# Heavy Metals Concentration inSardina pilchardus (Walbaum, 1792) from the Moroccan Mediterranean Coast and Potential Human Health Risk Assessment

Authors: Khaoula Kasmi; Kamal Belhaj; Hanae Nasri; Douaa Slimani; Larbi Allai; Farid Mansouri; Souad Aissioui; Souad Abdellaoui; Mohamed Addi; Abdelhafid Chafi
Journal: Journal of Food Quality
 (2023)

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## Abstract

Due to their toxicity, long persistence, bioaccumulative nature, and biomagnification in the food chain, heavy metals pose a serious hazard. The aim of this study was to evaluate the health risks associated with the consumption ofSardina pilchardus (Walbaum, 1792) and to study the spatiotemporal dynamics of four potentially toxic metallic elements. Three hundred and sixty specimens of sardine were collected between December 2020 and October 2021 at three Mediterranean coast sites, Beni-Ensar, Ras el Ma, and El-Houceima, in the northeast region of Morocco. The toxic metallic elements were evaluated by assessing the contamination level of cadmium (Cd), mercury (Hg), lead (Pb), and arsenic (As) in the muscle, liver, and gills of sardine from the Mediterranean Moroccan coast. The results showed a significant effect of the study area, organ, and season (p>0.05) on Moroccan Mediterranean sardine heavy metal contamination levels. Regardless the location and season, the liver presents the higher concentration of the studied metals (p<0.05). The highest heavy metal concentrations of Cd (0.408 μg·g−1 wet weight (ww)), Hg (0.044 μg·g−1 ww), and As (6.74 μg·g−1 ww) were found in winter while the concentration of Pb was the highest in autumn (0.056 μg·g−1 ww). Furthermore, the lowest contamination of metal levels was found in the spring. El-Houceima region contains the highest values for Hg (0.093 μg·g−1 ww), Pb (0.018 μg·g−1 ww), and As (7.73 μg·g−1 ww). However, the highest values for Cd (0.172 μg·g−1 ww) were recorded in Beni-Ensar. Regarding the assessment of possible risks to human health, the results showed that the indices are below the established safety values in the case of estimated weekly intake, and target hazard quotient (THQ). In contrast, the carcinogenic risk index and total THQ were above the threshold limits and thus represented a potential carcinogenic risk to human health.


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## Body

## 1. Introduction

Fish is one of the most popular parts of the Mediterranean diet. It is very appreciated among the populations, and its consumption has widely increased in many countries owing to its high nutritional value [1, 2]. It contains, among others, essential fatty acids, indispensable amino acids, and minerals which are crucial for the cell growth and the brain function; it also prevents stroke and heart diseases [3, 4]. However, the aquatic environment is often subject to excessive contamination and noxious effects from industrial discharges, urbanization, and extensive agricultural activities [5, 6]. The metal contamination is an alarming concern given that heavy metals are persistent bioaccumulative toxic contaminants and could be, once entered the food chain, transferred to people through seafood consumption. The metallic elements are divided into essential and nonessential metals [7]. Zinc, copper, iron, and manganese play a significant part in biological systems and typical biological processes in living organisms [8, 9]. Metals and metalloids such as arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) derived from both natural and anthropogenic sources are considered as toxic substances and their presence in excessive amounts can cause serious health issues. They enter the aquatic environment and consequently the food chain by bioamplification and biomagnification to ultimately reach the human [10]. Several authors have studied fish contamination levels by potentially toxic elements by examining the effect of species, location, season, and organ. According to the literature, element content varies within groups of species. This variability suggests a complex interaction of several factors such as ecological needs, metabolic regulation, physiological processes, feeding source, reproduction, food supply, and environmental conditions [11–13]. As a result of the heavy metal accumulation in both freshwater and marine environments, heavy metal contaminated fish are now a threat to human health. Fish is a great bioaccumulator and the assimilation can occur in the bones, heart, kidney, operculum, liver, and flesh, and heavy metals can proceed by ingestion, ion exchange across the surface of the gills or membranes, and tissue absorption in fish. One of the most significant commercial species in the Mediterranean Sea is Sardina pilchardus [14]. Moreover, S. pilchardus makes for approximately 45% of the biomass for all small pelagic species captured in Northwest Africa’s commercial fisheries in 2017 [15]. Furthermore, in Morocco, this pelagic species is one of the highly appreciated species by the consumers as well as fishermen with an annual production of 968 477 tons representing more than half of the total Moroccan fisheries production [16]. To assess the environmental pollution and the consumer health risk associated with Mediterranean S. pilchardus consumption, this investigation was carried out on three Moroccan Mediterranean coastlines. Beni-Ensar Sea and Ras el Ma Sea are the two main ports in the eastern region of Morocco, and that of El-Houceima in the northeast. The latter is subject to strong anthropogenic pressures including a large urban population (generating significant urban discharges), a developed industrial chain, and the airport pollution effect. The Beni-Ensar Sea site is relatively subject to high anthropogenic pressure with a middle-developed urban fabric, an industrial chain, and the airport pollution effect. The Ras el Ma Sea site is subject to less anthropogenic pressure than the first two sites, but it is the mouth of the Moulouya wadi which drains discharges from various industries in particular the agricultural rejects.Since there have been very few studies on the heavy metal contents inS. pilchardus from the Moroccan Mediterranean Sea, the objectives of this study were to examine potential health hazards related to the consumption of this species, evaluate the dynamics of four contaminants (As, Cd, Hg, and Pb) in relation to spatiotemporal and biological characteristics of S. pilchardus, and finally look into the habitat quality of this fish using multivariate analysis to process several variables simultaneously, in order to highlight relationships of dependence between studied factors and contaminants.


## 2. Materials and Methods

### 2.1. Study Site

S. pilchardus samples were taken at the level of three ports in the Mediterranean Sea that extends over a coastline of 512 km (El-Houceima Sea (35.250000, −3.933333), Beni-Ensar Sea (35.2569, −2.93417), and Ras el Ma Sea (35.1407, −2.42455), Figure 1).Figure 1
The geographical site of the three studied zones in the Mediterranean coast of northeast Morocco.


### 2.2. Sampling and Samples Preparation

A total of 360 specimens ofS. pilchardus (n = 360) were collected using artisanal fishing (nets) throughout four seasons from December 2020 until November in three targeted sites according to a factorial schema 4 × 3 × 3, respectively, for the season, area, and organ. The sampling was carried out during each season, 10 specimens per month to obtain 30 samples per season: December, January, and February for winter; March, April, and May for spring; June, July, and August for summer; and September, October, and November for autumn. The sardines were divided into four groups for each area (n = 30 for each area and each organ in each season). In the laboratory, the samples were cleaned with distilled water and were dissected using stainless steel scissors. On each individual, three parts were taken separately, muscle, gills, and liver. The materials were dried to a consistent weight at 103 ± 2°C. Then, they were crushed with an agate mortar, to avoid any external contamination by heavy metals, and to generate a homogenous meat powder that was stored under a vacuum in sachets sterilized at −18°C, until subsequent analyses. During sample preparation and analysis, acid-cleaned laboratory materials were utilized to avoid any possible contamination.


### 2.3. Chemical Analyses

A quantity varying from 1 to 2 grams of dry biological material was used for the analysis of the following metals: Cd, Hg, Pb, and As. The mineralization was realized by wet digestion using a biacid attack (15 ml of HNO3 and 5 ml of H2SO4) combined with the heating on a hot plate (60°C, 120°C, and 150°C). Then, when the samples went dark, the beaker was taken off the hot plate. The content was filtered (0.45 µm) and transferred into a 50 ml volumetric flask and then diluted with double distilled water. Finally, the samples were stored in polypropylene bottles at 4°C until the moment of analysis. The metal concentrations were measured using inductively coupled plasma mass spectrometry (ICP-MS, Agilent, Series 7500). The results are presented as μg·g−1 wet weight (ww).Quality control was performed using certified reference material (CRM) for Sigma-Aldrich Nos. 1.70384.0100 and 1.11355.0100: MERCK 1.70384, Mercury ICP standard 10000 mg/l Hg Certipur®, diluted nitric acid Suprapur® 10%, for Hg; MERCK 1.1355.0100 ICP multi-element standard solution IV 1000 mg/l Certipur®, diluted nitric acid Suprapur® 6.5% for Cd, Pb, and As. The limit of detection (LOD) and lowest limit of quantification (LOQ) were calculated: Cd, Hg, and As—LOD = 5 × 10−3 mg/kg, LOQ = 10−2 mg/kg and for Pb—LOD = 5 × 10−3 mg/kg, LOQ = 5 × 10−2 mg/kg. The uncertainty was estimated between 10 and 15%. The accuracy recovery for the analytical methods used in the examination of Hg, Cd, Pb, and As contents in CRM samples were 92.2%, 99.9%, 100.1%, and 103.0%, respectively.


