Total Mercury (THg) Content in Red Mullet (Mullus barbatus) from Adriatic Sea (Central Mediterranean Sea): Relation to Biological Parameters, Sampling Area and Human Health Risk Assessment

Abstract

Mercury (Hg) is a pollutant that has toxic effects on ecosystems and biota. As it biomagnifies in the food chain, its presence in edible fish poses a high risk to human health. Herein, total Hg (THg) content was quantified in 2018–2019 using thermal decomposition amalgamation atomic absorption spectrometry in muscle tissue of red mullet (Mullus barbatus), a commercially important species throughout the Adriatic Sea (Central Mediterranean Sea). Specimens were grouped into 16 pools based on sex, reproductive stages, and sampling area. The overall mean value of THg content was 0.20 ± 0.15 mg kg⁻¹ in terms of wet weight. THg levels in males and females showed no statistically significant differences, whereas specimens that were captured in open sea showed a higher THg content than coastal samples. Statistically significant differences between THg content and the reproductive stages of fish were found in females. However, neither lipid content nor fish length were statistically correlated with THg content. The analyzed specimens were considered to be safe food according to EU directives, but it is necessary to exercise caution and further investigate Italian people in the 0–18 age group, because they were found to be exposed to a higher dose of methylmercury than the safety threshold set by the EFSA.

1. Introduction

Mercury (Hg) is one of the most toxic elements. In its methylated form, it can biomagnify along the food chain and reach concentrations up to milligrams per kilogram of tissue, in top marine predators [1]. It is naturally present in the Earth’ crust, and the Mediterranean Sea is particularly rich in Hg because it is characterized by a natural content of cinnabar (HgS) deposits [2]. Furthermore, the northern area of the Adriatic Sea is known for mercury input phenomena, due to transport by run-off of the water coming from the dismissed Idrija mine in Slovenia [3,4]. In this basin, Hg is distributed in the various environmental matrices in different concentrations: from ng L⁻¹ in seawater [5] and µg kg⁻¹ in sediments [6], up to mg kg⁻¹ in biota tissues [7,8]. Exposure to this element, even at low concentrations, causes toxic effects at the level of the blood–brain barrier, the central nervous system, the cellular level, in protein synthesis, in enzymatic activity, as well as at the neurophysiological level [9].

Red mullet, Mullus barbatus Linnaeus, 1758, is a common demersal fish that is widely distributed along the coasts of the Mediterranean Sea, the Black Sea, northwest Africa, and northern Europe, living on sandy and muddy bottoms of the continental shelf at depths down to 200 m [10]. In the Adriatic Sea (central Mediterranean), it is one of the most important edible fishes, and is one of the principal target species of trammel net and bottom trawl fisheries. This species accounted for an average landing in Italy of 5888.5 tons in 2013–2015 [11]. M. barbatus is considered a sentinel species in the Mediterranean Sea because it accurately reflects contaminant bioavailability and concentration in the marine environment, and has extensively been used as a bioindicator for pollution monitoring [12,13,14,15,16].

Many studies have been conducted on the total mercury (THg) content in M. barbatus tissue in areas of the Mediterranean Sea [17,18,19,20,21,22,23,24,25]. However, none of the studies took into consideration the reproductive stage, biological parameters such as sex and lipid content, and sampling area.

The present study was carried out to (1) assess the state of contamination in Adriatic red mullet; (2) identify a possible correlation between THg content, biological parameters, and sampling areas; and (3) evaluate human health risks due to the consumption of this product, according to EU standards.

2. Materials and Methods

2.1. Fish Sampling

In this study, 407 fish samples were obtained from commercial fish caught randomly by a commercial bottom trawler in a rich fishing ground spanning the northern and central Adriatic Sea (FAO-GFCM geographical sub-area 17; Figure 1), from May 2018 to January 2019. Fish samples were supplied by the same fishing vessel, and about 25 individuals were sampled monthly from two different areas, one close to the Italian coast, and the other from open sea.

