Water Research 151 (2019) 478e487

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Water Research
journal homepage: www.elsevier.com/locate/watres

Chronic effects of brine discharge from large-scale seawater reverse

osmosis desalination facilities on benthic bacteria
Hila Frank a, b, Katarina E. Fussmann a, b, Eyal Rahav b, **, Edo Bar Zeev a, *
a
Zuckerberg Institute for Water Research (ZIWR), The Jacob Blaustein Institutes for Desert Research (BIDR) Ben-Gurion University of the Negev, 84990,
Israel
b
Israel Oceanographic and Limnological Research, National Institute of Oceanography, Haifa, 8030, Israel

a r t i c l e i n f o a b s t r a c t

Article history: Seawater desalination facilities continuously discharge hyper-saline brine into the coastal environment
Received 6 August 2018 which often flows as a concentrated plume over the seafloor, hence possibly impacting benthic micro-
Received in revised form organisms. Yet, the effects of brine discharge from desalination plants on benthic bacteria, key players in
15 November 2018
biodegradation of organic material and nutrient recycling is unknown. In this study, we tested the
Accepted 8 December 2018
Available online 27 December 2018
chronic (years) effects of brine discharge from three large-scale desalination facilities on the abundance,
metabolic activity and community composition of benthic bacteria. To this end, four sampling campaigns
were carried at the outfall areas of the Ashkelon, Sorek and Hadera desalination facilities. The effects of
Keywords:
SWRO desalination
the brine were compared to corresponding reference stations which were not influenced by the brine
Brine effluent (i.e., water temperature and salinity). Our sampling data indicate that bacterial abundance and activity
Environmental impact that includes bacterial growth efficiency were 1.3e2.6-fold higher at the outfall area than the reference
Heterotrophic bacteria station. Concomitant analysis pointed out that the bacterial community structure at the brine discharge
Brine discharge method area was also different than the reference station, yet varied between each desalination facility. Our
results demonstrate that the impact of brine effluent from desalination facilities on benthic bacteria are
site-specific and localized (<1.4 Km2) around the discharge point. Namely, that the effects on benthic
bacteria are prominent at the brine mixing zone and change according to the discharge method used to
disperse the brine as well as local stressors (e.g., eutrophication and elevated water temperature). Our
results contribute new insights on the effects of desalination-brine to benthic microbes, while providing
scientifically-based aspects on the ecological impacts of brine dispersion for decision makers.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction (Missimer et al., 2015; Petersen et al., 2018).

Limitations in global freshwater supplies have stimulated the
application of desalination technologies with desalinized water
coming online worldwide at a rate of ~90 million m3 d
1
(Fritzmann et al., 2007; Greenlee et al., 2009; Malaeb and Ayoub,
2011). Currently, more than 60% of desalinated water is produced
via filtration of seawater through reverse osmosis (RO) membranes
(Greenlee et al., 2009; Shenvi et al., 2015). These large-scale
seawater reverse osmosis (SWRO) facilities continuously draw
large volumes of coastal water and discharge about half as
concentrated brine-effluent back to the marine environment
* Corresponding author.
** Corresponding author.
E-mail addresses: eyal.rahav@ocean.org.il (E. Rahav), barzeeve@bgu.ac.il (E. Bar
Zeev).
Brine-effluent from SWRO facilities contains a high concentra-
tion of salts and often various chemicals such as phosphonate-
based antiscalants and ferric (or alum) sulfate-based coagulants
that are added in the desalination process (Fritzmann et al., 2007;
Roberts et al., 2010). The discharge of the brine-effluent to the
coastal environment can be carried by a direct surface release on
the coastline or through a diffuser system away from the shore
(Fritzmann et al., 2007; Lattemann and Ho € pner, 2008; Voutchkov,
2011; Missimer et al., 2015). To maximize dilution, the brine is
typically mixed with cooling water of adjacent power-plants in
different ratios and methods. The result is a warm (~25% over
ambient temperature), saline (up to 10% over ambient salinity) and
buoyant brine plume that can be found up to a few kilometers away
from the discharge site (Missimer et al., 2015; Petersen et al., 2018).
Nevertheless, brine-effluents are occasionally found as a concen-
trated stream that flows on the seabed, which may accumulate