### 2.4. Health Risk Assessment

In order to analyze the risk to human health associated with Moroccan Mediterranean sardine consumption, the estimated weekly intake (EWI), estimated daily intake (EDI), target hazard quotient (THQ), and lifetime cancer risk (CR) were calculated for each analyzed metal. The EWI was estimated according to the equation (1) considering the studied metals content in sardine muscle and the weekly fish consumption.(1)EWI=Ci×IRwBw,where EWI is reported in μg/g wet weight; Ci is the metallic element content in seafood (μg/g ww); the weekly ingestion rate is known as IRw (g/week), 169 g/capita/week [16, 17]; and the Bw is the body weight, in Morocco, the mean adult body weight is 73.35 kg [18].The THQ was estimated according to equation (2), it is the ratio between the exposure and the reference dose (RfD) [19]. This index is calculated to evaluate the possibility of adverse health effects other than cancer [20]. Nonobvious risk is indicated by a ratio of less than 1. It is significant to highlight that if the dose is equivalent to or higher than the RfD, an exposed population will face health hazards [19].(2)THQ=EFxEDxFIRxCiRfDxBWxAT×10−3,where EF = the exposure frequency (365 days/year); ED = the average lifetime duration; 76.9 years for the Moroccan population [21]; FIR = the fish ingestion rate (38 g/person/day) [16, 17]; Ci = the metal concentration in the fish sample; RfD = the reference oral dose in μg·g−1·day−1 (Hg: 1 ∗ 10−4, As: 3 × 10−4, Cd: 1 × 10−3, and Pb: 4 × 10−3) [19]; BW = the average body weight of the adult consumer; 73.35 kg for Moroccan population [18]; AT = the mean time of exposure to noncarcinogens (365 days/year∗ED) [22].The daily dose was multiplied by the cancer slope factor (CSF), which was obtained from the response-dose curve for toxicant consumption, to determine the lifetime cancer risk (CR), following the formula [23]:(3)CR=EFxEDxFIRxCxCSFBWxAT,where CSF stands for the Integrated Risk Information System’s oral carcinogenic slope factor (μg/g/day). Only As (1.5 μg/g/day), Cd (6.3 μg/g/day), and Pb (8.5 × 10−3 μg/g/day) have CSF values available [24].Since consumers are frequently exposed to multiple contaminants with associated combinations or interacting effects, the total target hazard quotient (TTHQ), which is the sum of the THQ values, was also calculated in order to evaluate the danger of the four metallic elements collectively [25], the formula is as follows:(4)TTHQ=THQMetal1+THQMetal2+THQMetal3+THQMetal4.Assuming that the hazardous form of arsenic (inorganic arsenic) made up of 3% of the total concentration, the EWI, THQ, and CR for arsenic were determined [26, 27]. A TTHQ value greater than 1 is viewed as a sign of significant danger, or a serious health concern [28].


### 2.5. Statistical Analysis

Using the statistical analysis program SPSS version 20 and taking into account the location, season, organ, and their interaction as the source of variation, Shapiro–Wilk test for quantitative variables was used to examine the normal distribution. The Shapiro–Wilk test is a useful tool for testing the normality of data, which is an important assumption for many statistical tests such as the analysis of variance (ANOVA) and principal component analysis (PCA). If the data are not normally distributed, these assumptions may not hold, and the statistical analysis results may be biased or inaccurate. For means comparison, Tukey’s post hoc analysis was performed. Tukey’s test can be used to identify which groups in a dataset differ significantly from each other, making it a useful tool for hypothesis testing and exploring differences between groups. The data set underwent PCA to see whether it was possible to distinguish the samples based on the location, season, and organ, as well as to learn more about the factors that primarily influence the concentration of heavy metals. The PCA is useful for reducing the dimensionality of a dataset by identifying a smaller number of principal components that explain most of the variance, making it easier to visualize and analyze the data. It can also identify patterns and relationships among variables and provides a graphical representation of the data, which can be used for data exploration and visualization. Experimental results were presented as mean ± standard deviation of triplicate determinations. The difference was considered significant atp<0.05. All statistical analyses were applied to the data without transformation.


## 2.1. Study Site

S. pilchardus samples were taken at the level of three ports in the Mediterranean Sea that extends over a coastline of 512 km (El-Houceima Sea (35.250000, −3.933333), Beni-Ensar Sea (35.2569, −2.93417), and Ras el Ma Sea (35.1407, −2.42455), Figure 1).Figure 1
The geographical site of the three studied zones in the Mediterranean coast of northeast Morocco.


## 2.2. Sampling and Samples Preparation

A total of 360 specimens ofS. pilchardus (n = 360) were collected using artisanal fishing (nets) throughout four seasons from December 2020 until November in three targeted sites according to a factorial schema 4 × 3 × 3, respectively, for the season, area, and organ. The sampling was carried out during each season, 10 specimens per month to obtain 30 samples per season: December, January, and February for winter; March, April, and May for spring; June, July, and August for summer; and September, October, and November for autumn. The sardines were divided into four groups for each area (n = 30 for each area and each organ in each season). In the laboratory, the samples were cleaned with distilled water and were dissected using stainless steel scissors. On each individual, three parts were taken separately, muscle, gills, and liver. The materials were dried to a consistent weight at 103 ± 2°C. Then, they were crushed with an agate mortar, to avoid any external contamination by heavy metals, and to generate a homogenous meat powder that was stored under a vacuum in sachets sterilized at −18°C, until subsequent analyses. During sample preparation and analysis, acid-cleaned laboratory materials were utilized to avoid any possible contamination.


## 2.3. Chemical Analyses

A quantity varying from 1 to 2 grams of dry biological material was used for the analysis of the following metals: Cd, Hg, Pb, and As. The mineralization was realized by wet digestion using a biacid attack (15 ml of HNO3 and 5 ml of H2SO4) combined with the heating on a hot plate (60°C, 120°C, and 150°C). Then, when the samples went dark, the beaker was taken off the hot plate. The content was filtered (0.45 µm) and transferred into a 50 ml volumetric flask and then diluted with double distilled water. Finally, the samples were stored in polypropylene bottles at 4°C until the moment of analysis. The metal concentrations were measured using inductively coupled plasma mass spectrometry (ICP-MS, Agilent, Series 7500). The results are presented as μg·g−1 wet weight (ww).Quality control was performed using certified reference material (CRM) for Sigma-Aldrich Nos. 1.70384.0100 and 1.11355.0100: MERCK 1.70384, Mercury ICP standard 10000 mg/l Hg Certipur®, diluted nitric acid Suprapur® 10%, for Hg; MERCK 1.1355.0100 ICP multi-element standard solution IV 1000 mg/l Certipur®, diluted nitric acid Suprapur® 6.5% for Cd, Pb, and As. The limit of detection (LOD) and lowest limit of quantification (LOQ) were calculated: Cd, Hg, and As—LOD = 5 × 10−3 mg/kg, LOQ = 10−2 mg/kg and for Pb—LOD = 5 × 10−3 mg/kg, LOQ = 5 × 10−2 mg/kg. The uncertainty was estimated between 10 and 15%. The accuracy recovery for the analytical methods used in the examination of Hg, Cd, Pb, and As contents in CRM samples were 92.2%, 99.9%, 100.1%, and 103.0%, respectively.