Total lengths (cm) and weights (g) of all specimens were determined before dissection, and sex was evaluated. The gonadal development stage (maturity) was established macroscopically in order to assign individuals into one of four categories: juveniles, sexually immature; adults, individuals whose gametes were just beginning to develop; spawning, individuals whose gametes were ready for the spawning season; and post-spawning, individuals at the end of their reproductive cycle, sexually mature but inactive [26].

Edible tissue, muscle from the dorsal fin up to the tail, was extracted and stored at −20 °C until laboratory analysis. Then, samples were grouped into 16 pools based on the reproductive stage, sex, and sampling areas (coast and open sea).

2.2. Laboratory Analysis

All analytical steps were performed in an ISO 5 clean room laboratory under laminar flow. All scalpels, microspoons, and spatulas that came into contact with the samples were first decontaminated with a specific washing procedure that used an HCl (34–37% superpure, Carlo Erba, Milano, Italy) 1:10 (v/v) solution. The samples were weighed using a microanalytical balance (XS205 Mettler Toledo, Greifensee, Switzerland, readability, 0.01 mg; repeatability standard deviation, 0.015 mg). The variable volume micropipettes and neutral tips were from Brand (Transferpette, Wertheim, Germany), and the scalpels with sterile stainless-steel blades were from Granton (Mod. 91021, Sheffield, UK). Ultrapure water was obtained from a Milli-Q water system (Merck Millipore, Darmstadt, Germany). For the lipid extraction, acetone and petroleum ether were used (RS grade, Carlo Erba, Milano, Italy). Dogfish muscle DORM-2 (NRCC, Ottawa, ON, Canada) was used as certified reference material for Hg content.

2.3. Quantification of Total Mercury Content

At least 4 aliquots per pool were prepared for the analysis. The THg content of muscle tissue was determined with thermal decomposition amalgamation atomic absorption spectrometry (TDA AAS), using a direct mercury analyzer (DMA-1, FKV, Milestone, Sorisole, Italy). About 10 g of muscle tissue was minced and homogenized (homogenizer MZ 4110, DCG Eltronic, Monza, Italy). About 0.05 g of each minced sample was weighed into a quartz tube; then, the tubes were placed into an auto injector. The samples were heated with an ultrapure air stream (99.998% purity) passing over them at 600 °C. The Hg vapour passed through a catalytic tube, then the products of combustion were removed and trapped in a gold amalgamator. Desorption was performed at 850 °C, and the Hg content was quantified by determining the absorption at 253.7 nm. TDA AAS involved the following steps: drying at 250 °C for 60 s, and decomposition and desorption at 650 °C for 120 s and 60 s, respectively [8,27]. The detection cell ranges used were 0.03–200 ng and 200–1500 ng. The linearity of the instrumental response was tested on the first (5–200 µg kg⁻¹, R² = 1) and second (500–10,000 µg kg⁻¹, R² = 0.9999) cell. Accuracy was estimated at 0.7% by the use of DORM-2-certified reference material as analytical quality control. The mean experimental Hg value of 4.40 ± 0.06 mg kg⁻¹ dry weight (dw) was in line (p > 0.05) with the mean value of the certified reference material (4.43 ± 0.05 mg kg⁻¹ dw). Repeatability (<10%) was performed using replicate analyses of the same samples. Reproducibility (<10%) was tested by the analysis of the same sample for six days. Limit of detection (LOD) and limit of quantification (LOQ) were estimated according to [28] at 1.65 and 4.99 µg kg⁻¹, respectively. THg content was estimated using the calibration curve technique. In order to correct for possible Hg contamination during the analysis, the THg concentration of a blank was subtracted from the concentration that was measured in a sample. Results were expressed as arithmetic mean ± standard deviation (SD).