https://doi.org/10.1016/j.watres.2018.12.046
0043-1354/© 2018 Elsevier Ltd. All rights reserved.
 H. Frank et al. / Water Research 151 (2019) 478e487
within the sediment if the buoyancy of the plume is negative
(Bleninger et al., 2010; Fern andez-torquemada et al., 2009;
Fernandez - Torquemada et al., 2005).
Coastal sediments play central ecological and biogeochemical
roles in organic matter and inorganic nutrients recycling (Defeo
et al., 2009; Kemp et al., 2005; Lukkari et al., 2009; Nealson,
1997). Benthic habitats are usually biodiverse and are inhabited
by various organisms from multiple trophic levels, including
benthic prokaryotes (bacteria and archaea), microalgae, as well as
various meiofaunal and macrofaunal organisms (Gooday and
Turley, 1990; Lubinevsky et al., 2017; Titelboim et al., 2016).
Benthic organisms such as invertebrates and seagrass were previ-
ously shown to be negatively affected by desalination brine. This
response was detected up to several hundred meters away from the
outfall of SWRO desalination facilities Begher Nabavi et al. (2013);
Fernandez-Torquemada et al. (2013); Gacia et al. (2007); Merwe
et al. (2014); Riera et al. (2012) and see review in (Petersen et al.,
2018). Yet, the effect of SWRO brine upon benthic heterotrophic
bacteria, which are the base of the microbial food-web (Fenchel,
1988), is currently limited despite their large ecological impor-
tance (Forehead et al., 2013; Frank et al., 2017; Petersen et al., 2018).
Currently, five large-scale desalination facilities are operating
along a relatively short (~100 km) stretch of the Israeli Mediterra-
nean coast. These facilities draw seawater at a rate of 1.8 M m3 d
1
and provide ~80% of Israel's potable needs. The Israeli coast is
located at the southeast corner of the Mediterranean Sea, and is
characterized by a simple bathymetry (Rosentraub and Brenner,
2007) and long-shore currents from south to north (Kit and
Kroszynski, 2014). Seawater temperature range from ~31  C dur-
ing summer to ~15  C during the winter (Rahav et al., 2018; Raveh
et al., 2015). Salinity is relatively constant throughout the year and
range from 38.5 to 39.8 (Raveh et al., 2015). This coastal region is
considered oligotrophic, with low autotrophic biomass (<1 mg
chlorophyll. a L
1), low primary production rates (~5 mg C L
1 d
1),
basal level of inorganic nutrients (~300 nM NO2þNO3 and
10e50 nM PO4) and dominated by heterotrophic microbial pop-
ulations (usually 107-109 cells L
1) (Azov, 1986; Drami et al., 2011;
Kress et al., 2016a,b; Rahav et al., 2018; Rahav and Bar-Zeev, 2017;
Raveh et al., 2015).
In this study, we focused on the chronic effects (5e13 years) of
SWRO desalination brine on benthic heterotrophic bacteria. To this
end, we sampled the sediments around the outfalls of three large-
scale SWRO desalination facilities along the Israeli coast on a
seasonal-basis between 2016 and 2017 (Fig. 1). Sediment samples
from each of these sites were tested for different physiochemical
characteristics as well as bacterial abundance, production (i.e.,
carbon assimilation), respiration and community composition. On
discrete event (November 2016), meiofaunal abundance and com-
munity composition was also investigated. To the best of our
knowledge, this study provides the first indication on the possible
effects of operating SWRO desalination on benthic bacteria.
2. Materials and methods
2.1. Sampling strategy
Sediment samples were collected in-situ aboard the R.V Medi-
terranean Explorer in proximity to three SWRO desalination facil-
ities that have been operating for 5e13 years and located along the
eastern Mediterranean coastline: Hadera, Sorek and Ashkelon
(Fig. 1). Sampling campaigns were carried during November 2016
(autumn), January 2017 (winter), April 2017 (spring) and
September 2017 (summer). Each sampling cruise included sedi-
ment collection in close proximity to the brine outfall (<50 m) and a
reference station at respective depth located several kilometers
479
away from the discharge point (Fig. 1, Table 1). The reference sta-
tions were not influenced by the physicochemical characteristics of
the brine, namely the water temperature and salinity were similar
to the ambient values. Water temperature and salinity profile were
measured in real-time by an MS5 probe (Hydrolab MS5 water
quality multiprobe, USA), focusing on the values below the surface
and above the bottom at the outfall and reference stations. Addi-
tionally, a survey of the spatial and vertical dispersion of the brine
was conducted in several campaigns (Winter, Spring and Summer).
Sediments were collected from each site by a Van-Veen grab.
The grab was cleaned using a pressure-hose with seawater between
each sampling to minimize cross-contamination. Sediment sub-
samples were analyzed for bacterial abundance (BA), bacterial
productivity (BP) which measures the assimilate rate of carbon,
bacterial respiration (BR) and bacterial community structure.
Meiofaunal abundance was analyzed in the November 2016 sam-
pling campaign.
2.2. Analytical approaches
Bacterial abundance (BA): Sediment samples (2e5 gr) were
placed in cryo-vials containing sterile seawater (0.2 mm), and son-
icated for 7 min (Symphony, VWR) in a bath sonication to remove
all the bacterial cells associated with the sand grains into the sterile
water (Danovaro, 2010). The bacteria containing water was trans-
ferred into new sterile cryovials, fixed with 6 ml of 50% glutaralde-
hyde (Sigma, G7651), snap-freeze in liquid nitrogen and kept
at
80  C until analysis within <1 month. Prior to analysis, the
samples were defrosted in a 37  C water-bath, stained with 1 ml
SYBR green (Applied Biosystems cat #S32717) per 100 ml sample,
and incubated for 10 min in the dark. Stained samples were
analyzed by an Attune® acoustic focusing flow cytometer (Applied
Biosystems) as described in (Vaulot and Marie, 1999; Frank et al.,
2017). Filtered seawater (cutoff of 0.22 mm) was analyzed as
blanks, while beads with a nominal size of 0.93 mm (Polysciences,
USA) were used as a size standard.
2.2.1. Bacterial production (BP)
Collected sediment samples (1e4 gr) were re-suspended in
3.5 ml of filtered seawater (0.2 mm) and spiked with 500 nmol L
1
(final concentration) of [4,5-3H]-leucine (Perkin Elmer USA, specific
activity 160 Ci mmol
1) as previously described for sediments
(Danovaro, 2010). The samples were incubated in the dark for 4 h
under in-situ temperatures. At the end of the incubation, the
assimilation of leucine was halted by the addition of 100 ml of 100%
trichloroacetic acid (TCA) cold solution. The sediment samples
were then sonicated (Symphony, VWR) for 10 min to remove all the
bacterial biomass from the grain particles. The biomass extract was
divided into three 1 ml aliquots, and the micro-centrifugation
technique was applied (Smith and Azam, 1992). Disintegration
per minute (DPM) from each sample were read using a liquid
scintillation counter (Packard Tri carb 2100). A conversion factor of
1.5 kg C mol
1 with an isotope dilution factor of 2.0 was used to
calculate bacterial carbon assimilation rate (Simon and Azam,
1989). Blank samples included sediments added with radioiso-
tope and TCA (to kill all bacterial cells) at T0 (prior spiking) and
incubated under the same conditions as described above.
2.2.2. Oxygen concentration and bacterial respiration (BR)
Collected sediments were placed in custom-made air-tight
Perspex cores (three replicates for each station) (height: 20 cm,
diameter: 6 cm), covered with aluminum foil and incubated with
filtered (0.2 mm) seawater for 24 h. The core samples were incu-
bated under ambient conditions in a 0.3 m3 container with running
seawater. Oxygen concentrations were measured at T0 and T24
480 H. Frank et al. / Water Research 151 (2019) 478e487