## 2.4. Health Risk Assessment

In order to analyze the risk to human health associated with Moroccan Mediterranean sardine consumption, the estimated weekly intake (EWI), estimated daily intake (EDI), target hazard quotient (THQ), and lifetime cancer risk (CR) were calculated for each analyzed metal. The EWI was estimated according to the equation (1) considering the studied metals content in sardine muscle and the weekly fish consumption.(1)EWI=Ci×IRwBw,where EWI is reported in μg/g wet weight; Ci is the metallic element content in seafood (μg/g ww); the weekly ingestion rate is known as IRw (g/week), 169 g/capita/week [16, 17]; and the Bw is the body weight, in Morocco, the mean adult body weight is 73.35 kg [18].The THQ was estimated according to equation (2), it is the ratio between the exposure and the reference dose (RfD) [19]. This index is calculated to evaluate the possibility of adverse health effects other than cancer [20]. Nonobvious risk is indicated by a ratio of less than 1. It is significant to highlight that if the dose is equivalent to or higher than the RfD, an exposed population will face health hazards [19].(2)THQ=EFxEDxFIRxCiRfDxBWxAT×10−3,where EF = the exposure frequency (365 days/year); ED = the average lifetime duration; 76.9 years for the Moroccan population [21]; FIR = the fish ingestion rate (38 g/person/day) [16, 17]; Ci = the metal concentration in the fish sample; RfD = the reference oral dose in μg·g−1·day−1 (Hg: 1 ∗ 10−4, As: 3 × 10−4, Cd: 1 × 10−3, and Pb: 4 × 10−3) [19]; BW = the average body weight of the adult consumer; 73.35 kg for Moroccan population [18]; AT = the mean time of exposure to noncarcinogens (365 days/year∗ED) [22].The daily dose was multiplied by the cancer slope factor (CSF), which was obtained from the response-dose curve for toxicant consumption, to determine the lifetime cancer risk (CR), following the formula [23]:(3)CR=EFxEDxFIRxCxCSFBWxAT,where CSF stands for the Integrated Risk Information System’s oral carcinogenic slope factor (μg/g/day). Only As (1.5 μg/g/day), Cd (6.3 μg/g/day), and Pb (8.5 × 10−3 μg/g/day) have CSF values available [24].Since consumers are frequently exposed to multiple contaminants with associated combinations or interacting effects, the total target hazard quotient (TTHQ), which is the sum of the THQ values, was also calculated in order to evaluate the danger of the four metallic elements collectively [25], the formula is as follows:(4)TTHQ=THQMetal1+THQMetal2+THQMetal3+THQMetal4.Assuming that the hazardous form of arsenic (inorganic arsenic) made up of 3% of the total concentration, the EWI, THQ, and CR for arsenic were determined [26, 27]. A TTHQ value greater than 1 is viewed as a sign of significant danger, or a serious health concern [28].


## 2.5. Statistical Analysis

Using the statistical analysis program SPSS version 20 and taking into account the location, season, organ, and their interaction as the source of variation, Shapiro–Wilk test for quantitative variables was used to examine the normal distribution. The Shapiro–Wilk test is a useful tool for testing the normality of data, which is an important assumption for many statistical tests such as the analysis of variance (ANOVA) and principal component analysis (PCA). If the data are not normally distributed, these assumptions may not hold, and the statistical analysis results may be biased or inaccurate. For means comparison, Tukey’s post hoc analysis was performed. Tukey’s test can be used to identify which groups in a dataset differ significantly from each other, making it a useful tool for hypothesis testing and exploring differences between groups. The data set underwent PCA to see whether it was possible to distinguish the samples based on the location, season, and organ, as well as to learn more about the factors that primarily influence the concentration of heavy metals. The PCA is useful for reducing the dimensionality of a dataset by identifying a smaller number of principal components that explain most of the variance, making it easier to visualize and analyze the data. It can also identify patterns and relationships among variables and provides a graphical representation of the data, which can be used for data exploration and visualization. Experimental results were presented as mean ± standard deviation of triplicate determinations. The difference was considered significant atp<0.05. All statistical analyses were applied to the data without transformation.


