Metallic Contamination of theMuscles of Three Fish Species from the Moulouya River (Lower Moulouya, Eastern Morocco)

%is study introduces a spatiotemporal evaluation of the metallic contamination with three trace metals (mercury, lead, and cadmium) in the muscles of three fish species (Lepomis macrochirus, Barbus callensis, and Barbus nasus), of which the samples were taken from three stations in the Moulouya River: the confluence of the Moulouya River and the Sebra River (station 1), the neighbourhood of the farms in the region of Aklim downstream from confluence of Lakhmis river (station 2), and the level of ancient bridge of Ras El Ma-Moulouya (station 3), during the period from July 2017 to May 2018. %e results have allowed us to highlight rather high contents of lead and mercury in the fish muscles, mainly in station 1, which receives domestic and industrial discharges. %e contents of cadmium in all fish species in different stations are very low. Moreover, all concentrations assessed in different fish species do not exceed the maximum limit recommended by European Community (EC) Commission Regulation No. 1881/2006.


Introduction
e Moulouya River, which extends over a 520 km length, is considered to be the blue artery of the eastern region of Morocco. It has a major socioeconomic importance; it is used for irrigation. It is also a source of drinking water and has a big wealth of flora and fauna aquatic [1]. Fishery resources are among the most present animals in the river. e study of Melhaoui and Boudo in 2009 [2] revealed the presence of 29 species. e 29 species are distributed in the upper, middle, and lower regions of Moulouya. e abundance of fishery resources is more in the lower part, with an important activity of fishing along the river. On the contrary, at the higher and medium level, fish is not much varying, is very disseminated, and has weak density. is shows us that the fishing is mainly focused at the level of lower Moulouya. e local industrial (oil mills, the Sucrafor sugar factory in Zaio), agricultural (the plain of Triffa and of Sebra), and urban activities (the cities: Taourirt, Zaio, Aklim, Berkane) can affect the quality of waters in this river by the discharge of many contaminants [3][4][5], making them inappropriate to use. is contamination will be able to impact negatively on biodiversity of fauna and flora of this river, which leads to intoxication and mortality of fauna, as in the case of July 15th, 2011, when thousands of fish were found dead and floating at the level of lower Moulouya [6].
Among the possible contaminants that can constitute a danger to aquatic life in general and the fish fauna especially are trace metals. ey are very toxic even in weak concentrations and are characterized by their resistance to deterioration by organisms (not biodegradable), which causes their accumulation in the living organisms (the bioaccumulation) [7,8].
erefore, the consumption of fishes contaminated with greater levels of trace metals can pose risks to health of humans. Among the wide range of trace metals contaminating the aquatic ecosystem, a major concern has been focused on cadmium, lead, and mercury. ey are nonessential elements occupying top positions in all lists of toxicants [9,10].
To the best of our knowledge, there is only one single study on the contamination of the fish of the Moulouya River by trace metals [11]; this study is limited to single species (Anguilla anguilla) at the level of single site, that is, the mouth of the Moulouya.
From this official report, we have gotten the idea of doing this study again, but on different types of fish species of the Moulouya River and in different stations. We led a new study on three different fish species and in a larger zone. is study introduces three main objectives: the first is to use the fish as indicator of the metallic pollution of Moulouya River, the second is to assess the largeness of the consumers' health risks, and the last is to know which fish species are most contaminated with trace metals.
is study is interested in lower Moulouya, from the confluence of the Moulouya and the Sebra River up to the mouth of Moulouya. is part of the river is chosen because it is the richest in fish in comparison with other zones, and it is known for fishing activities. is study also introduces a spatiotemporal evaluation of the metallic pollution of the fish fauna of lower Moulouya, by determining the most alarming and dangerous concentrations of trace metals (cadmium, lead, and mercury (for the first time)) in the muscles of three fish species.  e fish samples were captured using a combination of fishing rods and gill nets of various mesh sizes; we determined the species to which every fish belonged using keys by Azeroual (2003) [12]. A total of 121 individuals representing 3 species (Lepomis macrochirus, Barbus callensis, and Barbus nasus) were collected. e standard length (cm) and weight (g) of each fish were determined using a vernier caliper and an analytical pocket balance, respectively. e individuals belonging to the same species were conditioned in the same polyethylene bags, which contained a chip of identification assuring their marking, then stocked in a cool box where the temperature is about 4°C; after that, they were routed in the laboratory, where they were frozen at −25°C until the instant of the analysis.

