A Multibiomarker Approach to Assess the Health State of Coastal Ecosystem Receiving Desalination Plants in Agadir Bay, Morocco

The present study aims to evaluate the initial health status of two stations receiving seawater desalination plants in Agadir Bay (Tifnit-Douira and Cap Ghir) and to assess their potential environmental impact on the marine ecosystem health. Six pairs of mussels (Mytilus galloprovincialis) were collected at six sampling sites on a monthly basis over two years. Each pair was homogenized to obtain the postmitochondrial fractions (S9). Toxicological effects were measured using a multibiomarker approach based on either acetylcholinesterase (AChE), glutathione S-transferase (GST), catalase (CAT), and malondialdehyde (MDA) rate. The results show a seasonal variation of the biomarkers: their activities increase in summer and decrease in spring and winter. High activities were recorded during summer in Cap Ghir (17.94 ± 0.88; 5.91 ± 052 nmol/min/mg of protein) for CAT and MDA, respectively. In Tifnit-Douira, low activities were recorded during winter for GST (3.74 ± 0.52 nmol/min/mg of protein) and during spring for the CAT (3.52 ± 0.45 nmol/min/mg of protein). The fluctuations in the activities of measured biomarkers could be attributed to different factors including the changes in environmental parameters, the influence of seasonal variation, and the contamination of the aquatic ecosystem. The data obtained in this study should be taken into account in the monitoring and management of the health of the ecosystems when the desalination plants are established.


Introduction
e national and regional sectoral strategy makers (Green Morocco Plan, Plan Azur, Halieutis, etc.) were planned to realize many development programs and projects in the Souss-Massa region [1][2][3][4][5]. However, water availability is a limiting factor for the implementation of these projects and for any regional development [6,7], particularly in regions that are characterized by dry climate conditions and overexploitation of water resources (dam and groundwater).
To overcome these problems, the regional council, as a part of its strategy, has invested in the installation of seawater desalination plants, in Agadir Ida Outanane (Cap Ghir) and Chtouka Ait Baha Province (Tifnit-Douira), to produce water and to meet the requirements of development projects [8,9].
Besides the fact that scientific data of Tifnit-Douira and those of Cap Ghir are not available, the importance of ecological coastal ecosystems and the potential impact of the desalination plants prompted us to launch a new study to assess the initial health status of the ecosystems.
is study was established by using a multidisciplinary approach to assess the coastal ecosystem health by several environmental monitoring programs such as Mussel Watch Program [10][11][12][13]. It will be useful to follow the environmental changes of these ecosystems after the implementation of projects. Our research laboratory has often used this approach for several years to evaluate and to follow-up the environmental quality of Agadir Bay ecosystems [14][15][16][17][18][19][20][21][22]. It is based on the use of many biomarkers, such as acetylcholinesterase, catalase, glutathione S-transferase, and malondialdehyde, as pollution indicators of seawater quality in the sentinel species (Mytilus galloprovincialis, Perna perna, Donax trunculus, Nereis diversicolor, Cerastoderma edule, etc.). e aims of this work are to study the initial health state of the coast (Tifnit-Douira and Cap Ghir) receiving desalination plants of the seawater in Agadir Bay and to evaluate the potential environmental impact of the installation of these plants on the coastal ecosystem. Our study combined classical research using biodiversity, population structure, and analysis of water and sediment parameters.

Studied Areas and Sampling
Sites. Our investigations were conducted in two stations (Cap Ghir and Tifnit-Douira), with three sampling sites for each station (South, Instal, and North). e first one (Cap Ghir) is located 50 km north of Agadir, and it is characterized by ecological and socioeconomic importance due to upwelling phenomenon [23][24][25]. e second one (Tifnit-Douira) is located 60 km south of Agadir Bay in the center of Souss Massa National Park (Figure 1). e choice of these sampling stations and sites was based on their implementation near the desalination plants and also because of the north-south direction of the marine currents [20,26]. In fact, the southern sites (according to the currents) will receive more environmental impact than Instal and North sites.