Fig. 1. Locations of the Hadera, Sorek and Ashkelon desalination facilities (top, middle and lower images, respectively) along the Israeli coastline (southeastern Mediterranean Sea).
The location of the outfall (Out.) and reference (Ref.) stations at each desalination facility are shown as red and white circles, respectively (specific locations are detailed in Table 1).
Insert include the year of initial operation (in brackets), the annual desalinated freshwater produced (approximated), and the discharge method of the brine for each facility. The
distance between Hadera and Sorek desalination facilities is ~60 km, while the distance between Sorek and Ashkelon is ~40 km. The map was modified from Google Earth. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Table 1
Summary of the sampling locations near the three desalination facilities along the Israeli coast. In each site, two stations were sampled; the brine outfall and an uninfluenced-
reference station.

Desalination facility Site Location Distance from outfall (km)

Lat. (N) Lon. (E)

Ashkelon Outfall 31.3795 34.3096 <0.05

Reference 31.4057 34.3276 5e6

Sorek Outfall 31.5648 34.4126 0

Reference 31.5557 34.4102 1e2

Hadera Outfall 32.2791 24.5298 <0.1

Reference 32.2367 34.5153 2e8

using a WTW oxygen optode (Multi 3430). Respiration was calcu-
lated by subtracting the oxygen values at T0 with the oxygen values
at T24, and divided by the incubation time. The results were
normalized to the dry weight of the sediment sample. Air-tight
Perspex core per station was incubated under the same condi-
tions with filtered sea water only and used as blank.
2.2.3. Bacterial carbon demand (BCD) and bacterial growth
efficiency (BGE)
Oxygen respiration was converted into carbon consumption
assuming a respiratory quotient (RQ) of 1 (Anesio et al., 2003;
Smith and Prairie, 2004; del Giorgio and Williams, 2005). BCD was
calculated as the sum of BP and BR. BGE was calculated as the ratio
between the utilized carbon through bacterial production and BCD
(Bar-Zeev and Rahav, 2015).
2.2.4. DNA extraction and bacterial community structure
Sediment samples were probe-sonicated (CL-188, QSONICA) for
1 min within 1 ml lysis buffer (EDTA 0.5 M pH 8, sucrose and Tris-
HCl pH 8) and incubated with lysozyme (4.8 mg ml
1) for 30 min
at 37  C. SDS 20% and proteinase K (20 mg ml
1) were then added
and the samples were incubated for additional 1 h at 55  C. Next,
samples were purified with phenol pH 8 and then with phenol:
chloroform: IAA (Bar-Zeev et al., 2008). DNA extracts were kept
 H. Frank et al. / Water Research 151 (2019) 478e487
in
20  C for further analysis in RNAase DNAase free water.
DNA samples were sequenced by the Illumina MiSeq platform.
The primers 515F and 806R were used to amplify the V4 region of
the rRNA 16S gene. Sequences in 250 bp length were trimmed from
adaptors and merged by the CLC bio-genomic workbench (Qiagen
bioinformatics). Operational Taxonomic Units (OTUs) picking was
done with QIIME (Caporaso et al., 2010) by an open reference OTU
picking method, followed by a b-diversity analysis with the default
parameters of QIIME. The b-diversity results are presented in a
PCoA plot, generated based on the weighted UniFrac distance ma-
trix. A comparison was made among all samples from a certain
desalination facility sampled along all seasons. The results from
each season for each desalination facility were collapsed to over-
come the seasonal variation (presented in Fig. 5). Samples were
collapsed by summing the number of reads of each phylum from
each sample into the final representative combination (merging
was done by QIIME). In order to statistically compare the commu-
nity structure composition among the different stations in each site
(outfall, and reference combined from all seasons) an analysis of
similarity (ANOSIM) was performed by the R ‘vegan' package
(Oksanen et al., 2017) with 1000 permutations. Only phyla that
exceeded 5% are presented.
2.2.5. Meiofauna abundance
Sediment samples from the outfall and reference stations were
collected in November 2016 in all locations (Fig. 1) and stained with
a Rose Bengal solution onboard (2 g of Rose Bengal per 1 L of
ethanol) following (Schonfeld et al., 2012). Meiofaunal species
abundance, including nematodes, rotifers, gastrotrichs, platy-
helminthes, annelids, tardigrades, arthropods and Mollusca, as well
as their taxonomic discrimination (phylum level) were performed
using an Olympus epi-fluorescent microscope (BH2-RFCA). Species
abundance was counted into 1 ml of sample volume for each
sediment sample converted to individuals per gram of dry sand.
2.2.6. Statistical analyses
Data from each desalination facility was first merged between
all seasons (i.e., the 4 cruises) for each parameter (BA, BP, BCD, BGE).
This pre-procedure was done since the similar differences/trends
were observed for BA, BP or BR in the reference vs. outfall sites
(Table S1) along all sampling times. This allowed us to discuss the
overall effects caused by the brine on the benthic microbial com-
