Mussels as Bioindicators for the Rapid Detection of Heavy Metal Fluctuations in Marine Coastal Waters: A Case Study of Seasonal Bioaccumulation Monitoring and Assessment of Perna viridis from the Gulf of Tonkin Coastline, Hai Phong, Vietnam

Abstract

This study aims to evaluate the feasibility of using the mussel as a bioindicator for the rapid detection of heavy metal (such as Cd, Pb, Hg, Ni, Cr, Cu, As, and Zn) fluctuations in aquatic environments and the sensitivity of the bioaccumulation of heavy metals in muscle tissues over time. The seasonal bioaccumulation patterns of heavy metals within Asian green mussels (Perna viridis), from Vietnamese coastal waters of Hai Phong were investigated using inductively coupled plasma mass spectrometry (ICP-MS). Additionally, the health risks from the consumption of P. viridis by local people were assessed. Mussels of varying sizes were sampled on a monthly basis between March (dry season) and July 2024 (wet season). The results revealed that the hepatopancreas had substantially higher concentrations of metals at all times relative to their corresponding muscle tissues, confirming its appropriateness as a bioindicator organ. The concentrations of heavy metals in mussels were recorded as significantly lower than the guideline levels, except for arsenic (As). Zinc (Zn) showed the highest concentrations, while mercury (Hg) had the lowest concentrations. There were strong seasonal and monthly differences, with peak levels of Pb, Cr, and As during the dry season, and high levels of Cs and Cu during the rainy season. It was found that the condition index, physiological factors, and shell size all had major impacts on the absorption of specific heavy metals. It was indicated that Pb, Cr, As, Cs, and Cu bioaccumulation are both biologically and environmentally responsive and can be used as proxies for environmental contamination, while the accumulation of these metals correlated with biological traits (shell length, weight, and CI), which is useful in modeling efforts. Health risk assessments using target hazard quotients (THQs) and the total hazard index (THI) identified Pb in the hepatopancreas as a primary contributor to the non-carcinogenic risk (THQ > 1), particularly during the dry season. The findings revealed the suitability of P. viridis, particularly hepatopancreatic tissue, as a short-term biomonitoring tool for detecting spikes and rapid fluctuations of certain heavy metals and assessing related human health risks in coastal aquatic systems.

1. Introduction

Heavy metal contamination in aquatic environments has emerged as a major global issue due to the dangerous and persistent nature of these pollutants, which can remain in ecosystems for thousands of years [1,2]. The toxicity, long-lasting presence, bioaccumulation in organisms, and magnification through the food chain make heavy metal pollution particularly harmful to ecosystems and human health [3]. Over the past decades, human activities have significantly increased pollution levels in the marine environment [4,5]. It is estimated that more than 80% of marine pollutants originate from land-based sources, including substantial amounts of solid and liquid wastes from agricultural, industrial, and urban development. These pollutants are transported by rivers, surface flow, underground water, or wind before being deposited in coastal areas [6,7,8,9,10]. Among coastal pollutants, trace heavy metals are of particular concern due to their accumulation capacity in marine organisms and their harmfulness to human health and marine ecosystems [8,11,12,13,14]. Numerous studies have been conducted to estimate the human health risks associated with consuming seafood contaminated with heavy metals [13,15,16,17,18].

Heavy metals such as Cd, Pb, Hg, Ni, Cr, Cu, As, and Zn are of particular concern in the marine environment due to their stability, persistence, and non-biodegradable nature [16,19,20]. Non-essential metals like As, Cd, Cr, Hg, and Pb are poisonous even in minute concentrations, while essential metals like Cu, Fe, Mn, and Zn are vital for biological processes, especially human physiology [21]. Moreover, aquatic sediments act as depositional sinks and secondary sources of heavy metals. Because contaminants attach themselves to sediment particles and integrate into the sediment matrix, aquatic systems may become permanently contaminated [22]. Through dietary intake, these pollutants can accumulate in the human body, causing numerous adverse health effects, including impaired kidney function, a reduced reproductive capacity, liver damage, neurological disorders, carcinogenic effects, and even death [19,23,24,25]. To mitigate these undesirable health effects, it is essential to assess the risks associated with the consumption of seafood contaminated with heavy metals.