Metal Analysis.
e fish samples were taken to the Regional Laboratory of Analysis and Research of National Office of Food Safety (RLAR, ONSSA) in Tangier, where they were dissected using stainless steel scalpels. We were interested in the edible parts of the fish that were ground and homogenized by a domestic food blender. At least three fish individuals were used as samples for trace metal analysis at each station, and each sample was analyzed three times to get the average value of metal content in fish. Mineralization was performed according to the technique described by AOAC Official Method [13], where a quantity varying between 0.5 g and 0.6 g of the homogenate mixture was treated, in a closed Teflon vessel, with 5 ml of Suprapur (69%) nitric acid (HNO 3 ) for mercury, and 5 ml of Suprapur (69%) HNO 3 and 2 ml of Suprapur (30%) hydrogen peroxide (H2O2) for  lead and cadmium. e digestion flasks were then put on a microwave oven (Berghof speedwave MWS-2) and gradually heated (for 45 min up to 185 C°) until all the materials were dissolved. After digestion and cooling to room temperature, the samples were diluted by 50 ml with ultrapure water in polyethylene tubes. e contents of trace metals were determined for lead and cadmium by a Graphite Furnace Atomic Absorption Spectrometry (GF-AAS) facility (Varian Perkin Elmer, ACE 800), equipped with a fully automated autosampler system. 2.5% NH 4 H 2 PO 4 and 1% Mg(NO 3 ) 2 were the applied matrix modifiers. Mercury was quantified by Cold Vapour Atomic Absorption Spectrometry (CV-AAS) (VARIAN FIMS 100). e reducing agent used is stannous chloride (SnCl 2 ) at 2.5%, and the carrier solution is hydrochloric acid (HCl) at 3%. For the two spectrometry techniques, using high purity argon as the carrier gas, the flow rate was 50 ml/min.

Quality Assurance and Quality Control.
All the tools used have been cleaned by soaking overnight in HNO 3 (10%), rinsed with ultrapure water, and dried before each use. In addition to HNO 3 , the Teflon vessels have been cleaned with acetone. All of the reagents employed in this study are of analytical grade. e calibration curve demonstrates good linearity for the three trace metals, with correlation coefficients (r) greater than 0.999 (Table 1). e limit of quantification (LQ), the wavelength, and the standard calibration concentration of the present study are presented in Table 1.
According to ISO 17025, the accreditation laboratories that perform analytical service must have quality control procedure for monitoring the validity of tests undertaken [14]. e methods of GF-AAS and CV-AAS were accredited in laboratory (RLAR, ONSSA), and the accuracy of the analytical methods was evaluated by participation to proficiency test schemes. e test materials distributed were canned fish at different concentrations of cadmium, lead, and mercury, obtained from the Food Analysis Performance Assessment Scheme (FAPAS). Replicate analysis of these proficiency tests showed good accuracy, with recovery rates for trace metals between 97.67% and 100.46% (Table 2).

