soluble phosphorous, respectively, in saline aquaculture wastewater using salt tolerant plants grown in 
a sand biofilter. Further, Gloger et al. [38] reported a decrease of PO43- concentration in fish 
wastewater circulated in a hydroponic lettuce tank of 4.9 mg/L. On the other hand, Mant et al. 
documented that 90.6% phosphorous removal was achieved using S. viminalis grown in a gravel 
hydroponic system to treat primary settled sewage wastewater [33]. 

Potassium concentration in each compartment decreased with time during the growth period. The 
concentrations listed in Tables 2 and 3 indicate a potassium removal of about 25 and 60 in the 50% 
and 100% brine treatments, respectively. The total amount of K+ removed by plants from 50% and 
100% brine treatments was 15 g and 33 g, respectively. Our results are in agreement with those 
obtained by Dontje and Clanton [39], which reported 25%�71% potassium removal in circulating 
aquacultural systems using cattails, reed canary grass, and tomatoes grown in sand beds. Furthermore, 
Mant et al. reported 25% potassium removal using S. viminalis grown in a gravel hydroponic system to 
treat primary settled sewage wastewater [33]. The removal efficiency of potassium achieved in this 
and other studies could be attributed to plant uptake. 

The brine wastewater used in our system, was rich in potassium, which resulted in potassium over 
fertilization for all plants (Table 4). It was indicated that when potassium supply is abundant, �luxury 
consumption� of potassium often occurs, which may have a possible interference with physiological 
availability and uptake of magnesium and/or calcium [35]. It is worthy to note, that it is well known 
that high K+ level in soil and fertilizer is considered as good character for high productivity [40,41]. 

3. Experimental Section 
3.1. Wastewater Treatment System and Site 
The wastewater treatment plant at Al-Quds University was selected to perform this study. 
The details of the treatment plant were described in previous investigations [42,43]. Briefly, it consists 
in a sequence of activated sludge unit, hollow fiber ultra-filtration filters (HF-UF), spiral wound 
ultra-filtration filters (SW-UF), granular activated carbon (GAC) and RO filters. The RO system 
consists of a 1 � 4 inch pressure vessel constructed from composite material having a pressure resistance 
up to 400 psi. The vessel holds two 4 inch separation membranes composed of thin polyamide film 
with pH range 1�11 (model BW30-4040 by DOW Filmtec, Edina, MN, USA). A membrane-antiscaler 
(product NCS-106-FG) solution (phosphoric acid disodium salt) is continuously dosed to the RO feed 
at a concentration of 4 mg L-1 in order to prevent deposition of divalent ions in the RO membrane. The 
system is designed to remove major ions and heavy metals. The intended RO permeate capacity of the 
system is 0.45�0.50 m3 h-1. The quantity of RO brine generated from the plant is estimated as 25% of 
the total flow. It is collected in special storage tank for further treatment and reuse. 


Int. J. Mol. Sci. 2013, 14 13820 

3.2. Epuvalisation System 
The experiment was conducted in a greenhouse from April to June 2012 at Al-Quds University 
under semi-controlled conditions (day and night temperatures were maintained at 25 .C and 18 .C, 
respectively, and relative humidity of 50%�60%. Epuvalisation system (Figure 8) is composed of two 
equivalent sectors; in the first, brine was used as hydroponic water, and the second was adopted as a 
control device employing only fresh water. Each system (brine treatment or fresh water treatment) 
consists of two 0.5 m3 storage tanks (one for influent and the other for effluent water) and 5 cropping 
channels. For both treatments (brine and fresh water), the influent tank is placed one meter higher than 
the epuvalisation tracks to let water flow by gravity through five channels. Each channel (made of 
galvanized metal) is 2-meter long, 40-cm wide and 11-cm deep. The five channels are placed 
consecutively with 10-cm height difference between each channel. The slope of each channel is about 
(1%�1.5%). The effluent storage tank is located under the channel placed in the bottom. The effluent 
was continuously pumped to the influent tank to close the cycle. To enhance the dissolution of oxygen, 
the channels were continuously aerated by means of a pump and thin aeration plastic pipes. 

Figure 8. Graphical design of the epuvalisation system. 


3.3. Plant Selection 
Basilicum (Ocimum Basilicum L.) is an annual herb plant from the Lamiaceae family; it is 
considered a medicinal plant and used often as a spice. The plant contains an essential oil, which can 
be used in manufacturing perfumes and flavors for food and beverages [44,45]. Basilicum has 
restricted requirements of water and minerals [31]. Basilicum was used in epuvalisation systems by 
other researchers along with different plant species such as: Metha, Mint, Peppermint, Alfalfa, Sudax, 
Salvia, Gerbera, Turf Grass, Celery, Cyperus, Water Cress and Iris [15,19]. 