munities at each site without the seasonal variations. The differ-
ences between the reference and outfall sites were then analyzed
by a t-test using the Excel add-in XLSTAT.
3. Results and discussion
3.1. Physicochemical characteristics at the outfall discharge and
reference sites
The three SWRO desalination facilities tested in this study
exhibited large physicochemical variability between the outfall and
the reference sampling locations. These differences were mainly
triggered by the brine discharge method undertaken (coastal
discharge vs. diffuser system), as well as by local release of other
anthropogenic stressors such as sewage and well amelioration
brines (i.e., concentrated aquifer water with high concentrations of
nitrate) at the outfall sites (Rahav et al., 2018).
3.1.1. Ashkelon desalination facility
The plant has been operating since 2005 with desalinated water
coming online at a rate of 115 M m3 y
1. The desalination brine is
discharged via an open-coastal channel (Fig. 1). The concentrated
brine from the seawater desalination process is mixed with cooling-
481
water of the adjacent power-plant to reduce the salinity level and
increase the buoyancy of the brine before its discharge back into the
coastal environment. Consequently, in-situ measurements of salinity
and water temperature at the outfall location indicated that these
values were higher than the receiving environment by 10% and 34%,
respectively (Table 2, Figs. 2 and S1). Our results indicate that in two
out of the four sampling campaigns undertaken, a brine plume
(defined as 2e5% salinity over the ambient levels) was observed near
the seafloor (Table 2, Fig. 2). Under these conditions, the brine plume
spread up to 1.4 Km2 around the outfall site (Fig. 2). Elevated salinity
levels of <2% were considered as the natural dial or seasonal vari-
ability of the eastern Mediterranean Sea coastal zone (Gefen - Glazer,
2014a; Glazer, 2015; Abramson and Shafir, 2016a). In addition to the
SWRO brine effluents, desalinated brine from brackish-water well-
amelioration facilities was also mixed with the brine from the Ash-
kelon desalination facility (Drami et al., 2011). Brine from these well-
amelioration facilities was enriched with nitrate and silicic-acid
(150 mM and 97 mM, respectively), hence discharging it with the
Ashkelon desalination brine often increases the concentrations of
these nutrients at the discharge site (Shafir and Ben - Yosef, 2017a).
Nutrient amendments in this area may lead to eutrophication and
prompt phytoplankton growth as has been recently reported by
Rahav et al. (2018). Furthermore, the brine from the Ashkelon
desalination facility is occasionally mixed with sewage carried with
the northern current from the south (Elmanama et al., 2005; Herut
et al., 2007). A recent study from the eastern Mediterranean
coastal water indicated that this type of sewage discharge may lead
to abrupt microbial blooms (Rahav and Bar-Zeev, 2017). Hence, we
surmise that the outfall area of the Ashkelon desalination facility was
affected from multi-stressors that could result in a synergistic effect
on the benthic microbial community.
3.1.2. Sorek desalination facility
The plant has been operating since 2013 and is located north-
ward from the Sorek stream (Fig. 1). The Sorek plant is currently the
largest SWRO desalination facility in the world, producing
~150 M m3 of freshwater per year. During our measurements the
brine was discharged directly without any prior dilution via a
diffuser system located 1.9 km away from the shore at ~20 m depth.
The measured salinity near the discharge point reached up to 6%
over the ambient values (~41e42), while water temperature
remained unchanged (Table 2, Fig. 2). The brine was consistently
found over the seabed with an affected (salinity of 2e5% above the
ambient) area of up to ~0.9 km2 (Table 2). During our sampling
campaigns the dispersion of the brine around the outfall site was
efficient, namely that the salinity signal rapidly decreased from 5%
to around 2% above the ambient.
3.1.3. Hadera desalination facility
The Hadera desalination facility was constructed near the
outflow of the Hadera stream (Fig. 1). The plant has been operating
since 2009, producing ~127 M m3 desalinated water per year
(Fig. 1). The brine is mixed with the power-plant cooling water prior
its discharge via an open trench (Fig. 1), which resulted in an
increased temperature of 10e26% above ambient levels (Table 2,
Fig. S1). Additional dilution of the brine is achieved by mixing the
brine with the Hadera stream that may contain additional nutrients
(up to ~6 mg L
1 NH4 and ~3 mg L
1 PO4) (Barinova et al., 2010).
Therefore, salinity increase over the ambient values was overall
low, ranging from <1% to 7% (Table 2, Fig. 2). Our measurements
indicate that the brine signal (2e5% increase in salinity above
ambient level) was detected near the seabed at two out of the four
sampling campaigns undertaken in this study and spread south-
ward from the discharge site (Fig. 2).
482 H. Frank et al. / Water Research 151 (2019) 478e487