Specimen Sampling.
Our study was conducted on samples of standardized mollusks with the same shell length (40 to 50 mm) of Mytilus galloprovincialis. ey were collected from February 2015 to March 2017, quickly transferred to laboratory, and stored immediately at − 80°C until use. e choice of this mollusk was based on many reasons: it is a good sentinel species with sedentary lifestyle, it can be collected easily, and it accumulates pollutants by filtration. Moreover, it has often been used as a sentinel species in such studies, particularly in the Mussel Watch Program [27].

Preparation of Postmitochondrial Fraction.
All procedures and preparations were conducted at 4°C. Mussels were thawed, dissected, and drawn out of the shells; the whole soft tissues of each two individual mussels (pool of six pairs) were homogenized for 1 min in a ULTRA-TURRAXT25 homogenizer at 9500 rpm with 1:3 w/v of 100 mM Tris buffer (pH 7.4). e homogenates were then centrifuged for 30 min at 9000g (Sigma, 3-16k), and the postmitochondrial supernatant S9 fraction was stored at − 80°C until use. All biochemical parameters are measured in this S9 fraction.

Acetylcholinesterase Activity.
e AChE activity was measured by the method described by Ellman et al. [28] with the use of acetylthiocholine iodide as the substrate. e enzyme kinetic measurements were performed at 412 nm every 15 seconds for 2 min at 25°C. e enzyme activity was expressed as nanomoles of acetylthiocholine/min/mg of S9 protein.

Glutathione S-Transferase Activity.
e GST activity was determined by the method described by Habig et al. [29] using glutathione (GSH) and 1-chloro-2-4-dinitrobenzene (CDNB) as the substrate. Enzyme activity was estimated by monitoring changes in the optical density (ΔOD/min) at 340 nm for 2 min at 25°C. e GST activity was expressed as nmol/min/mg of protein.

Catalase Activity.
e CAT activity was measured according to the method of Aebi [30] in which the decrease in absorbance at 240 nm caused by the consumption of hydrogen peroxide H 2 O 2 was quantified. e activity of CAT was expressed as nmol/min/mg of protein S9.

Malondialdehyde Rate.
e determination of MDA contents was estimated by the thiobarbituric acid, according to the method described by Sunderman et al. [31] using 1,1,3,3-tetraethyloxypropane as the standard. e absorbance was measured at 532 nm, and MDA content was expressed as nmol/mg of proteins.

Protein Assay.
Total protein concentration in the S9 fraction was determined as stated by Lowry et al. [32] using bovine serum albumin as the reference.

Statistical
Analysis. Data were expressed as mean-± standard deviation (m ± SD) and repeated in six replicates. Comparison of the means was done by the analysis of variance (ANOVA). e statistical significance of the differences between samples was determined using Statistica (version 6) software and Fisher's least significant difference (LSD) test. A p value less than 0.05 was considered statistically significant.

Biomarkers Response in Cap Ghir Station.
e seasonal variation of biomarkers in Mytilus galloprovincialis from three sites of Cap Ghir station is illustrated in Figure 2. e results showed that the AChE activity (Figure 2(a)) displays the same tendency of seasonal variations in the three sites of Cap Ghir station. e highest value was observed during summer (7.87 ± 0.38 nmol/min/mg of protein in 2016), and the lowest one was recorded in spring (1.94 ± 0.37 nmol/ min/mg of protein in 2015). e statistical test regarding the three sites does not show any significant difference, except in summer 2015 and spring 2016 using AChE biomarkers.  e statistical analysis of CAT in the three sites of Cap Ghir station did not show any significant difference during the studied period. However, the results show that, in Cap Ghir, the CAT induction was more significant in 2016 compared to 2015.
For the MDA rate ( Figure 2(d)), we recorded the maximum value in summer 2015 (5.91 ± 0.52 nmol/mg of proteins) and the minimum one in spring 2015 (1.13 ± 0.04 nmol/mg of protein). e statistical test did not show any significant difference between the three sites in Tifnit-Douira and Cap Ghir.