## 3. Results and Discussion

Tables1–3 show the average levels of the four heavy metals (Cd, Hg, Pb, and As) found in the muscle, liver, and gill of S. pilchardus from the Moroccan Mediterranean coast. The results are expressed in μg·g−1 wet weight (ww). As and Pb were detected in all the analyzed samples. In contrast, the Cd and Hg values were less than 0.001 μg·g−1 ww in the same samples. Therefore, the statistical analysis of As and Pb content has been run using analysis of variance, while Cd and Mg were analyzed using the test “t.” The results show a significant effect of the study area, organ, and season (p<0.05, Table 4). These results are in agreement with the literature in this sense [27, 29–34]. Regardless of the sampling site and season, the metallic trace elements concentration is high in the liver than those recorded in the muscle and gill (Tables 1–3). The range in µg·g−1 ww obtained for the studied metals in analyzed samples are as follows: As (4.34–10.57 μg·g−1 ww), Pb (0.002–0.046 μg·g−1 ww), Cd (<0.001–0.401 μg·g−1 ww), and Mg (<0.001–0.026 μg g−1 ww). Mean concentrations of As show a significant difference between the studied sites, seasons, and organs (p<0.05). The levels of As contamination recorded show a wide range of distribution, with higher average values recorded in the liver of specimens from Ras el Ma site in the different seasons of the year (6.437 μg·g−1–14.80 μg·g−1 ww). As is a natural component of the earth’s crust and is widely present in the environment, whether in the air, water, or soil. Seafood is the major source of dietary exposure to arsenic. It should be taken into account that arsenic is generally accumulated by living organisms in the organic form (arsenobetaine). Its inorganic form (iAs) is more hazardous to humans and is classified as carcinogenic. In the edible tissue of fish, most countries’ legislation does not include legal limits for As contamination. No maximum levels are currently set by the European Commission for As. The highest permissible content of iAs in fish flesh is specified only by some countries such as the United States (0.26 mg/kg), Switzerland (0.1 mg/kg), Australia-New Zealand (2 mg/kg), and Canada (5 mg/kg). Knowing that the toxic form of As (iAs) represents only 3% of the total As concentration [22, 35, 36], our results do not exceed the acceptable value for Australia-New Zealand and Canada legislation, while they exceed that of Switzerland. Moreover, the recorded values in edible tissues for specimens of winter and autumn exceed the legal limit of the United States, however, those registered in spring and summer do not. The obtained results are lower than those reported by Traina et al. [27] in the edible tissue of sardine from the Mediterranean Sea in Italy, lower than those found by Bonsignore et al. [31] in sardine from northern Italy, and higher than those reported by Olmedo et al. [33] and Falcó et al. [34] in the Mediterranean Sea of Spain (Table 5). This difference could be explained by the natural or anthropogenic contamination. Furthermore, the explanation of the recorded difference between studies is complicated because their geographical origins are undetermined, and the fish diet may exhibit variations in predation patterns and specificity. We believe that differences in the trace elements’ content are due to their accumulation in marine organisms and is related not only to the presence of the contaminants but also to several biological environmental factors.Table 1
Mean ± standard deviation of cadmium, mercury, lead, and arsenic concentrations in the muscle ofSardina pilchardus from the Mediterranean coast of Morocco.
Metals (µg g−1)SiteWinterSpringSummerAutumnCadmiumBeni-Ensar0.292a ± 0.02<0.001 ± 0.000.194 ±a0.030.20a ± 0.03Ras el Ma0.008 ± 0.0010.005 ± 0.00<0.001 ± 0.00<0.001 ± 0.00El-Houceima0.012a ± 0.001<0.001 ± 0.000.401b ± 0.030.005a ± 0.001MercuryBeni-Ensar<0.001 ± 0.000.016a ± 0.000.016a ± 0.0010.026b ± 0.00Ras el Ma<0.001 ± 0.000.013 ± 0.001<0.001 ± 0.00<0.001 ± 0.00El-Houceima0.207d ± 0.0010.012a ± 0.0010.093c ± 0.0010.060b ± 0.001LeadBeni-Ensar0.010 ± 0.0010.013 ± 0.0020.006 ± 0.0010.004 ± 0.001Ras el Ma0.009 ± 0.0010.006 ± 0.0010.002 ± 0.000.039 ± 0.001El-Houceima0.004 ± 0.0010.017 ± 0.0010.005 ± 0.0010.046 ± 0.001ArsenicBeni-Ensar7.41ab ± 1.185.06a ± 1.555.80a ± 1.869.53b ± 1.25Ras el Ma10.57b ± 0.554.79a ± 1.444.50a ± 1.2410.00b ± 0.25El-Houceima10.43b ± 0.314.34a ± 1.296.29ab ± 1.079.83b ± 0.37The results are expressed inμg·g−1 wet weight; significant differences (p<0.05) between means are shown with different lowercase letters (a-b).Table 2
Mean ± standard deviation of cadmium, mercury, lead, and arsenic concentrations in the liver ofSardina pilchardus from the Mediterranean coast of Morocco.
Metals (µg g−1)SiteWinterSpringSummerAutumnCadmiumBeni-Ensar0.609c ± 0.0030.224a ± 0.0010.805d ± 0.0040.321b ± 0.001Ras el Ma0.868d ± 0.0040.354a ± 0.0020.504c ± 0.0020.463b ± 0.002El-Houceima1.347d ± 0.0070.107a ± 0.0010.311b ± 0.0010.653c ± 0.003MercuryBeni-Ensar0.038c ± 0.000<0.001 ± 0.0000.012b ± 0.000<0.001 ± 0.000Ras el Ma0.036b ± 0.000<0.001 ± 0.0000.166d ± 0.0010.058c ± 0.001El-Houceima0.080c ± 0.001<0.001 ± 0.0000.010b ± 0.000<0.001 ± 0.000LeadBeni-Ensar<0.001 ± 0.0000.081a ± 0.0000.107b ± 0.0000.186c ± 0.000Ras el Ma0.076a ± 0.0010.153b ± 0.0010.173 ± 0.0010.114b ± 0.000El-Houceima0.104 ± 0.0010.087 ± 0.0010.081 ± 0.0010.156 ± 0.001ArsenicBeni-Ensar5.510a ± 0.0275.898b ± 0.29511.53d ± 0.0588.259c ± 0.041Ras el Ma6.437a ± 0.03214.613c ± 0.07314.80d ± 0.07412.23b ± 0.000El-Houceima13.36d ± 0.0672.38a ± 0.0126.15b ± 0.036.30c ± 0.03The results are expressed inμg·g−1 wet weight; significant differences (p<0.05) between means are shown with different lowercase letters (a–d).Table 3
Mean ± standard deviation of cadmium, mercury, lead, and arsenic concentrations in the gill ofSardina pilchardus from the Mediterranean coast of Morocco.
Metals (µg g−1)SiteWinterSpringSummerAutumnCadmiumBeni-Ensar<0.001 ± 0.000<0.001 ± 0.0000.313b ± 0.004<0.001 ± 0.000Ras el Ma0.263b ± 0.010<0.001 ± 0.0000.312c ± 0.015<0.001 ± 0.000El-Houceima0.274b ± 0.0100.310c ± 0.0150.226a ± 0.0150.309c ± 0.015MercuryBeni-Ensar0.013b ± 0.001<0.001 ± 0.000<0.001 ± 0.000<0.001 ± 0.000Ras el Ma0.016c ± 0.000<0.001 ± 0.000<0.001 ± 0.0000.011b ± 0.015El-Houceima0.013b ± 0.001<0.001 ± 0.000<0.001 ± 0.000<0.001 ± 0.000LeadBeni-Ensar<0.001 ± 0.000<0.001 ± 0.000<0.001 ± 0.000<0.001 ± 0.000Ras el Ma<0.001 ± 0.000<0.001 ± 0.000<0.001 ± 0.000<0.001 ± 0.000El-Houceima<0.001 ± 0.000<0.001 ± 0.000<0.001 ± 0.000<0.001 ± 0.000ArsenicBeni-Ensar1.086b ± 0.0100.807a ± 0.0111.62d ± 0.0151.336c ± 0.010Ras el Ma2.52d ± 0.0101.037a ± 0.0011.62c ± 0.0011.34b ± 0.010El-Houceima1.408c ± 0.0080.959a ± 0.0091.129b ± 0.0091.887d ± 0.009The results are expressed inμg·g−1 wet weight; significant differences (p<0.05) between means are shown with different lowercase letters (a–d).Table 4
Analysis of variance for the concentration of heavy metals in different organs ofSardina pilchardus (Walbaum, 1792) from the Mediterranean coast of Morocco.
dfMean squaresF-valuep valueCadmium (µg/gww)Area20.1006.223<0.01Organ22.134133.210<0.001Season30.46829.219<0.001Area∗ organ40.0763.7370.002Area∗ season60.0643.972<0.01Organ∗ season60.22113.803<0.001Area∗ organ ∗ season120.1348.341<0.001Error720.016Mercury (µg/gww)Area20.00820212.19<0.001Organ20.01129315.735<0.001Season30.00821327.245<0.001Area∗ organ40.01538860.560<0.001Area∗ season60.00615549.203<0.001Organ∗ season60.0025673.968<0.001Area∗ organ ∗ season120.00411308.215<0.001Error723.843E−007Lead (µg/gww)Area20.0015.035<0.01Organ20.131602.938<0.001Season30.00523.945<0.001Error40.0027.437<0.001Area∗ organ60.00210.435<0.001Area∗ season60.00418.324<0.001Organ∗ season120.00211.201<0.001Area∗ organ ∗ season720.001Arsenic (µg/gww)Area234.33744.914<0.001Organ2572.999749.499<0.001Season329.38538.436<0.001Area∗ organ427.11735.469<0.001Area∗ season620.86027.286<0.001Organ∗ season624.98532.680<0.001Area∗ organ ∗ season1220.44626.744<0.001Error720.765ww: wet weight; df: degrees of freedom.Table 5
Comparison of the obtained results with the literature data of cadmium (Cd), mercury (Hg), lead (Pb), and arsenic (As) in the muscle ofSardina pilchardus (Walbaum, 1792).
LocationMetals (μg·g−1 wet weight)ReferenceCdHgPbAsBeni-Ensar, Morocco0.1720.0150.0086.95Current studyRas el Ma, Morocco0.0040.0040.0057.47Current studyEl-Houceima, Morocco0.1040.0930.0187.73Current studyAlgiers, Algeria0.130.10.23—[30]Dellys, Algeria0.0150.130.03—[30]Kafr El-Sheikh, Egypt—0.720.33—[38]Porticello, Italy<0.0010.12<0.00111.39[27]S. Agata, Italy0.030.100.0910.36[27]Tuscany, Italy0.030.02–0.710.0510.6–39.2[31]Mersin, Turkey<0.0004—0.91—[32]Boumerdès, Algeria0.00—0.99—[39]Agadir, Morocco0.0060.0290.023—[29]Essaouira, Morocco0.0050.0330.020—[29]Laayoune, Morocco0.008ND0.021—[29]Mediterranean Sea, Spain0.000.030.000.56[33]Mediterranean Sea, Spain0.002–0.010.07–0.090.01–0.083.53–3.94[34]ND: not detected.Concerning the Hg, the recorded values were significantly higher in specimens from the El-Houceima region regardless the organ and season (<0.001–0.207μg·g−1). Individuals from El-Houceima had the highest average Hg contents (0.207 μg·g−1 in the liver); however, the lowest levels were obtained in the muscle of sardine from the Ras el Ma region (<0.001 μg·g−1). No recorded values of Hg concentration exceeded the recommended values set by European Commission regulation (EC) N629/2008 (0.5 mg/kg for fish muscle) [37]. In the case of Beni-Ensar and Ras el Ma Sea, our results of sardine Hg contamination (Table 5) are lower than others found in other Mediterranean coasts [27, 30, 31, 38]. In contrast, results for Hg contamination recorded in individuals from El-Houceima Sea (Table 5) are higher than those reported in fish from Algeria and the Italian Mediterranean Sea [27, 30], and lower than those found by Shokr et al. [38] in the Mediterranean Sea of Egypt (Table 5).Concerning Pd and Cd, their content in the different specimens’ organs show a significantly higher difference (p<0.05). Cd average content ranges between 0.224 and 1.347 μg·g−1, <0.001–0.401 μg·g−1, and <0.001–0.313 μg·g−1, respectively, in the liver, muscle, and gill of studied samples. Regardless of the organ or season, the specimens from El-Houceima port had the highest level of Cd (<0.001–1.347 μg·g−1ww) followed by the decreasing order by Ras el Ma port (<0.001–0.868 μg·g−1) and Beni-Ensar port (<0.001–0.805 μg·g−1). However, the recorded values in the edible tissue of specimens from Beni-Ensar and El-Houceima Sea exceeded the regulatory threshold value for Cd in the fish muscle set by the EC N 629/2008 (0.10 mg/kg), while those recorded in individuals from Ras el Ma Sea do not exceed the regulatory limit. Cadmium (when measurable) values registered in sardine are higher than those reported by numerous studies in specimens from the Mediterranean Sea [27, 33, 34]; however, they are close to those recorded by Aissioui and Poirier [30] in the Algiers Sea, except for our result obtained in Ras el Ma Sea samples (Table 5). For the latter, their results are comparable to those of [29], lower [27, 31] or higher [32, 39] than values previously reported in sardine muscles. The high contamination level of specimens from the El-Houceima Sea could be explained by the strong anthropogenic pressure including a large chain of industrial activities because of the high urbanization in comparison with the other studied sites.The Pb mean concentration recorded in the studied fish was 0.04μg·g−1 ww, with a maximum level of 0.173 μg·g−1 ww in the liver of individuals from Ras el Ma port. In opposition, the lowest Pb levels in the muscle were found in sardine sampled in the Ras el Ma region. This suggests that recent ingestion of Pb, via water and/or food, has occurred. In addition, the concentration measured in the muscle is far from the threshold value of 0.3 mg/kg for edible fish tissues, set by the EC N 629/2008 [37]. This recorded contamination could be explained by the presence of a port, moreover the proximity of an airport, in this region. The repeated aircraft takeoff whose gasoline contains lead could generate air contamination in this area. This heavy metal (lead) is a very reactive element in the environment, the atmosphere is its main vector toward the oceans, and consequently ends up on the coast via rivers and the atmosphere. Furthermore, antifouling, considered as the coating or the paint of the ship that prevents the attachment of unwanted organisms, contains very harmful chemical substances and is therefore likely to cause acute and chronic pollution of the environment because of the metals that it may contain metals such as copper and lead. The mean value recorded in this study for samples from Beni-Ensar and Ras el Ma is lower than several values found in the edible tissue of sardine in other areas [27, 29–32]. For the El-Houceima Sea, the results are comparable to those reported by Falcó et al. [34] in the Mediterranean Sea of Spain and by Chahid et al. [29] in specimens from the Laayoune Sea, Atlantic Ocean of Morocco.