Fig. 2. Spatial distribution of the brine plume (% change in salinity relative to background-uninfluenced water) at the surface layer (AeC) and near the seabed (DeF) measured in
April 2017. The dispersion of the brine plume was measured around the outfall site and up to a reference station (Ref.) at the Ashkelon (A, D), Sorek (B, E) and Hadera (C, F)
desalination facilities. The corresponding temperatures (as % change) are shown in Fig. S1. The maps were extrapolated by the Ocean Data View software. Black dots indicate the
different sampling locations around the desalination outfall sites.

Table 2
Temperature and salinity measurements around the three desalination facilities along the Israeli coast (southeastern Mediterranean Sea) at the outfall (brine discharge site)
and in reference (uninfluenced) areas.

Facility Sampling season Temperature ( C) Salinity

Outfall Reference Outfall Reference

Surface Bottom Surface Bottom Surface Bottom Surface Bottom

Ashkelon Summer 34.1 34.1 30.9 30.8 41.8 43.9 40.2 40.3
Autumn 31.8 31.8 24.9 24.9 42.2 42.3 39.5 39.6
Winter 20.7 20.5 17.3 16.8 42.9 42.8 39.3 39.4
Spring 26.3 26.7 19.7 19.8 41.5 42.8 39.0 39.5
Sorek Summer 30.7 30.7 30.7 30.7 40.2 42.1 40.2 40.2
Autumn 24.7 24.9 24.8 24.4 39.5 41.0 39.6 39.7
Winter 16.8 16.9 16.8 16.8 39.5 41. 7 39.6 39.6
Spring 19.0 18.8 18.8 18.7 39.7 42.2 39.9 39.8
Hadera Summer 31.3 31.3 31.0 31.0 40.3 40.2 40.2 40.1
Autumn 25.3 25.2 22.5 22.5 40.9 41.0 39.5 39.6
Winter 19.2 19.4 16.8 16.8 39.8 39.7 39.1 39.5
Spring 23.8 24.0 19.1 19.0 41.9 42.5 39.6 39.7