Biomarkers Response in Tifnit-Douira Station.
e AChE activity was high in summer (8.85 ± 0.61 nmol/min/mg of protein in 2016) and low in spring (1.99 ± 0.37 nmol/min/mg of protein in 2015) (Figure 3(a)). Similar seasonal variations were recorded in the three sites of Tifnit-Douira station. e only significant differences were recorded in the North site of Tifnit-Douira during the summer of 2016. e results obtained for the GST activity in Tifnit-Douira (Figure 3

Discussion
e AChE activity has a seasonal profile, i.e., higher activity in summer and lower one in spring. e difference in AChE activity is noted in the southern sites during the summer of 2015 and spring of 2016 in Cap Ghir and during the summer of 2016 in the North site of Tifnit-Douira. Such a difference is probably related to the change in environmental conditions. e measured values in Mytilus galloprovincialis during the spring of 2015 show that this biomarker of neurotoxicity was significantly inhibited in mussel populations in these two stations. is inhibition is actually attributed either to the presence of contaminants in the environment, physiological change state, environmental parameters, or to the reproductive cycle of mussels. e results of the seasonal variations of AChE activity in the present study are in concordance with those reported by Abbassi et al. [22], using the same mollusk as a sentinel  AChE activity is inhibited by the contaminants such as carbamate and organophosphate pesticides used in agriculture [19]. is inhibition was also observed in other species of polychaete Nereis diversicolor [17] and in Diopatra neapolitana [33]. Other authors associate this activity in mussels to the reproductive cycle [34]; however, the maximum of AChE activity coincides with spawning period, and the minimum one overlapped with the sexual rest [35,36]. e contamination by heavy metals can also cause AChE inhibition during spring. Moukrim et al. [37] reported that the highest level of contamination by Cd, Cu, and Zn was observed in winter and spring, and the lowest one was in summer and fall in Agadir Bay. is contamination is imported by seawater mass of currents upwelling, which took place in these sites' coasts. Similar results are reported by Banaoui et al. [38] and De Lima et al. [39]. Nadir et al. [21] reported that Mercury contamination has a direct influence on biomarkers in Donax trunculus. Other types of pollution can affect AChE activity, like wastewater [22], contamination by polycyclic aromatic hydrocarbons (PAHs), and organic chemicals [14,[40][41][42]. Our results corroborate those of Lagbouri [43] and Moukrim et al. [16] in Anza beach (Agadir Bay) using the other sentinel species Donax trunculus. ey measure higher value in summer and lower one in spring in the polluted site.   e Scientific World Journal e lowest AChE activity in soft mass of organisms in the South site of Cap Ghir could be due to the influence of hydrodynamics. e same activity was observed in Tifnit-Douira because of the very important dose of fertilizers and pesticides that arrived to the sea by drainage of Oued Souss and Oued Massa; they are then sprinkled by the marine currents (direction North to South) and they affect organisms, especially in spring, which overlaps with maximum inhibition of AChE enzyme activity. e organochlorine pesticide (OCP) concentration is high in the sediment and in the sentinel species Nereis diversicolor during spring in Oued Souss estuary according to Agnaou et al. [19]. Banaoui et al. [20] showed that the AChE activity revealed a significant inhibition in three mollusk species (D. trunculus, P. perna, and M. galloprovincialis) following a contamination by organophosphorus and organochlorinated pesticides. e reported decrease in the AChE activity in Tifnit-Douira station may also be related to the presence of sand dunes in the coastal zone that could be brought into the sea by downwind transport in the region that import sprayed pesticides in the agricultural zone accumulated in the sediment. e presence of the army field in Tifnit-Douira may have some consequences; the metals and chemical residues of ammunition can reach the sea by sand movements and cause inhibition of AChE activity. e results obtained in this work showed that GST activity varies following a seasonal tendency, with high values during summer and lowest ones during spring and winter. At the same time, we have registered a significant difference between stations for GST activity during spring, autumn, and winter of 2015. is kind of variations could only be due to the change in the environmental conditions during the seasons of the studied period. e peak of