Hai Phong, a coastal city is home to Hai Phong Port, one of Vietnam’s most critical hubs for international and domestic trade. Recently, the area has garnered increasing attention for marine environmental issues associated with human activities [26]. Industrial activities such as coal mining, metallurgy, food processing, and shipbuilding, alongside agricultural and residential wastes, have caused alarming consequences for the marine environment [27,28,29]. It is estimated that substantial quantities of heavy metals are discharged into this area annually, including approximately 382 tons of Zn, 53 tons of Cu, 27.9 tons of As, 20 tons of Pb, 3 tons of Hg, and 2 tons of Cd, due to human activities [30]. Notably, this area is impacted not only by city activities but also by other provinces in the northern Vietnam [27]. However, studies of pollutants, particularly heavy metals, in this area remain limited [27,31]. Consequently, investigating, assessing, and monitoring heavy metal pollution in the marine environment has become a critical task. The main source of metal contamination is the bioaccumulation of trace metals in seafood, such as mussels, which could put seafood consumers at risk [32]. Enhancing efforts to investigate, assess, and monitor heavy metal changes to implement effective mitigation actions is necessary. Marine species serve as important biological indicators for evaluating the impact of heavy metals on marine ecosystems [11,33,34,35]. Through the process of biomagnification along food chains and webs, the presence of these pollutants not only threatens the health of marine life but also poses a serious hazard to non-target groups, including humans [32]. Due to the ability to accumulate pollutants, exhibit sensitive biological responses, have a wide distribution, remain sedentary, easy sampling, and tolerate chemical pollution, mussels are widely recognized as bioindicators of heavy metal pollution [6,8,17,35,36,37,38]. Numerous studies have been conducted on the use of mussels as bioindicators across different regions of the world [6,8,17,37,39,40]. According to those results, differences in metal concentrations, food availability, climate, and the biological traits of individual species in different locations have substantial impacts on metal bioaccumulation. To provide accurate scientific data, it is crucial to assess the extent to which bioindicators work in each area. In addition, most mussel monitoring programs rely on annual sampling; however, heavy metal concentrations in bivalve mollusks can vary throughout the year due to environmental fluctuations and biological responses to pollutants [37,40,41,42,43].

The inclusion of sentinel species in monitoring programs frequently makes it more difficult to evaluate field data due to factors including temporal and spatial variability. It is crucial to investigate mussels’ sensitivity to metals over time, taking into account seasonal variations and changes within a single season, in order to assess their suitability for heavy metal monitoring programs in a particular area. Notably, the distribution of heavy metals varies among different mussel organs, reflecting the distinct functions of these organs within the mussel body [8,44]. Understanding these distribution patterns provides valuable insights into the accumulation characteristics of mussels, forming a basis for environmental monitoring applications. Therefore, the objectives of this study are (1) to investigate the distribution characteristics of heavy metals in two organs of the Asian green mussel (Perna viridis); (2) to monitor the seasonal/monthly fluctuations of certain heavy metal concentrations; and (3) to assess the potential health risks to local people through the target hazard quotients (THQs) and hazard index (HI). The results will provide a database for assessing heavy metal pollution in the area. Additionally, insights into heavy metal concentrations in mussels will support coastal environmental pollution monitoring programs in Hai Phong.

2. Materials and Methods

2.1. Sampling

The Hai Phong coastal area is located in the northeastern part of the Gulf of Tonkin, Vietnam (Figure 1). This region features a dense river network, with an average river density of 0.6–0.8 km per km². Hai Phong Port in the study area is one of the most critical ports for both international and domestic trade. Rapid industrialization and urbanization in the area have significantly impacted the marine coastal environment [26,27]. Due to its geographical location and specific climatic conditions, the studied area has become a hub for aquaculture, supplying various seafood species, including Asian green mussels (Perna viridis). In terms of climate, the study area is located in a subtropical region characterized by two distinct seasons: a rainy season (from May to September) with temperatures ranging from 15 to 23 °C, and a dry season (from October to April next year) with temperatures between 25 and 29 °C [29]. The rainy season receives substantial precipitation, with an annual rainfall of approximately 1800 mm. In contrast, the dry season is marked by low rainfall, often resulting in drought conditions and significantly reduced river water levels.

Mussels of varying sizes and weights (ten individuals) were collected monthly from March (dry season) to July (rainy season) in 2024. Due to the wild nature of our samples and sampling conditions, only ten individuals with significantly different body sizes were collected. This study could serve as a baseline and preliminary investigation of representative individuals for further studies. After collection, the mussels were frozen and promptly transported to the laboratory, where they were separated into muscle tissues and hepatopancreas glands. Morphometric parameters, including body weight and shell size, were measured and recorded for each mussel. The condition index (CI), which reflects the physiological condition and nutritional status of the mussel, was calculated by dividing the wet weight of the soft tissue (in grams) by the wet weight of the shell (in grams), and then multiplying the result by 100.

The samples were washed with deionized water to remove surface contaminants and then frozen at −20 °C until further analysis.