Results and Discussion
e number of fish individuals caught for each station and the biometric parameters (weight and length) of the fish species are summarized in Table 3.
e results of the spatiotemporal measure of metallic contents in the muscles of the different captured fish species are presented in Table 4 and Figure 3.
At all the stations, the results of cadmium concentration in the analyzed fish species are low. ey vary from 0.001 mg/kg of wet weight in Barbus callensis and Lepomis macrochirus of station 2 to 0.006 mg/kg of wet weight in Barbus callensis captured in station 1 (Table 4).
Similar results were found in the muscles of Barbus meridionalis of the Ripoll River in Catalonia [15], of Luciobarbus graellsii, Rutilus rutilus, and Lepomis gibbosus in station 1 from the Llobregat River to Spain [16], and in Lepomis gibbosus from theŠalek Lakes in Slovenia [17]. A weak concentration of cadmium was revealed in the muscles of Barbus callensis and Liza ramada by El Morhit et al. [18] and in the muscles of Barbus barbus and Anguilla anguilla by Boscher et al. [19]. On the contrary, high concentrations of cadmium were obtained by Rajotte and Couture [20], Yi and Zhang [21], and Arantes et al. [22] (Table 5).
No significant correlation (P < 0.05) was found between cadmium and other trace metals in different species of fish in all stations (Table 6). e order of the bioaccumulation of cadmium in the muscles of the studied fish is slightly high in Barbus nasus ( Table 7).
Note that the concentration of cadmium in the muscles of studied fish species is much less than the regulation limit of cadmium in the flesh of fish (0.05 mg/kg of wet weight) according to Commission Regulation (EC) No. 1881/2006 [28] (the same standard is adopted by Morocco). e concentration of lead (Table 4) revealed in the muscles of the fish studied varies from 0.016 mg/kg of wet weight in Barbus callensis gotten in station 2 to 0.200 mg/kg of wet weight in Lepomis macrochirus of station 3. Similar levels were found by El Bouhali et al. [26], Merciai et al. [16], El Morhit et al. [18], and Djedjibegovic et al. [23]. However, low concentrations of lead were found by Maceda-Veiga et al. [15], Boscher et al. [19], Wariaghli et al. [27], and International Journal of Ecology     Petkovšek et al. [17] compared to our results. On the other hand, some studies showed high contents of lead in the muscles of fish, such as those of Yi and Zhang. [21] and Arantes et al. [22] (Table 5). Very high positive correlations were recorded between the bioaccumulation of lead and mercury at a level of signification (P < 0.05) for the fish in these stations, whose correlation coefficient is 0.9997 (Table 6). e order of the bioaccumulation of lead in the muscles of the studied fish species is relatively high in Lepomis macrochirus (Table 7). is signals also that lead concentration in the muscles of studied fish species is much less than the regulation limit of lead in the flesh of fish (0.3 mg/kg of wet weight) according to Commission Regulation (EC) No. 1881/2006 [28].
e results of mercury measured in the fish muscles show that the lowest value is 0.009 mg/kg of wet weight in Barbus callensis captured in station 2, and the highest value is 0.187 mg/kg of wet weight in Lepomis macrochirus captured in station 2 (Table 4).
Close values were found by Boscher et al. [19] in Barbus barbus from the Sûre River in Luxembourg, Petkovšek et al. [17] on Lepomis gibbosus from theŠalek Lakes in Slovenia, and Djedjibegovic et al. [23] in the muscles of different fish species from Neretva River in Bosna and Herzegovina. However, low concentrations of mercury were found in the muscles of different fish species from the Yangtze River in China [21]. On the contrary, high concentrations of mercury were found in the muscles of Barbus meridionalis from the Ripoll River in Spain [15], in the muscles of Micropterus salmoides from the Sipsey River in USA [24], and in the muscles of different fish species from the Cecina River in Italy [25]. In addition, high concentrations were found in the muscles of Pseudoplatystoma corruscans from the Paraopeba River in Brazil [22] (Table 5).
Mercury and lead are strongly correlated at a level of signification (P < 0.05) for the fish in these stations, whose correlation coefficient is 0.9997 (Table 6). e order of the bioaccumulation of mercury in the muscles of the studied fish species is relatively high in Lepomis macrochirus (Table 7).    [28]. e muscles of Lepomis macrochirus contain high concentrations of mercury and lead, attaining a value of 0.187 mg/ kg of wet weight for mercury and 0.200 mg/kg of wet weight for lead. is can be attributed to the nature of the food of this fish species, as it is considered very voracious eater feeding on almost everything, thus having a strong accumulation of trace metals in its body [12,29]. e relatively high concentrations of lead and mercury in the muscles of Barbus Nasus and Barbus callensis captured in station 1 may be due to significant bioaccumulation, probably related to the high concentrations of lead and mercury in the station itself.