### 3.1. Organ Variation

In terms of metabolism and accumulation and from a toxicological point of view, the muscles and the liver are the primordial organs to evaluate the contamination dangers. Overall, the levels of the four toxic metals were greater in the liver than in the muscle and gills. The highest values for the studied metals recorded in the liver were As (8.95μg·g−1), Cd (0.547 μg·g−1), Pb (0.110 μg·g−1), and Mg (0.037 μg·g−1), Tables 1–3. Similar results were found by numerous authors [29, 30]. These results could be related to the role of this functional organ as a site of accumulation, detoxification, and distribution [40, 41]. Furthermore, this finding could be explained by the fact that the gills constitute provisional target organs for toxic substances accumulation before being transferred afterward to the digestive organs such as the liver, kidneys, and intestine then the metals reach the bloodstream where they will bind to the hemoglobin then be transported to target tissues. The liver is known for its functions of storing and regulating elements. The gills’ contamination reflects the composition of the ambient environment, in particular, if the pollution is recent and/or occasional. [42].


### 3.2. Seasonal Variation

The obtained values for Cd and Hg show a significant difference (p<0.05) according to the sampling period (season, Tables 1–4). Comparable findings in the Mediterranean Sea were recorded [27, 30].Winter was the season with the greatest Cd value (0.408μg·g−1 ww) followed by descending order in summer (0.340 μg·g−1 ww), autumn (0.217 μg·g−1 ww), and spring (0.111 μg·g−1 ww). Similarly, for Hg, specimens sampled in winter (0.044 μg·g−1 ww) show the highest contamination level followed by summer (0.033 μg·g−1 ww), autumn (0.017 μg·g−1 ww), and spring (0.001 μg·g−1 ww). Concerning the As content, the individuals of winter (6.74 μg·g−1 ww) and autumn (6.52 μg·g−1 ww) show a higher contamination level than those of spring (4.43 μg·g−1 ww) and summer (5.93 μg·g−1 ww). However, a nonsignificant difference was recorded between studied samples according to the season in terms of Pb (p>0.05). This difference could be linked to the variation in the water physicochemical conditions, according to the sampling period, which has a significant impact on solubility, bioavailability, and distribution of metals affecting the physiological state of the aquatic system’s biota on the one hand [43–46]. On the other hand, it could be explained by the intensity of the diet which is important in winter and autumn favoring the bioaccumulation of toxic elements [47]. Garrido et al. [48] have reported that in the winter season, S. pilchardus has an intense diet feeding exclusively on zooplankton. In general, the Mediterranean region experiences rainy winters and dry, hot summers; these variations are noticed between sampling periods (seasons) in metal content could be related to the physicochemical characteristics’ difference depending on the season, as well as anthropic activities along the area of the study [49]. The higher value recorded in sardine sampled during the winter period could be attributed, mainly, to the anthropogenic activities (such as industrial, agricultural, and domestic effluents) from the studied area, which is probably connected with the drainage of soil during the wet season. Contaminants can enter the seawater from feeder rivers, and therefore, they can be absorbed and accumulated by fish [50]. Besides the ecotoxicological risk assessment methods that were applied in this study, there are novel and promising approaches to assess to investigate spatiotemporal and biological characteristics of fish, such as interacting effects and metal stable isotope techniques (e.g., copper, lithium, and zinc) [51–53]. These approaches allow us to monitor and quantify the bioaccumulation of anthropogenic metals (the past, current, and future status), and consequently, provide guidance for pollution recovery and monitoring programs and support the sustainable development of fisheries and human well-being in these areas [51–53].