2.4. Lipid Content Determination

Lipid content was determined according to Truzzi et al. (2018) and Zarantoniello et al. (2020) [29,30]. About 1 g of muscle tissue from each pool was minced, homogenized, accurately weighed, and freeze-dried (Edwards EF4 Modulyo, Crawley, Sussex, UK) to reach a constant weight (± 0.2 mg). Total lipid content was determined in 3 aliquots per pool. About 0.5 g of each pool was loaded into Teflon-coated liners to perform a microwave-assisted extraction (MAE) using a icrowave-accelerated reaction system (MARS-5, 1500 W; CEM, Mathews, NC, USA). A mixed solution of 10 mL petroleum ether and 5 mL acetone was added on each liner. The process involved the use of an extraction program that consisted of a first step of heating up to 90 °C for 10 min, and a second step of maintaining this temperature for 20 min. After a 10-min cooling process, the solution was filtered through Whatman GF/C filter papers (Ø 90 mm, GE Healthcare Life Sciences, Buckinghamshire, UK) that were filled with anhydrous sodium sulphate (Carlo Erba) to prevent water interference. Sample residues in the liner were recovered with three aliquots of 2 mL mix solution (petroleum ether:acetone mixture, 2:1 v/v). The lipid extract was evaporated under a stream of N₂, and the lipid content was measured gravimetrically.

2.5. Risk Assessment

The risk assessment was based on 1 mg kg⁻¹ content in fresh fillet of the genus Mullus, which was set by the European Union for Hg [31]. Furthermore, the risk associated with the average consumption of fish by the Italian population was evaluated on the basis of the recommended provisional tolerable weekly intake (PTWI) for inorganic mercury and methylmercury, established by the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives [32].

2.6. Statistical Analysis

Statistical analyses were performed using Statgraphics 19 (Statgraphics Technologies Inc., The Plains VA, USA). We compared each group using one-way analysis of variance (ANOVA) and the multiple range test. In order to test the homogeneity of the variance, Levene’s test was applied. Significant differences were evaluated at the 95% confidence level. A principal component analysis (PCA) was applied to the data set, in order to investigate multivariate relationships among our variables; significant components were obtained through the Wold cross-validation procedure [33].

3. Results

Hg was found in all samples, regardless of the sex and the sampling area (coast and open sea). The concentrations are reported in

Table 1. Concentrations of total mercury (THg) and total lipids in muscle tissues of M. barbatus samples from the Adriatic Sea. Reproductive stage, sex, sampling area, and biometric data of investigated fish. n, number of fish samples; mean value ± standard deviation; range (min–max) of length, weight, THg, and total lipids.

Reproductive Stage	Sex	Sampling Area	n	Length, cm (Min–Max)	Weight, g (Min–Max)	Total Hg mg kg−1 ww (Min–Max)	Total Lipids g/100 g (Min–Max)
Juvenile	Female	Open sea	6	12 ± 3	16 ± 1	0.25 ± 0.05	3.27 ± 0.05
		Coast	9	11 ± 1	12 ± 2	0.03 ± 0.01	1.15 ± 0.09
	Male	Open sea	4	10 ± 1	9 ± 3	0.15 ± 0.01	0.59 ± 0.02
		Coast	10	10 ± 1	13 ± 1	0.07 ± 0.01	1.12 ± 0.10
	Total		29	11 ± 1 (10–12)	13 ± 3 (9–16)	0.13 ± 0.09 (0.03–0.25)	1.53 ± 1.19 (0.59–3.27)
Adult	Female	Open sea	19	17 ± 2	49 ± 12	0.41 ± 0.03	5.11 ± 0.49
		Coast	44	16 ± 2	43 ± 13	0.19 ± 0.01	7.85 ± 0.74
	Male	Open sea	30	13 ± 1	21 ± 5	0.23 ± 0.01	2.97 ± 0.17
		Coast	7	13 ± 1	26 ± 4	0.07 ± 0.01	6.49 ± 0.27
	Total		100	15 ± 2 (13–17)	35 ± 13 (21–49)	0.22 ± 0.14 (0.07–0.41)	5.61 ± 2.08 (2.97–7.85)
Spawning	Female	Open sea	24	15 ± 2	36 ± 11	0.62 ± 0.02	1.62 ± 0.02
		Coast	31	15 ± 2	39 ± 12	0.17 ± 0.01	4.23 ± 0.06
	Male	Open sea	26	13 ± 1	26 ± 16	0.22 ± 0.01	1.85 ± 0.16
		Coast	8	13 ± 1	21 ± 7	0.11 ± 0.01	3.63 ± 0.30
	Total		89	14 ± 1 (13–15)	31 ± 8 (21–39)	0.28 ± 0.23 (0.11–0.62)	2.84 ± 1.29 (1.62–4.23)
Post-spawning	Female	Open sea	31	16 ± 2	42 ± 17	0.12 ± 0.01	7.87 ± 0.07
		Coast	80	15 ± 2	35 ± 13	0.05 ± 0.01	2.59 ± 0.14
	Male	Open sea	58	13 ± 1	23 ± 9	0.23 ± 0.01	0.44 ± 0.03
		Coast	20	13 ± 1	22 ± 5	0.24 ± 0.01	1.82 ± 0.08
	Total		189	14 ± 2 (13–16)	31 ± 10 (22–42)	0.16 ± 0.09 (0.05–0.24)	3.18 ± 3.25 (0.44–7.87)
Grand total			407	13 ± 2 (10–17)	27 ± 12 (9–49)	0.20 ± 0.15 (0.03–0.62)	3.29 ± 2.43 (0.44–7.87)