3.4. Plantation and Growth 
Young plants of Basilicum were grown in aerated brine and fresh water supplied with additional 
nutrients. Nutrients added in the influent tanks of brine and fresh water treatments were 5.0, 4.0, 1.0, 
0.8, 0.7, 0.5 mM total nitrogen, total potassium, calcium, phosphorus, magnesium, and iron, 
respectively, using the following chemicals: K2SO4, KCl, KNO3, Ca(NO3)2�4H2O, NH4NO3, KH2PO4, 


Int. J. Mol. Sci. 2013, 14 13821 

MgSO4�7H2O, Fe�Na EDTA. Micronutrients were added in adequate amounts (�M): 2.97 
MnCl2�4H2O, 1.24 ZnCl2, 0.66 CuCl2�2H2O, 24.75 H3BO3, 0.083 (NH4)6 Mo7O24�4H2O, and 0.0413 
NiCl2 [40]. The plant cropping in the brine irrigated channels was performed following a time based 
program: fresh water, during the first 2 weeks; 50% brine, other 2 weeks for plant adaptation; 
100% brine to conclusion of experiment. In the second sector only fresh water added with fertilizers 
(macro- and micro-nutrients) was used as hydroponic water during the same experimental time of the 
first sector. 

The nutrients were added once at the beginning of the experiment in the inlet container. The flow 
rate in the system was maintained daily at 0.5 m3 influent per 24 h. Basilicum plants were put at 25 cm 
distance each other to permit roots to develop in a sufficient volume (seven plants in each channel), 
so 35 replicates were performed in each cropping system. 

3.5. Water Analysis 
Water samples were taken weekly from influent and effluent tanks up to the end of cropping period. 
Electrical conductivity EC, pH, total dissolved solids (TDS), chemical oxygen demand COD, biological 
oxygen demand BOD, and most important ions (Cl-, NO3 
-, NH4+, Na+, K+, Ca+2, Mg+2, PO4 
-3) were 
analyzed according to standard methods [46]. 

3.6. Harvesting and Analytical Procedures 
Plant growth parameters were monitored at the end of the growing period (plant height, fresh 
weight, and dry weight). Plants were harvested in flowering growth stage 57 days after planting 
(DAP). Each plant was harvested individually. Plants were separated into leaves, stems, and roots. 
All plant parts were dried at 70 .C in a drying oven until constant weight, grinded to pass a 1.5 mm 
sieve. After thorough mixing, a sub-sample of 5 g was milled to a fine powder. Plant materials were 
prepared to be analyzed for P, K and Na using dry ashing method, in which 50 mg of dried 
sample were ashed in a crucible at 450 .C in a muffle furnace overnight, then 1 mL of 0.35 M HNO3 
solution was added, swirled and left for at least 10 min. Pure water (18.2 MO cm-1) was added 
(9 mL) and then the sample was filtered through ashless filter paper (Whatman International Ltd., 
England) into polypropylene tubes [47]. Total P was measured using colorimetric method 
(Ammonium-Vanadate-Molybdate) according to Gericke and Kurmies [48]. Total N was determined 
using Kjeldahl method [49]. 

3.7. Statistics and Yield Component Analysis 
Results were expressed as means and standard deviations for three replicates. All statistical analyses 
were carried out using Statistical Analysis System (SAS) (SAS Institute Inc., Cary, NC, USA, Release 
8.02, 2001). Comparison among data was carried out using the GLM procedure. The Bonferroni 
procedure was employed with multiple-tests in order to maintain an experiment wise of 5%. 


Int. J. Mol. Sci. 2013, 14 
13822 

4. Conclusions 
Soil salinity, surface and ground water contamination are the major problems of brine water 
generated from a RO plant. The management of the brine water problem will help in minimizing 
health and environment risks. The reuse of brine in irrigation of high salt tolerant plants is considered 
among the most suitable disposal methods to solve the brine problem. In this work, we demonstrated 
that the newly developed epuvalisation technique has not only high potential in reducing the salt 
content of brine to a safe level, but also to produce plants of high economic value without any loss of 
yield. Basilicum was selected for this purpose and our results showed that it can be grown in brine 
generated from the RO system with high tolerance and good yield. Results show a significant decrease 
in the electrical conductivity in brine during the growing season. Plant growth parameters of Basilicum 
irrigated with brine and fresh water have shown insignificant difference between them. This work 
demonstrates that the epuvalisation system is simple, flexible, easily managed and low cost. It has a 
high potential for inland brine treatment if a proper salt tolerant plant, such as Basilicum, is used. 

Acknowledgments 

This work was supported by generous grants from USAID-MERC program, Sanofi Pharmaceutical 
Company (France) managed through Peres Center for Peace and Exo Research Organization, 
Potenza, Italy. 

Conflict of Interest 

The authors declare no conflict of interest. 

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