### 3.3. Health Risk Assessment

Generally, the Moroccan consumer of fish does not consume the liver and the lungs, the consumption is limited only to the muscles. As a result, the dietary health risk assessment for consumers has been studied only for the muscles. Information on the content and toxicity of each pollutant in relation to the exposure assessment is necessary to ascertain the possible danger to human health. According to the European Commission, arsenic, cadmium, mercury, and lead have been classified as a priority list of hazardous substances. We have calculated the estimated weekly intake (EWI), target hazard quotient (THQ), and cancer risk (CR) indices of Hg, Cd, Pb, and As for Moroccan consumers by taking into consideration an exposure time of 7 per week, an average adult weight of 73.3 kg, and a life expectancy of 76.9 years of Moroccan people. The mean daily fish intake in Morocco is 38 g/person, thus equal to 269 g/person/week. The calculated values were compared to the recommended threshold of provisional tolerable weekly intake (PTWI) preconized by the European Food Safety Authority for Hg, Cd, Pb, and As are 4, 7, 25, and 15µg/kg body weight, respectively [26, 54–56]. Regarding As, the toxicity is linked only to the inorganic form, since the organic As is not very harmful to humans. Consequently, in the calculation of the aforementioned indices (EWI, THQ, and CR), we have taken into consideration only 3% of the total concentration of arsenic which is considered as the toxic form (the inorganic form). The calculated THQ values for studied metals in the different study locations were lower than the permissible limit (1.0) indicating that the sardine concentration levels in the Mediterranean Sea of Morocco do not present an adverse effect on human health. As seen in Table 6, the EWI calculated values did not exceed the recommended value. The maximum EWI of Cd was 0.086 μg/kg bw/week or 0.012 μg/day representing 1.22% of the PTWI (Table 6). These values are comparable to those reported by Aissioui et al. [30] in sardine muscle from the Algiers Sea, lower than those found by Chahid et al. [29] and higher than those registered by Traina et al. [27] in sardine from the Mediterranean Sea of Italy. Concerning the Hg, we have compared our result to the PTWI defined for methylmercury (MeHg: 1.6 μg/kg/body weight) which is classified as possibly carcinogenic to humans [57, 58]. Several studies have reported that fish meat contains especially MeHg form (toxic form). The calculated EWIHg intake ranges between 0.044 and 0.759 μg/kg bw/week representing 2.75–47.43% of the PTWI (Table 6). Except for the EWIHg recorded in the El-Houceima Sea, our results of As are lower than those reported by numerous researchers in the Mediterranean Sea [27, 30, 59]. In opposition, the EWI values of Hg recorded in samples from the El-Houceima Sea are comparable to those reported by Özden and Erkan [59] in Turkey, lower than those reported by Chahid et al. [29] and higher than those reported by Aissioui et al. [30], and by Traina et al. [27] in Algerian and Italian Sea, respectively. The large intakes of Cd (EWI = 0.086) and Hg (EWI = 0.759) have been recorded in El-Houceima Sea. With regard to Pb, the EWI ranged from 0.005 to 0.06 μg/kg bw/week, which presents a relative percentage of 0.02–0.24% of the PWTI (Table 4). These results are lower than those recorded for the sardine muscle in various Mediterranean Seas. [27, 30, 59], indicating that the Moroccan Mediterranean Sea seems to be less contaminated by lead. For As, the maximum EWI was 1.16 μg/kg bw/week or 0.16 μg/kg bw/day representing 7.73% of the legal limit (15 μg/kg bw/week). The largest contribution for As (EWI = 1.16) was recorded in the Ras el Ma Sea. The EWIAs values for sardine sampled in the spring and summer seasons are comparable to those reported by Traina et al. [27] in sardine from the Italian Mediterranean Sea. However, those calculated in the winter and autumn seasons are higher than those reported in other Mediterranean Seas. This result indicates that the three seas are more contaminated by arsenic, especially in the autumn-winter season. We have also calculated the total THQ (TTHQ) to assess the risk of the contamination level including the four heavy metals (Cd, Hg, Pb, and As; Table 6). For Beni-Ensar and Ras el Ma, the TTHQ calculated values were below the recommended value (<1). As a result, no health risks can be associated with sardine consumption in these sites in the case of Beni-Ensar and Ras el Ma. Inversely, the total THQ of specimens from El-Houceima Sea exceed the preconized threshold (1) indicating a potential risk to human health with a consummation frequency of 7 days/week. The large intakes of Cd (EWI = 0.086) and Hg (EWI = 0.759) have been recorded in the El-Houceima Sea (Table 6). Based on the calculated TTHQ values, the El-Houceima Sea seems to be more contaminated followed by the decreasing order by Ras el Ma and Beni-Ensar Sea (Table 6).Table 6
Health risk assessment of the consumers for each analyzed metal in edible tissues ofSardina pilchardus (Walbaum, 1792) from the Mediterranean coast of Morocco.
WinterSpringSummerAutumnEWI (EDI)THQCREWI (EDI)THQCREWI (EDI)THQCREWI (EDI)THQCRCdBeni-Ensar0.065 ± 0.00 (0.009)0.009 ± 0.001.7E − 3 ± 0.01———0.060 ± 0.01 (0.008)0.009 ± 0.001.6E − 3 ± 0.010.046 ± 0.01 (0.006)0.007 ± 0.001.2E − 3 ± 0.01Ras el Ma0.029 ± 0.01 (0.004)0.004 ± 0.007.8E − 4 ± 0.010.018 ± 0.01 (0.002)0.003 ± 0.004.8E − 4 ± 0.00——————El-Houceima0.042 ± 0.01 (0.006)0.006 ± 0.001.1E − 3 ± 0.01———0.086 ± 0.01 (0.012)0.012 ± 0.002.3E − 3 ± 0.010.018 ± 0.01 (0.002)0.003 ± 0.004.8E − 4 ± 0.01HgBeni-Ensar———0.057 ± 0.00 (0.008)0.050 ± 0.00—0.058 ± 0.00 (0.008)0.051 ± 0.00—0.097 ± 0.002 (0.013)0.086 ± 0.00—Ras el Ma———0.047 ± 0.00 (0.006)0.042 ± 0.00———————El-Houceima0.759 ± 0.00 (0.107)0.670 ± 0.00—0.044 ± 0.00 (0.006)0.038 ± 0.00—0.341 ± 0.00 (0.048)0.301 ± 0.00—0.220 ± 0.00 (0.031)0.194 ± 0.003—PbBeni-Ensar0.035 ± 0.00 (0.005)0.0012 ± 0.001.2E − 6 ± 0.000.046 ± 0.01 (0.006)0.0016 ± 0.001.6E − 6 ± 0.000.020 ± 0.00 (0.002)0.0007 ± 0.007.3E − 7 ± 0.000.016 ± 0.00 (0.002)0.0005 ± 0.005.8E − 7 ± 0.00Ras el Ma0.028 ± 0.01 (0.004)0.001 ± 0.001.0E − 6 ± 0.000.023 ± 0.01 (0.003)0.0008 ± 0.008.3E − 7 ± 0.000.005 ± 0.00 (0.0007)0.0001 ± 0.002.0E − 7 ± 0.000.014 ± 0.00 (0.002)0.0005 ± 0.005.0E − 7 ± 0.00El-Houceima0.013 ± 0.00 (0.002)0.0004 ± 0.004.8E − 7 ± 0.000.06 ± 0.01 (0.008)0.0022 ± 0.002.2E − 6 ± 0.000.019 ± 0.00 (0.002)0.0007 ± 0.007.2E − 7 ± 0.000.16 ± 0.01 (0.023)0.0059 ± 0.006.0E − 6 ± 0.00AsBeni-Ensar0.815 ± 0.10 (0.11)0.38 ± 0.060.17 ± 0.020.557 ± 0.18 (0.07)0.26 ± 0.130.11 ± 0.050.638 ± 0.20 (0.09)0.30 ± 0.090.13 ± 0.041.04 ± 0.13 (0.14)0.49 ± 0.060.22 ± 0.02Ras el Ma1.16 ± 0.06 (0.16)0.54 ± 0.020.24 ± 0.010.52 ± 0.20 (0.07)0.24 ± 0.070.11 ± 0.890.49 ± 0.10 (0.07)0.23 ± 0.060.10 ± 0.961.10 ± 0.02 (0.15)0.51 ± 0.010.23 ± 0.00El-Houceima1.14 ± 0.03 (0.14)0.54 ± 0.010.24 ± 0.000.47 ± 0.15 (0.07)0.22 ± 0.100.10 ± 0.050.69 ± 0.10 (0.08)0.32 ± 0.050.14 ± 0.021.08 ± 0.04 (0.15)0.50 ± 0.010.22 ± 0.00TTHQBeni-Ensar0.3980.3150.3620.587Ras el Ma0.5560.2940.2370.522El-Houceima1.2180.2670.6400.712The results are presented as mean ± standard deviation; Cd: cadmium; Hg: mercury; Pb: lead; As: arsenic; EWI (μg kg−1 body weight): estimated weekly intake; THQ: target hazard quotient; CR: carcinogenic risk; TTHQ: total target hazard quotient; THQ values >1 are indicated in bold. The values indicated in bold exceed the established threshold.The probability of a person developing cancer over their lifetime was calculated using the cancer risk (CR) as a result of exposure to contaminants that could cause cancer. If the CR value is exceeding 10−5, it indicates a high probability (1/100 000) of having cancer in the consumer. The CR was not estimated for Hg since the cancer slope factor (CSF) for Hg is not determined by the United States Environmental Protection Agency. The values of the carcinogenic risk index for As and Cd exceed the established threshold (10−5) in all cases (site and season). In contrast, the calculated CRCd was below the legal limit in all sites and seasons (Table 6). Comparable outcomes have been reported by Traina et al. [27] in sardine muscle from the Mediterranean Sea of Italy. In the current study, the CR was calculated considering all the week as a frequency of ingestion, 7 days/week (maximalist theoretical approach). So, if we consider that the frequency of ingestion is 2 or 3 days/week, the values of these risk indices for contamination by heavy metals on public health will decrease significantly. This is the case for Morocco, since the consumption of fish is quite moderate because of the unaffordable price. Overall, 100% of the values for CR of As and Cd (when measurable) were above the recommended limits. Based on the calculated EWI and THQ, it may be concluded that the consumption of Moroccan sardine from the Mediterranean Sea does not represent a health risk for consumers. Inversely, As and Cd represent a potential risk on human health in terms of carcinogenic risk.


### 3.4. Principal Component Analysis

A principal component analysis (PCA) was carried out to identify the relationships among the studied groups of specimens and evaluated parameters. This multifactorial analysis was conducted using 12 variables, including heavy metals and health risk indices. This analysis allowed us to classify and process the relative information of the metals studied by establishing the possible correlations between all the evaluated variables. The first two principal components (PCs) represent 78.07% of the total information. The PC1 and PC2 accounted, respectively, for 55.60% and 22.90% of the total variance in the data set. Figure2 represents the projection of metals in the plane defined by the first two principal components. PC1 was correlated positively with As, Pb, and Cd (Table 7 and Figure 2). In opposition, the PC2 was defined mainly by Hg. Liver samples were clearly differentiated from the gill and muscle and are located on the right side of the plane (Figure 2), where all metals lie. According to PC2, the effect of the season is mainly manifested. The projection of results according to the season on the factorial map (Figure 2) shows discrimination between the studied sardine and allows summarizing the interpretations already mentioned above in a very simplified way. The samples of winter sampled from the El-Houceima Sea were clearly differentiated from other season samples indicating the high sardine contamination level especially by mercury. The correlation matrix analysis reveals the presence of a correlation between As and Pb (r = 0.61) and a correlation between Hg and As (r = 0.50).Figure 2
Graphical representation of principal component analysis of heavy metals based on the first two principal components (PCs) (factorial map of the seasons (a), sites (b), organs (c), and heavy metals (d)).
(a)(b)(c)(d)Table 7
First two principal components explaining 78.07% of the total variance of the sardine,Sardina pilchardus (Walbaum, 1792), contamination level from three coast sites in the Mediterranean Moroccan Sea.
VariablesPrincipal component12As0.8530.337Pb0.848−0.269Cd0.685−0.410Hg0.5040.729EWIAs0.8530.337EWIPb0.848−0.269EWICd0.719−0.490EWIHg0.5040.729THQAs0.8530.337THQPb0.848−0.269THQCd0.719−0.490THQHg0.5040.729CRAs0.85300.337CRPb0.848−0.269CRCd0.719−0.490As: arsenic; Pb: lead; Cd: cadmium; Hg: mercury; EWI: estimated weekly intake; THQ: target hazard quotient; CR: carcinogenic risk. The values represent the correlations of variables with the first two principal components.The PCA analysis exposed a significant effect of the location and season on the contamination level of the studied specimens (p<0.05). This variation can be probably the result of the difference in feeding source, growth stage, and environmental conditions. Similar results were reported by Bonsignore et al. [31] for fish, crustaceans, mollusks, and echinoderms and by Leonard et al. [60] for Oreochromis niloticus and Clarias gariepinus species.