.

A well-defined pattern of THg content was evident in female specimens in relation to reproductive stages, both in coastal and open sea sampling areas. The THg content increased, passing from the juvenile phase to the spawning phase, whereas the content drastically decreased in the post-spawning phase, in which the lowest value was recorded. Coastal samples showed a significant statistical decrease (p = 0.0017) overall in THg content compared to the open sea samples (0.11 ± 0.08 and 0.35 ± 0.22 mg kg⁻¹ ww, in coastal and open sea, respectively). The same trend was also observed at every reproductive stage of females. Therefore, comparing coastal and open sea fish, a significant statistical decrease in THg content was recorded in the coastal area in comparison with the open sea (Figure 2a).

Regarding male specimens, catches made in open sea, the values of THg content in muscle tissue were significantly lower in juveniles than in other reproductive stages of fish (p = 0.0003). In regards to coastal samples, the lowest THg level was recorded in juvenile and adult phases, with an exponential increase (p < 0.0001, Figure 2b), whereas the highest THg level was recorded in post-spawning phase.

Comparing both sampling areas, the males caught from the coastal area showed THg values that were significantly lower than those of the open sea (p = 0.034, 0.12 ± 0.08 and 0.21 ± 0.04 mg kg⁻¹ ww, respectively) (Figure 2b). Furthermore, coastal fish samples showed a significant statistical decrease in THg content compared to offshore samples for each reproductive stage, except for post-spawning (Figure 2b).

Comparing the sex of fish within the same reproductive stage and sampling area, the value of THg from open sea was significantly higher in females than in males; this finding was observed in all life stages, except in post-spawning, where females showed a lower THg content than males (p < 0.0001). With regards to fish that were sampled from coastal area, females showed a statistically significant higher THg content both in adult and spawning phases, compared to males. Otherwise, juvenile and post-spawning females showed a THg content that was significantly lower than in males (p < 0.0001).

The lipid content ranged from 0.44 (post-spawning males in open sea area) to 7.87 (post-spawning females in open sea area) g/100 g wet weight. The value of lipid content was significantly lower in males that were collected in open sea area, compared to other coastal related samples (p = 0.008). Taking into consideration the variations in total lipid content based on the reproductive stage of fish, all investigated samples highlighted a bell-shaped trend. This trend showed that lipid content grew in the spawning phase and decreased during the post-spawning phase, with the exception of post-spawning females caught from open sea (Table 1).

The relationship between THg content and lipid content was also investigated; no statistically significant correlation (p > 0.05) between the two parameters was found (Table 1). Although a positive linear trend between these two parameters was identified (r = 0.41, p = 0.118), fish length was not statistically correlated with THg content. A principal component analysis (PCA) was applied to the samples, considering the variables of Hg level, lipid content, sample length, and weight (Figure 3). This was made in order to reduce the dimensionality of the data matrix, and more easily visualize the results to interpret data.

The PCA extracted two significant, cross-validated principal components which account for 93.75% of the variability in the original data (

Table 2. Principal component analysis. Eigenvalues, explained and cumulative variance, loadings of the variables for the first two PCs.