3.2. The effects of SWRO brine on benthic bacteria: abundance,
respiration and activity
The overall responses (i.e., BP, BR and BA) of the benthic mi-
crobial community to brine (outfall vs. reference sites) were similar
between the 4 cruises undertaken at each desalination facility
(Table S1). We therefore pooled together the data of all the sam-
pling campaigns for each bacterial variable at each desalination
facility, resulting in the overall impact of the brine near a specific
desalination plant (Fig. 3). Information of the entire data-set from
all cruises is available in Table S1.
3.2.1. Ashkelon desalination facility
Bacterial abundance and production rates (BA and BP, respec-
tively) were ~2-fold higher at the outfall area compared to the
uninfluenced-reference stations (Fig. 3A and B). Bacterial carbon
demand (BCD¼BP þ BR), which sums the cellular anabolic (BP) and
catabolic (BR) requirements of bacteria, remained overall un-
changed, whereas bacterial growth efficiency (BGE ¼ [BP/BCD]
*100) was higher by 2-fold at the outfall area than the reference
(Fig. 3C and D). Concurrently to bacteria, the abundance of meio-
faunal organisms during the fall sampling campaign (i.e., nema-
todes, rotifers, gastrotrichs, platyhelminthes, annelids, tardigrades,
arthropods and Mollusca) decreased by 92% at the outfall station
 H. Frank et al. / Water Research 151 (2019) 478e487
Fig. 3. (A) Bacterial abundance, (B) bacterial production, (C) bacterial carbon demand, and (D) bacterial growth efficiency measured at the outfall (out.) and reference (ref.) stations
in the different desalination facilities along the Israeli (eastern Mediterranean Sea) coast. Data were pooled from all seasonal measurements. Whiskers represent the 5th and 95th
percentiles, the solid line represents the average. Asterisks represent the significance between stations, a ¼ 0.05, n ¼ 8e11.
compared to the reference station (Table S2). Meiofauna diversity
was lower by ~67% at the outfall compared to the reference stations
(Table S2). In agreement with our measurements, a significant
reduction in benthic meiofauna abundance was previously re-
ported in similar sites at the Israeli coast (Shafir and Ben - Yosef,
2017b). Benthic bacterivorous, such as nematodes and forami-
nifera are often susceptible to environmental stressors such as os-
motic stress and alterations in water temperature. A recent study
pointed that the abundance and richness of foraminifera were
highly reduced near power-plant cooling water and brine discharge
outfall zones (Titelboim et al., 2016). We suggest that the brine
(along with the other contaminants at the mixing zone) shifted
heterotrophic bacteria from being top-down controlled (i.e., govern
by bacterivorous abundance such as foraminifera and nematodes)
to being dominated by bottom-up interactions (i.e., exposure to
elevated salinity and temperature as well as the availability of nu-
trients such as N; P and C). Under this scenario, the significant
reduction in bacterivorous at the outfall may result in lower
predatory pressure on bacteria, hence increase in BA abundance at
the outfall site.
Enhanced bacterial activity (measured as BP) at the outfall area
could result from local eutrophication processes, namely chemicals
that were directly associated with the SWRO operation (e.g.,
phosphonates-based antiscalants) and indirectly by mixing the
desalination brine with nutrients from nearby well-amelioration
effluents (Abramson and Shafir, 2016; Shafir and Ben - Yosef,
2017a) and/or by sewage discharge from nearby Gaza (Powley
et al., 2016). It is also possible that the increase in water
483
temperature compared to the reference station due to mixing the
brine with power-plant cooling water (Table 2, Fig. S1) may also
spur bacterial production rates. These increased BP rates resulted in
high BGE at the outfall site compared to the reference station
(Fig. 3D). We suggest that enhanced BGE may be explained by the
increased synthesis of various high molecular weight osmo-
protective molecules such as glycine betaine, ectoine and glycerol
(Poli et al., 2017; Wilson et al., 2009). These molecules provide
electrolyte homeostasis and maintain the bacterial fluid balance
under osmotic stresses (Frank et al., 2017). This regulation mech-
anism can provide adaptive preference to benthic bacteria capable
assimilating/synthesizing such molecules, thus thrive in elevated
salinities that are often present near desalination outfall sites. We
stress that this subject should be reinforced in future dedicated
studies. Such information will be highly important to the under-
standing of how benthic microbial communities cope with chang-
ing environmental conditions due to multi-stressors from
anthropogenic sources.
3.2.2. Sorek desalination facility
BA in the sediment was ~1.3-fold higher at the outfall area than
the reference sites, whereas BP remained unchanged (Fig. 3A and
B). Yet, BCD was significantly different between the two sampling
stations. At the reference site, BCD was higher by 1.8-fold compared
to the outfall location (Fig. 3C). Concurrently, BGE was higher by 2-