## 3.1. Organ Variation

In terms of metabolism and accumulation and from a toxicological point of view, the muscles and the liver are the primordial organs to evaluate the contamination dangers. Overall, the levels of the four toxic metals were greater in the liver than in the muscle and gills. The highest values for the studied metals recorded in the liver were As (8.95μg·g−1), Cd (0.547 μg·g−1), Pb (0.110 μg·g−1), and Mg (0.037 μg·g−1), Tables 1–3. Similar results were found by numerous authors [29, 30]. These results could be related to the role of this functional organ as a site of accumulation, detoxification, and distribution [40, 41]. Furthermore, this finding could be explained by the fact that the gills constitute provisional target organs for toxic substances accumulation before being transferred afterward to the digestive organs such as the liver, kidneys, and intestine then the metals reach the bloodstream where they will bind to the hemoglobin then be transported to target tissues. The liver is known for its functions of storing and regulating elements. The gills’ contamination reflects the composition of the ambient environment, in particular, if the pollution is recent and/or occasional. [42].


## 3.2. Seasonal Variation

The obtained values for Cd and Hg show a significant difference (p<0.05) according to the sampling period (season, Tables 1–4). Comparable findings in the Mediterranean Sea were recorded [27, 30].Winter was the season with the greatest Cd value (0.408μg·g−1 ww) followed by descending order in summer (0.340 μg·g−1 ww), autumn (0.217 μg·g−1 ww), and spring (0.111 μg·g−1 ww). Similarly, for Hg, specimens sampled in winter (0.044 μg·g−1 ww) show the highest contamination level followed by summer (0.033 μg·g−1 ww), autumn (0.017 μg·g−1 ww), and spring (0.001 μg·g−1 ww). Concerning the As content, the individuals of winter (6.74 μg·g−1 ww) and autumn (6.52 μg·g−1 ww) show a higher contamination level than those of spring (4.43 μg·g−1 ww) and summer (5.93 μg·g−1 ww). However, a nonsignificant difference was recorded between studied samples according to the season in terms of Pb (p>0.05). This difference could be linked to the variation in the water physicochemical conditions, according to the sampling period, which has a significant impact on solubility, bioavailability, and distribution of metals affecting the physiological state of the aquatic system’s biota on the one hand [43–46]. On the other hand, it could be explained by the intensity of the diet which is important in winter and autumn favoring the bioaccumulation of toxic elements [47]. Garrido et al. [48] have reported that in the winter season, S. pilchardus has an intense diet feeding exclusively on zooplankton. In general, the Mediterranean region experiences rainy winters and dry, hot summers; these variations are noticed between sampling periods (seasons) in metal content could be related to the physicochemical characteristics’ difference depending on the season, as well as anthropic activities along the area of the study [49]. The higher value recorded in sardine sampled during the winter period could be attributed, mainly, to the anthropogenic activities (such as industrial, agricultural, and domestic effluents) from the studied area, which is probably connected with the drainage of soil during the wet season. Contaminants can enter the seawater from feeder rivers, and therefore, they can be absorbed and accumulated by fish [50]. Besides the ecotoxicological risk assessment methods that were applied in this study, there are novel and promising approaches to assess to investigate spatiotemporal and biological characteristics of fish, such as interacting effects and metal stable isotope techniques (e.g., copper, lithium, and zinc) [51–53]. These approaches allow us to monitor and quantify the bioaccumulation of anthropogenic metals (the past, current, and future status), and consequently, provide guidance for pollution recovery and monitoring programs and support the sustainable development of fisheries and human well-being in these areas [51–53].


## 3.3. Health Risk Assessment

Generally, the Moroccan consumer of fish does not consume the liver and the lungs, the consumption is limited only to the muscles. As a result, the dietary health risk assessment for consumers has been studied only for the muscles. Information on the content and toxicity of each pollutant in relation to the exposure assessment is necessary to ascertain the possible danger to human health. According to the European Commission, arsenic, cadmium, mercury, and lead have been classified as a priority list of hazardous substances. We have calculated the estimated weekly intake (EWI), target hazard quotient (THQ), and cancer risk (CR) indices of Hg, Cd, Pb, and As for Moroccan consumers by taking into consideration an exposure time of 7 per week, an average adult weight of 73.3 kg, and a life expectancy of 76.9 years of Moroccan people. The mean daily fish intake in Morocco is 38 g/person, thus equal to 269 g/person/week. The calculated values were compared to the recommended threshold of provisional tolerable weekly intake (PTWI) preconized by the European Food Safety Authority for Hg, Cd, Pb, and As are 4, 7, 25, and 15µg/kg body weight, respectively [26, 54–56]. Regarding As, the toxicity is linked only to the inorganic form, since the organic As is not very harmful to humans. Consequently, in the calculation of the aforementioned indices (EWI, THQ, and CR), we have taken into consideration only 3% of the total concentration of arsenic which is considered as the toxic form (the inorganic form). The calculated THQ values for studied metals in the different study locations were lower than the permissible limit (1.0) indicating that the sardine concentration levels in the Mediterranean Sea of Morocco do not present an adverse effect on human health. As seen in Table 6, the EWI calculated values did not exceed the recommended value. The maximum EWI of Cd was 0.086 μg/kg bw/week or 0.012 μg/day representing 1.22% of the PTWI (Table 6). These values are comparable to those reported by Aissioui et al. [30] in sardine muscle from the Algiers Sea, lower than those found by Chahid et al. [29] and higher than those registered by Traina et al. [27] in sardine from the Mediterranean Sea of Italy. Concerning the Hg, we have compared our result to the PTWI defined for methylmercury (MeHg: 1.6 μg/kg/body weight) which is classified as possibly carcinogenic to humans [57, 58]. Several studies have reported that fish meat contains especially MeHg form (toxic form). The calculated EWIHg intake ranges between 0.044 and 0.759 μg/kg bw/week representing 2.75–47.43% of the PTWI (Table 6). Except for the EWIHg recorded in the El-Houceima Sea, our results of As are lower than those reported by numerous researchers in the Mediterranean Sea [27, 30, 59]. In opposition, the EWI values of Hg recorded in samples from the El-Houceima Sea are comparable to those reported by Özden and Erkan [59] in Turkey, lower than those reported by Chahid et al. [29] and higher than those reported by Aissioui et al. [30], and by Traina et al. [27] in Algerian and Italian Sea, respectively. The large intakes of Cd (EWI = 0.086) and Hg (EWI = 0.759) have been recorded in El-Houceima Sea. With regard to Pb, the EWI ranged from 0.005 to 0.06 μg/kg bw/week, which presents a relative percentage of 0.02–0.24% of the PWTI (Table 4). These results are lower than those recorded for the sardine muscle in various Mediterranean Seas. [27, 30, 59], indicating that the Moroccan Mediterranean Sea seems to be less contaminated by lead. For As, the maximum EWI was 1.16 μg/kg bw/week or 0.16 μg/kg bw/day representing 7.73% of the legal limit (15 μg/kg bw/week). The largest contribution for As (EWI = 1.16) was recorded in the Ras el Ma Sea. The EWIAs values for sardine sampled in the spring and summer seasons are comparable to those reported by Traina et al. [27] in sardine from the Italian Mediterranean Sea. However, those calculated in the winter and autumn seasons are higher than those reported in other Mediterranean Seas. This result indicates that the three seas are more contaminated by arsenic, especially in the autumn-winter season. We have also calculated the total THQ (TTHQ) to assess the risk of the contamination level including the four heavy metals (Cd, Hg, Pb, and As; Table 6). For Beni-Ensar and Ras el Ma, the TTHQ calculated values were below the recommended value (<1). As a result, no health risks can be associated with sardine consumption in these sites in the case of Beni-Ensar and Ras el Ma. Inversely, the total THQ of specimens from El-Houceima Sea exceed the preconized threshold (1) indicating a potential risk to human health with a consummation frequency of 7 days/week. The large intakes of Cd (EWI = 0.086) and Hg (EWI = 0.759) have been recorded in the El-Houceima Sea (Table 6). Based on the calculated TTHQ values, the El-Houceima Sea seems to be more contaminated followed by the decreasing order by Ras el Ma and Beni-Ensar Sea (Table 6).Table 6
Health risk assessment of the consumers for each analyzed metal in edible tissues ofSardina pilchardus (Walbaum, 1792) from the Mediterranean coast of Morocco.
WinterSpringSummerAutumnEWI (EDI)THQCREWI (EDI)THQCREWI (EDI)THQCREWI (EDI)THQCRCdBeni-Ensar0.065 ± 0.00 (0.009)0.009 ± 0.001.7E − 3 ± 0.01———0.060 ± 0.01 (0.008)0.009 ± 0.001.6E − 3 ± 0.010.046 ± 0.01 (0.006)0.007 ± 0.001.2E − 3 ± 0.01Ras el Ma0.029 ± 0.01 (0.004)0.004 ± 0.007.8E − 4 ± 0.010.018 ± 0.01 (0.002)0.003 ± 0.004.8E − 4 ± 0.00——————El-Houceima0.042 ± 0.01 (0.006)0.006 ± 0.001.1E − 3 ± 0.01———0.086 ± 0.01 (0.012)0.012 ± 0.002.3E − 3 ± 0.010.018 ± 0.01 (0.002)0.003 ± 0.004.8E − 4 ± 0.01HgBeni-Ensar———0.057 ± 0.00 (0.008)0.050 ± 0.00—0.058 ± 0.00 (0.008)0.051 ± 0.00—0.097 ± 0.002 (0.013)0.086 ± 0.00—Ras el Ma———0.047 ± 0.00 (0.006)0.042 ± 0.00———————El-Houceima0.759 ± 0.00 (0.107)0.670 ± 0.00—0.044 ± 0.00 (0.006)0.038 ± 0.00—0.341 ± 0.00 (0.048)0.301 ± 0.00—0.220 ± 0.00 (0.031)0.194 ± 0.003—PbBeni-Ensar0.035 ± 0.00 (0.005)0.0012 ± 0.001.2E − 6 ± 0.000.046 ± 0.01 (0.006)0.0016 ± 0.001.6E − 6 ± 0.000.020 ± 0.00 (0.002)0.0007 ± 0.007.3E − 7 ± 0.000.016 ± 0.00 (0.002)0.0005 ± 0.005.8E − 7 ± 0.00Ras el Ma0.028 ± 0.01 (0.004)0.001 ± 0.001.0E − 6 ± 0.000.023 ± 0.01 (0.003)0.0008 ± 0.008.3E − 7 ± 0.000.005 ± 0.00 (0.0007)0.0001 ± 0.002.0E − 7 ± 0.000.014 ± 0.00 (0.002)0.0005 ± 0.005.0E − 7 ± 0.00El-Houceima0.013 ± 0.00 (0.002)0.0004 ± 0.004.8E − 7 ± 0.000.06 ± 0.01 (0.008)0.0022 ± 0.002.2E − 6 ± 0.000.019 ± 0.00 (0.002)0.0007 ± 0.007.2E − 7 ± 0.000.16 ± 0.01 (0.023)0.0059 ± 0.006.0E − 6 ± 0.00AsBeni-Ensar0.815 ± 0.10 (0.11)0.38 ± 0.060.17 ± 0.020.557 ± 0.18 (0.07)0.26 ± 0.130.11 ± 0.050.638 ± 0.20 (0.09)0.30 ± 0.090.13 ± 0.041.04 ± 0.13 (0.14)0.49 ± 0.060.22 ± 0.02Ras el Ma1.16 ± 0.06 (0.16)0.54 ± 0.020.24 ± 0.010.52 ± 0.20 (0.07)0.24 ± 0.070.11 ± 0.890.49 ± 0.10 (0.07)0.23 ± 0.060.10 ± 0.961.10 ± 0.02 (0.15)0.51 ± 0.010.23 ± 0.00El-Houceima1.14 ± 0.03 (0.14)0.54 ± 0.010.24 ± 0.000.47 ± 0.15 (0.07)0.22 ± 0.100.10 ± 0.050.69 ± 0.10 (0.08)0.32 ± 0.050.14 ± 0.021.08 ± 0.04 (0.15)0.50 ± 0.010.22 ± 0.00TTHQBeni-Ensar0.3980.3150.3620.587Ras el Ma0.5560.2940.2370.522El-Houceima1.2180.2670.6400.712The results are presented as mean ± standard deviation; Cd: cadmium; Hg: mercury; Pb: lead; As: arsenic; EWI (μg kg−1 body weight): estimated weekly intake; THQ: target hazard quotient; CR: carcinogenic risk; TTHQ: total target hazard quotient; THQ values >1 are indicated in bold. The values indicated in bold exceed the established threshold.The probability of a person developing cancer over their lifetime was calculated using the cancer risk (CR) as a result of exposure to contaminants that could cause cancer. If the CR value is exceeding 10−5, it indicates a high probability (1/100 000) of having cancer in the consumer. The CR was not estimated for Hg since the cancer slope factor (CSF) for Hg is not determined by the United States Environmental Protection Agency. The values of the carcinogenic risk index for As and Cd exceed the established threshold (10−5) in all cases (site and season). In contrast, the calculated CRCd was below the legal limit in all sites and seasons (Table 6). Comparable outcomes have been reported by Traina et al. [27] in sardine muscle from the Mediterranean Sea of Italy. In the current study, the CR was calculated considering all the week as a frequency of ingestion, 7 days/week (maximalist theoretical approach). So, if we consider that the frequency of ingestion is 2 or 3 days/week, the values of these risk indices for contamination by heavy metals on public health will decrease significantly. This is the case for Morocco, since the consumption of fish is quite moderate because of the unaffordable price. Overall, 100% of the values for CR of As and Cd (when measurable) were above the recommended limits. Based on the calculated EWI and THQ, it may be concluded that the consumption of Moroccan sardine from the Mediterranean Sea does not represent a health risk for consumers. Inversely, As and Cd represent a potential risk on human health in terms of carcinogenic risk.