GST activity and the significant difference between sites was registered in the Instal site of Cap Ghir during spring 2016 compared to Tifnit-Douira station. is stimulation in the two stations could be explained by the overlap of sampling with a period of high precipitations during 2016. ey can bring different types of pollutants to the sea by Oued Souss and Oued Massa. ose pollutions contribute to the stimulations of the detoxification processes. e GST is the second enzyme phase involved in the metabolism of the detoxification of lipophilic organic contaminants [44,45]. e apparent increase in the GST activity may be attributed to the presence of different types of pollution in aquatic ecosystems, which stimulates the cellular detoxification process of xenobiotics [44][45][46]. It is also considered like an adaptive response to altered environment reported by Abbassi et al. [22]. Many other studies have described a similar relationship between environmental pollution and GST activity in mussels and other sentinel organisms in marine ecosystems.
High GST activity levels are caused by exposing molluscs to pesticides [20], heavy metal pollutants [21,47], wastewater [15][16][17][18], and some other toxins such as polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs) [48]. e same results of seasonal evolution of GST activity were already obtained by many studies in Moroccan coasts and Estuary (Agadir Bay), like in Mytilus galloprovincialis and Perna perna [15], in Donax trunculus [20,21], in Nereis diversicolor [17], in Scrobicularia plana and Cerastoderma edule [18]. In addition, the CAT activity shows the same seasonal tendency with an increase in summer and a decrease in spring and winter.
No significant difference was observed between stations and between sites during 2015 and 2016 seasons except for the northern site of Tifnit-Douira during the summer of 2016. When the maximum was observed during the two years of the study, the induction would be attributed to the change in the seawater temperature in the summer and probably to the presence of the same oxidative stress factors like environmental and physiological changes.
e CAT activity is considered as the first line of defense against oxidative stress of organisms [49]. e seasonal tendency of CAT activity obtained in our study confirms the results of Mytilus galloprovincialis living in the coast of Sidi Ifni, South Morocco [22], and in other sentinel species like Donax trunculus from Anza and Aghroud beaches (Agadir Bay) [16]. Authors reported high CAT levels especially in impacted sites [18,43], and others confirmed a positive correlation between inductions of CAT activity and some heavy metal concentrations [21].
MDA levels showed high values during all periods of our study, and the only significant difference was registered in the South site comparing the two stations. e same results were revealed by many studies using Donax trunculus in Agadir Bay [21], Tunis Golf [50], and Mirleft and Sidi Ifni coast line (South of Morocco) in Mytilus galloprovincialis [22]. Such variations have also been reported by El Jourmi et al. [51] in Perna perna along the coast of the big Casablanca. Several other studies have described that MDA levels may be positively correlated with the level of certain pollutants such as heavy metals and organic compounds [52,53]. Bendjoudi et al. [54] reported that biological contamination by Staphylococcus aureus causes induction of the GST, CAT, and MDA. e results show that the investigated biomarkers present the same tendency: increase in summer and decrease in spring and winter excluding the spring of 2016. We observed high stimulation of GST, CAT, and MDA, accompanied by the perturbation in environmental parameters (T, pH, salinity, etc.) in seawater due to high pluviometry registered in this period.

Conclusion
e results obtained during our investigations contribute to the assessment of the initial health status of the sites that will receive the two desalination plants of seawater in Agadir Bay. e use of biochemical parameters to assess the health status of both studied ecosystems showed that these parameters present variations that can be attributed to natural factors, while others may be affected due to the presence of contaminants in the environment.
is study will be of great use for the monitoring and management of the two ecosystems during and after the installation of the two seawater desalination plants. Furthermore, it will minimize the impact as much as possible in these two stable ecosystems. 6 e Scientific World Journal

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
ere are no conflicts of interest.