fold at the outfall in respect to the reference site (Fig. 3D). These
changes were mostly attributed to the higher respiration rates
measured at the reference stations (2.85 ± 0.73 mg C gr
1 d
1)
484 H. Frank et al. / Water Research 151 (2019) 478e487
compared with the outfall area (1.31 ± 0.50 mg C gr
1 d
1), and, to a
lesser extent, by the BP rates that remained overall similar (Fig. 3B).
Additionally, phosphonates (organic phosphate-based molecules)
which are often used as antiscalants in the desalination process
(Drami et al., 2011; Belkin et al., 2017; Petersen et al., 2018) can be
found at higher concentrations near the outfall area of Sorek
(0.4e0.7 mM) compared to the typical-background levels (<0.1 mM)
(Kress et al., 2016a,b). It is possible that BR rates as well as
concomitant BCD and BGE can be affected due to higher phos-
phonate concentrations at the outfall site compared to the ambient
environment. We stress that biodegradation of phosphonates by
benthic microbes should be studied in relation to the desalination
process, and especially in oligotrophic, P-limited systems such as
the eastern Mediterranean Sea (Krom et al., 2010). Meiofauna
abundance and community composition (i.e., number of phyla)
were overall similar between the outfall and reference stations,
with ~30% higher abundances at the outfall (Table S2). The mod-
erate difference between the outfall and reference stations implies
that the brine dispersion was more efficient than the coastal
discharge at the Ashkelon desalination facility.
3.2.3. Hadera desalination facility
Benthic BA was similar between the reference and outfall sites
(Fig. 3A). However, BP (Fig. 3B) and BR (Table S1) were significantly
higher by 1.6 fold and 1.7 fold, respectively, at the reference site
relative to the outfall station. Hence, BCD was up to 2-fold higher at
the reference site than the discharge point (Fig. 3C). Despite these
changes in BCD, no significant trends were found in BGE (Fig. 3D).
Total meiofauna abundance and especially platyhelminthes (flat
worm) counts were higher (2 and 6-fold, respectively) at the outfall
area than the reference station (Table S2). Overall, our results
suggest that the discharged brine near the Hadera desalination
plant had a local and moderate environmental effect on the benthic
bacterial biomass and activity. We suggest that these moderate
effects resulted from efficient dilution of the brine with the cooling
water of the adjacent power plant, which occurs on land prior its
discharge as well as additional mixing of the outlet with the Hadera
stream. This is in contrary with the Ashkelon desalination plant
where the brine is discharged directly to the coastal environment
and is being diluted in-situ with cooling seawater of the nearby
power plant. We therefore find that the discharge method applied
in Hadera desalination facility inflicted less environmental impact
on the benthic microbes (i.e., on BA, BP and community structure)
than the one used in Ashkelon, despite both being a coastal
Fig. 4. PCoA plots showing the distance in bacterial diversity (derived from 16S rRNA analyses) between the outfall and reference stations in (A) Ashkelon; (B) Sorek; and (C) Hadera
desalination facilities (n ¼ 8e10).
discharge.
3.3. Changes in bacterial community structure in respect to the
outfall sites of large scale SWRO facilities
3.3.1. Ashkelon desalination facility
PCoA analyses indicated a significant change between the bac-
terial diversity at the outfall and the reference stations (Fig. 4A,
ANOSIM, p ¼ 0.049). These differences are in accordance with the
changes found for the biomass and metabolic rate variables at this
area (Fig. 3). We suggest that bacterial succession occurred toward
halophytic species in the outfall area compared to the native
communities that inhabit the unaffected reference station. Indeed,
members of Planctomycetes such as Rhodopirellula baltica that
increased in their relative OTUs abundance from the reference
(~1%) to the outfall (12%) areas (Fig. 5) are known to be highly
adaptive to high salinities of up to 60‰ (Wecker et al., 2009).
Further, Proteobacterial phylotypes were higher in the outfall vs.
the reference stations (46% vs. 29%, Fig. 5). Members of this phylum
have the genetic capabilities to express osmoregulated periplasmic
glucans and thus cope with hyper-saline environments (Bontemps-
Gallo et al., 2013). Moreover, Actinobacteria, which are important
nutrient recyclers in the benthos (including of polyphosphates)
(Hupfer et al., 2007; Kong et al., 2005), were higher in the outfall
(9%) than reference (2%) stations (Fig. 5). Since polyphosphates are
often used for the SWRO desalination process (Lattemann and
Ho€ pner, 2008; Prihasto et al., 2009) it is not surprising that this
phylum exhibited high relative OTU abundance near the outfall.
Interestingly, the ubiquitous archaea Euryarchaeota were found in
the reference stations (17%) but were almost absent (~0.4%) from
the outfall area (Fig. 5) although it was reported that this phylum
can survive at high salinities (Yakimov et al., 2013).