## 3.4. Principal Component Analysis

A principal component analysis (PCA) was carried out to identify the relationships among the studied groups of specimens and evaluated parameters. This multifactorial analysis was conducted using 12 variables, including heavy metals and health risk indices. This analysis allowed us to classify and process the relative information of the metals studied by establishing the possible correlations between all the evaluated variables. The first two principal components (PCs) represent 78.07% of the total information. The PC1 and PC2 accounted, respectively, for 55.60% and 22.90% of the total variance in the data set. Figure2 represents the projection of metals in the plane defined by the first two principal components. PC1 was correlated positively with As, Pb, and Cd (Table 7 and Figure 2). In opposition, the PC2 was defined mainly by Hg. Liver samples were clearly differentiated from the gill and muscle and are located on the right side of the plane (Figure 2), where all metals lie. According to PC2, the effect of the season is mainly manifested. The projection of results according to the season on the factorial map (Figure 2) shows discrimination between the studied sardine and allows summarizing the interpretations already mentioned above in a very simplified way. The samples of winter sampled from the El-Houceima Sea were clearly differentiated from other season samples indicating the high sardine contamination level especially by mercury. The correlation matrix analysis reveals the presence of a correlation between As and Pb (r = 0.61) and a correlation between Hg and As (r = 0.50).Figure 2
Graphical representation of principal component analysis of heavy metals based on the first two principal components (PCs) (factorial map of the seasons (a), sites (b), organs (c), and heavy metals (d)).
(a)(b)(c)(d)Table 7
First two principal components explaining 78.07% of the total variance of the sardine,Sardina pilchardus (Walbaum, 1792), contamination level from three coast sites in the Mediterranean Moroccan Sea.
VariablesPrincipal component12As0.8530.337Pb0.848−0.269Cd0.685−0.410Hg0.5040.729EWIAs0.8530.337EWIPb0.848−0.269EWICd0.719−0.490EWIHg0.5040.729THQAs0.8530.337THQPb0.848−0.269THQCd0.719−0.490THQHg0.5040.729CRAs0.85300.337CRPb0.848−0.269CRCd0.719−0.490As: arsenic; Pb: lead; Cd: cadmium; Hg: mercury; EWI: estimated weekly intake; THQ: target hazard quotient; CR: carcinogenic risk. The values represent the correlations of variables with the first two principal components.The PCA analysis exposed a significant effect of the location and season on the contamination level of the studied specimens (p<0.05). This variation can be probably the result of the difference in feeding source, growth stage, and environmental conditions. Similar results were reported by Bonsignore et al. [31] for fish, crustaceans, mollusks, and echinoderms and by Leonard et al. [60] for Oreochromis niloticus and Clarias gariepinus species.


## 4. Conclusion

Sardine metallic contamination from three regions of the Moroccan Mediterranean Sea (Beni-Ensar, Ras el Ma, and El-Houceima) was assessed using inductively coupled plasma mass spectrometry. The results show that cadmium (Cd), mercury (Hg), lead (Pb), and arsenic (As) were present only in trace concentrations and do not exceed the legal limit of 0.09, 0.037, 0.01, and 7.38μg·g−1 ww, respectively. Concerning the health risks for consumers, the results show that the indices are below the established safety values in the case of estimated weekly intake, and target hazard quotients. However, the cancer risk index and the total target hazard quotients were above the threshold limits and thus present a potential carcinogenic risk to human health. This study gives details on the possible risks to human health from exposure to Hg, Cd, Pb, and As through sardine intake in high-level fish consumers in the food chain. More research is required to gain a comprehensive understanding of the spatiotemporal dynamics of potentially toxic metals, evaluating the contamination degree by other toxic elements, in particular polycyclic aromatic hydrocarbons, polychlorinated biphenyls, iron, chromium, copper, nickel, and zinc, in order to participate in drawing up an evaluation of the Moroccan population’s exposure (adults and children) to inorganic and mineral contaminants.



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*Source: 1455410-2023-03-18.xml*