	Principal Components
	1	2
Variance explained
Eigenvalues	2.6673	1.0828
% of variance	66.683	27.070
Cumulative %	66.683	93.754
Factor loadings
Length	0.5990	0.0387
Weight	0.5985	0.0127
Hg	0.2571	0.8452
Lipid	0.4657	−0.5328

). The first principal component (PC1, 66.68%) is dominated by biometric parameters and lipid content (Table 2). Juvenile specimens showed the lowest values of weight, length, and lipid content (negative scores), both for male and female. Considering the other reproductive stages, females (positive scores) were associated with higher biometrics values and higher lipid content than males (mainly negative scores). The second principal component (PC2, 27.07%) separated the samples mainly on the basis of Hg content (positive loadings): the coastal samples (mainly negative scores) were associated with lower Hg content than open sea samples (mainly positive scores).

4. Discussion

To our knowledge, this is the first study that investigated the mercury content in red mullet from the Adriatic Sea, in relation to some biological parameters, sampling area, and human health risk assessment. In the present study, THg content was similar to the findings of previous studies that were carried out in the Mediterranean Sea [17,18,19,20,21,22,24,25]. Generally, in comparison with previous research carried out in the Adriatic Sea, this study showed similar Hg values to those recorded by di Lena et al. (2017) [34], Martínez-Gómez et al. (2012) [23], and Sulimnec Grgec et al. (2020) [19], whereas other studies recorded greater Hg concentrations [17,25]. Our results were also similar to those found for the Gulf of Lions [24]. Additionally, specimens from Sicily and the Spanish fish market recorded similar Hg values [21,22]. However, mercury concentration in the central Tyrrhenian was higher in comparison with our findings [34] (

Table 3. THg concentrations (mg kg ⁻¹ w.w.) recorded in other areas of the Mediterranean Sea. n, number of fish samples; mean ± standard deviation, range (min–max).

Sampling Area	n	Total Length, cm (Weight, g)	THg, mg kg−1 ww (Min–Max)	Reference
Adriatic Sea	407 (16 pool)	10–17 (9–49)	0.20 ± 0.15 (0.03–0.62)	This study
Adriatic Sea			(0.08–1.74)	[17]
Central Adriatic Sea	14	13.75 ± 0.44 (34.96 ± 3.71)	0.48 ± 0.9	[25]
Eastern Adriatic Sea	60	9.4–15.5 (10–62)	(0.178–0.996)	[19]
Central Adriatic Sea	25	15 ± 0.4 (37 ± 5)	0.236 ± 0.102	[34]
Central Tyrrhenian Sea	58	18 ± 2 (73 ± 29)	0.702 ±0.834	[34]
Spanish market	20		(0.14–0.36)	[22]
Gulf of Lions	132	8.5–24.5	0.244 ± 0.253 (0.004–1.962)	[24]
Sicily	48		0.138 ± 0.107	[21]
Ionian Sea			(nd–1.50)	[17]
Ionian Sea			(nd–1.50)	[18]
Spanish Mediterranean	36	13.5–14.5	(0.074–0.121)	[23]

).

Almost all previous studies focused on Hg and other metal content in different biological tissues of M. barbatus only in relation to biometric indexes, without considering any important parameters such as habitats of this species, the reproductive stage, or the lipid content, as investigated in the present study. To the best of our knowledge, only Martinez-Gòmez et al. [23] simultaneously investigated several environmental and biological parameters, including lipid content, in Spanish Mediterranean fish. They found a similar lipid content (1.16–5.65 g/100 g, min–max) as that in the present study, with a marked difference between males and females, with higher levels in females. However, no reference to a correlation with Hg content was mentioned by the authors.

Our results demonstrated that the distribution of Hg was influenced by several factors, such as sampling area, sex, and reproductive stage. The specimens collected from the open sea showed higher contamination levels than the samples taken from coastal areas. Our findings showed that there was a difference between coastal and open sea samples. Therefore, our findings suggest that sampling area may influence Hg levels in red mullet tissues. Moreover, our findings could be explained by the fact that M. barbatus spends its juvenile life stage mainly in the coastal waters before migrating to open deeper seas [35]. Thus, higher levels of Hg found in open sea specimens could be explained by a higher average age of these fish than coastal specimens, and a consequently longer exposure time to this metal. This is also confirmed by another study carried out in the Adriatic Sea, where lower Hg values were recorded in coastal areas [6]. Since mercury accumulates over time in fish tissues, it is probable that older fish will have absorbed higher levels of this metal. Moreover, M. barbatus lives close to the sea floor, so they are probably exposed to higher concentrations of dissolved methylmercury produced from the methylation activity of microorganisms living in the sediment.