3.3.2. Sorek desalination facility
Analysis of the bacterial relative OTUs indicated that the bac-
terial diversity was insignificantly different between the outfall and
reference sites in the Sorek desalination plant (Fig. 4B, ANOSIM,
p ¼ 0.799). This was despite the significant differences in the
metabolic rate measurements (Fig. 3), as well as the evident dif-
ferences in the relative OTUs abundance of most bacterial phylum
(Fig. 5) that exhibited some seasonal variations. Relative abundance
of Firmicutes was 9-fold higher in the outfall stations vs. the
reference stations. This bacterial phylum is a known member in
trace-metals contaminated sediments (Dell’Anno et al., 2012). It is
 H. Frank et al. / Water Research 151 (2019) 478e487
Fig. 5. Bacterial dominant OTUs (taxonomic level: phylum) measured near the Ashkelon (A,D), Sorek (B,E) and Hadera (C,F) desalination facilities at the reference (AeC) and outfall
(DeF) stations. Data were pooled from all seasonal measurements (i.e., four cruises). Bacterial groups were chosen if they exceeded 5% of the OTUs.
possible that the use of Fe-based coagulants in the desalination
process (Prihasto et al., 2009) may favor the abundance of this
bacterial phylotype. Actinobacteria and Euryachaeota phyla also
differed between sites where the relative abundance was 3e6-fold
higher in the outfall area compared to the reference station.
Differently, Planctomycetes which have a central role in benthic
carbon cycling (Fuerst and Sagulenko, 2011), was abundant in the
reference stations and nearly disappeared at the outfall sites
(Fig. 5).
3.3.3. Hadera desalination facility
Both the PCoA (Fig. 4C, ANOSIM, p ¼ 0.568) and the relative
OTUs analyses (Fig. 5C) point that the benthic microbial commu-
nities in the reference and outfall sites of Hadera were highly
similar. Proteobacteria dominated the bacterial phylum (46e50%)
followed by Acidobacteria (~8%), Planctomycetes (7e9%) and Bac-
teroides (~5%). The only notable difference was for Firmicutes,
where the relative abundance reduced from 9% at the reference
stations to 3% at the outfall site. The similar bacterial community
structure suggests that the brine-discharge method, namely addi-
tional mixing of the brine with the Hadera stream (Fig. 1) reduced
the environmental impact compared to the Ashkelon desalination
facility. This was also in agreement with the moderate changes
observed for BA, BP and BGE (Fig. 3).
4. Conclusions
Extensive construction of large-scale SWRO desalination facil-
ities is highly common in many arid and coastal regions worldwide.
Yet, only limited environmental research was done to estimate the
impact of desalination brine on adjacent coastal ecosystems and
especially benthic bacteria that inhibit these areas. Our results
indicate that benthic heterotrophic bacteria are affected by desa-
lination brine. These effects are often characterized by changes in
bacterial abundance and bacterial activity resulting in altered
bacterial carbon demand and growth efficiency. In addition, brine
discharge may change the benthic bacterial community at the
outfall area. Nevertheless, we found that these impacts were site
specific and highly local. Namely, that the effects of SWRO brine on
485
benthic bacteria were a combination of the discharge method used
to disperse the brine at each site and the local stressors including:
(i) eutrophication from different anthropogenic sources (i.e.,
amelioration wells and/or sewage runoffs) and (ii) possibly due
elevated water temperatures after mixing with cooling water from
power plants. We also surmise that SWRO brine can indirectly
affect the abundance of heterotrophic bacteria by reducing the
number of higher-trophic organisms such as bacteriovorus due to
osmotic stress. The reduction in bacterivorous abundance such as
nematodes and foraminifers may lead to bacterial proliferation and
changes in microbial food-web structure.
We propose that long-term effects of desalination brine on
benthic bacteria could be minimized by discharging the concen-
trate via diffuser systems, thus removing the dependency of brine
dilution on power plant operation and cooling water capacity. In
addition, reducing the use of power plants cooling water will
decrease the differences between water temperature at the outfall
area and the receiving environment. Additionally, the impact of
SWRO brine on benthic meiofauna as well as of eukaryotic algae
should be studied in future research to better evaluate the
ecological effects of the growing desalination industry on coastal
ecosystems.
Acknowledgments
We would like to thank the captain and crew of the R.V Medi-
terranean Explorer for assistance at sea. The work was funded by
grants awarded by the Israel Ministry of Science, Technology, and
Space (contract number 3-12376), the Israeli water authority
(contract number 4501284678) to E.B-Z and E.R, and the by the
Mediterranean Sea Research Center of Israel (MERCI)
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.watres.2018.12.046.
486 H. Frank et al. / Water Research 151 (2019) 478e487

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