Comparison of the mean concentrations of trace metals detected in the muscles of the fish species studied shows that lead contents are the highest compared to those of mercury and cadmium (Table 8). From these results, we can establish the general order of bioaccumulation of the trace metals measured in the muscles of the different fish species which is as follows: Pb > Hg > Cd. According to reported results [30][31][32], trace metals bioaccumulation is related to fish species, type of trace metals, length/age, and physical and chemical characteristics of water. Several studies have demonstrated that cadmium has a low tendency to accumulate in muscles, where the concentrations are usually very low [10,33]. Unlike cadmium, mercury preferentially accumulates in muscles due to their affinity for the sulfhydryl groups of proteins [15]. e analysis of the physicochemical parameters shows that the water of the Moulouya River has high dissolved organic matter values and an alkaline pH [3,34], which may decrease the bioavailability of trace metals in water column [35], therefore inducing a decrease in the rate of absorption and accumulation of trace metals in fish muscles. Otherwise, the high levels of dissolved organic matter may also increase microbial activity which is responsible for the release of trace metals from sediment to the water column [15,35]. In addition, the high water temperatures of the Moulouya River recorded during the summer season [3] lead to an increase in fish metabolism that could also increase pollution effects on fish [15].
Tissue concentrations found in dry weight were converted to wet weight by multiplying by a factor of 0.2 (considering an average water content in fish tissues of 80%) [36]. e space analysis of trace metals in the muscles of the studied fish species shows a positive correlation between the intensity of trace metals and some stations. erefore, as illustrated in Table 9, it was noted well that station 1 contains fish more contaminated with trace metals than other stations, which can be attributed primarily to the urban discharges from the city of Zaio which are loaded in trace metals and secondly to the industrial discharges of the refinery of sugar. Several studies have shown that the sugar industries wastewaters are loaded with organic matter and have a negative impact on the physicochemical parameters of the water [37][38][39]. In addition, some other studies have shown that the effluents of sugar factories can contain relatively high levels of trace metals [39][40][41]. Concerning stations 2 and 3, we note that the values of the trace metals in the muscles of the studied fish species are less than those in station 1, because these two stations (2 and 3) do not receive domestic and industrial discharges, even though they are located below the confluence point of the Moulouya with Charaâ river for station 3 and Lakhmis river for station 2, but their low flows do not help them reach Moulouya River. e explanation that can be given in terms of sources of trace metals in these two stations (station 2 and station 3) is the leaching of agricultural land during rainy periods (mainly in winter) and maybe also the road traffic mainly for station 3 located near ancient bridge of Ras El Ma-Moulouya. It is also potentially related to high evaporation of river water, which caused an increase in the concentrations of trace metals in the water. e seasonal variation of trace metals in the muscles of the studied fish species has slightly high concentrations during summer season (Figure 3), in view of the fact that the weak flow of Moulouya River during this season does not seem to dilute the industrial and domestic discharges of tributaries. However, the winter is the season when contents of trace metals are very low, so strong flow of currents leads to the dispersion of trace metals. Moreover, the very high temperatures in the summer could also lead to higher metabolic rates, which could induce an increase in fish food activity, and this in turn increases the concentration of metals in fish [42,43].
us, the concentration of trace metals in a fish is the product of a balance between the concentration of the metal in its environment and its rate of ingestion and excretion by fish [44,45].
Although concentration of trace metals in the muscles of the studied species does not exceed the proposed limit values, it is correlated with other contaminants (pesticides, PCB) likely to increase toxic effects on fish-farming fauna [48].
erefore, further studies are needed to identify all pollutants that may threaten the health of fish and fish consumers in this area and to determine the relationships between them.

Conclusions
According to the present results, we have noted that mercury and lead are both trace metals most concentrated in the muscles of the studied fish (it attained 0.187 mg/kg of wet Pb > Hg > Cd Table 9: e order of enrichment of stations for each trace metals in the fish muscles.

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International Journal of Ecology weight for mercury and 0.200 mg/kg of wet weight for lead), while cadmium is the metal with the lowest concentration (does not exceed 0.006 mg/kg of wet weight). Station 1 (confluence of Moulouya River with Sebra River) includes fish most contaminated with trace metals, because of their exposure to the domestic discharges of the city Zaio and industrial discharges from the Sucrafor sugar factory in Zaio.
Lepomis macrochirus is the species with the highest values of trace metals, while the season that records the highest concentrations of trace metals is the summer. All concentrations of three traces metals (lead, cadmium, and mercury) found in the muscles of the different studied fish species have values not exceeding the regulation limit of the European Community (EC), so the consumption of the fish in lower Moulouya of Morocco is not dangerous for human health.

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

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this work.