In addition, food habits represent an important pathway for the transport of heavy metals, such as mercury. M. barbatus lives in close contact with the marine sediment, feeding on benthic prey such as crustaceans and polychaetes. It is known that benthic preys are richer in mercury than pelagic organisms, and this has been found in the Adriatic Sea as well as in other areas [36,37]. Thus, an important contribution to the different bioaccumulations of Hg could due to the different feeding strategies of specimens that migrate to deeper waters.

Males showed a non-statistically significant lower average Hg content, and this could be due to a lower growth rate in biomass than for females [38]. The decrease in Hg levels in edible tissue for females after the spawning phase was also confirmed by several studies for other contaminants [16,39]. This could derive both from Hg bioaccumulation inside the egg cells (and therefore a release of Hg during the spawning phase), and from a reduction in predatory activity that occurs in post-spawning specimens. In fact, this pattern does not occur in males, in which the bioaccumulation of this element increases during the reproductive stages.

The lipid content was higher in females than males, and during the spawning phase it did not influence Hg levels, as demonstrated by Martínez-Gómez et al., 2012 [23], for red mullet, and for other species by Łuczyńska et al., 2017, and Soares et al., 2018 [40,41].

Considering the safety threshold set by EU regulations [31,42], which sets the limits for the mercury content in fresh tissue of M. barbatus at 1 mg kg⁻¹, all the samples analyzed in this study were found to be well below this threshold. Therefore, considering the toxic effects due to the intake of Hg, the average consumption of this product does not compromise human health.

The Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives has established regulatory guidelines regarding dietary mercury intake. It recommends a provisional tolerable weekly intake (PTWI) for methylmercury of 1.6 μg kg⁻¹ body weight (b.w.), and of 4 μg kg⁻¹ b.w. for inorganic mercury [32].

For fish meat, fish products, fish offal, and unspecified fish and seafood, the EFSA suggests evaluating the exposure assessment, and applying a conversion factor of 1.0 for methylmercury, and 0.2 for inorganic mercury. Based on the EFSA’s Comprehensive European Food Consumption Database (fish, seafood, amphibians, reptiles, and invertebrates) [43], we assessed the intake of inorganic Hg and methylmercury, based on gender and age, resulting from consumption of this species in the Italian population (Figure 4).

Although there were no exceedances for inorganic Hg intake, the results showed that methylmercury intake in the lower age groups always exceeded the limit set by the EFSA. In particular, toddlers were the most at risk, and showed an intake that is three to four times (4.9 and 6.2 μg kg⁻¹ b.w. per week, respectively) above the EFSA’s limit of 1.6 μg kg⁻¹ b.w. per week. However, the dietary consumption statistics for infants and toddlers were performed on a number of individuals that totaled less than 60; thus, these findings may not be statistically robust, as also indicated by the EFSA. Furthermore, these result were based on the assumption that all fish intake was actually coming from the samples examined in this study.

5. Conclusions

This first study on the M. barbatus species in the Adriatic Sea that focused on mercury content in relation to different biological, geographical, and dietary risk variables according to the EU directive and EFSA scientific opinion limits. Concentration trends were identified, with maximum values occurring during the adult and spawning phases for females, and during the post-spawning phase for males. The specimens captured in open sea areas were found to have a higher content of this element, while the percentage of lipids in the muscle tissue did not influence its distribution.

Considering the Hg limit levels set by the EU, no sample exceeded the threshold. However, Italian consumers in the 0–18 age group showed a value that was above the methylmercury intake limit set by the EFSA.

Although the analyses were carried out on a single chemical target, this study provides preliminary results to the problem of mercury contamination in edible fish species, using an approach that considered several variables of the target species. Studies on the speciation of this element and the distribution of other toxic elements and compounds are necessary in order to gain a better overview of this global environmental challenge.