2.2. Analytical Methods

Following the internal laboratory digestion procedures (adapted from open-vessel aqua regia digestion with H₂O₂ cleanup), approximately 0.5 g of each sample (wet weight) was digested with concentrated HNO₃ and HCl (1:3 ratio; total of 12 mL) using a hot plate method. The solution was heated at 95–120 °C to near dryness. Then, about 3 mL of 30% H₂O₂ was added dropwise while reheating to ensure complete digestion and oxidize residual organic matter. The digested samples were separately transferred to the labeled flasks and diluted to 25 mL with ultrapure water. The concentrations of Cd, Pb, Hg, Ni, Cr, Cu, As, and Zn were determined using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7900, Agilent Technologies, Tokyo, Japan). The instrument was calibrated with standard solutions of each metal at known concentrations. Quality assurance and quality control were maintained by processing blanks and reference standard materials DORM 3 (National Research Council, Ottawa, ON, Canada), achieving ~95% accuracy. The recovery rates were greater than 85% for all elements. The limits of detection (LOD) for Cs, Cd, Hg, Ni, Pb, Cr, Cu, As, and Zn were determined under optimal analytical conditions and found to be 0.001, 0.003, 0.013, 0.008, 0.001, 0.001, 0.003, 0.012, and 0.038 ppb, respectively.

2.3. Estimates of Target Hazard Quotients (THQ) and Hazard Index (HI)

The target hazard quotient (THQ) is used to assess the potential risk of adverse health effects caused by exposure to a single metal. THQ is calculated using Formula (1) [35,45,46]:

$$THQ={\displaystyle \frac{C\times CR\times EF\times ED}{AET\times ABW\times ORG}}\times {10}^{-3}$$

(1)

where, C, CR, EF, and ED are the concentrations of heavy metals (mg/kg ww), consumption rate (28.6 g/person/day [13]), exposure frequency (365 days/year), and exposure duration (the average life expectancy of Vietnamese people, 70 years [47]), respectively. AET is the average exposure time for non-carcinogens (365 days/year × ED), ABW is the average body weight (60 kg for adults), and ORD is the oral reference dose. The ORDs for Cd, Hg, Ni, Cr, Cu, and Zn are 1, 3, 20, 940, and 300 μg/kg/day, respectively [48], and for As, it is 0.3 μg/kg/day [49]. The value 10⁻³ is a unit conversion factor. This index may overestimate the significance of the hepatopancreas, as while muscle is often eaten separately for large bivalves, like oysters or scallops, cooking methods for small bivalves (cooking them whole in a stew or soup) will probably result in consuming the muscle and hepatopancreas together.

Since the ORG for Pb has not been established, the THQ for Pb is calculated using Formula (2) [35,50]:

$$TH{Q}_{Pb}={\displaystyle \frac{C}{MRL}}$$

(2)

where C and MRL are the concentration of the heavy metal (mg/kg ww) and the maximum regulation limits (1.50 mg/kg ww for bivalve mollusks), respectively.

The total hazard index (HI) was used to assess the cumulative risk of multiple metals found in mussels. The HI is calculated using Formula (3) [35,50]:

$$HI={\displaystyle \sum _{i=1}^{n}TH{Q}_I}$$

(3)

where THQ represents the target hazard quotient of each metal for two organs (muscle tissue and hepatopancreas gland), and n is the total number of heavy metals analyzed in this study (n = 10).

2.4. Statistical Analysis

Statistical analyses were conducted to examine the relationships between the biological characteristics of mussels and heavy metal accumulation, as well as to explore seasonal trends in metal distribution across different tissues (Supplementary Table S3). In addition, one-way ANOVA was performed to assess seasonal (Supplementary Table S4) and monthly (Supplementary Table S5) variations in heavy metal concentrations, after using Levene’s test for homogeneity of variances. Furthermore, the post hoc test (Tukey’s HSD test) was conducted to identify specific pairwise differences between groups. All data, with the exception of the muscle Hg values, passed the Shapiro–Wilk test of normality. Pearson’s correlation analysis and ANOVA were conducted using SPSS 2020 (Statistical Package for the Social Sciences). All statistical analyses were conducted at a 95% confidence level, with p-values less than 0.05 considered statistically significant. To investigate seasonal variability in metal accumulation, principal component analysis (PCA) was applied separately to the metal concentrations in the muscle and hepatopancreas tissues. Prior to PCA, all continuous variables were standardized by z-score transformation—each variable was centered to a mean of 0 and scaled to a standard deviation of 1. This multivariate approach enabled the identification of key metals contributing to the temporal variation and allowed for the grouping of sampling months based on similar accumulation profiles. Monthly variations and distribution patterns of individual metal concentrations in both tissues were analyzed using OriginPro 2023.

3. Results and Discussion

3.1. Characteristics of the Selected Heavy Metal Distributions in Two Organs of Asian Green Mussel

Table 1. Heavy metal concentrations in the muscle tissue (Muscle. T.) and hepatopancreas gland (Hepato. G.) of Asian green mussel (Perna viridis).

Heavy Metals	Organs	Heavy Metal Concentrations (mg/kg Wet Weight)					Guidelines (mg/kg)	Ref.
(mg/kg)		March (n = 10)	April (n = 10)	May (n = 10)	June (n = 10)	July (n = 10)
Cs	Muscle. T.	0.0108	0.0153	0.0174	0.0202	0.0275
	Hepato. G.	0.0525	0.0975	0.0394	0.0962	0.1207
Cd	Muscle. T.	0.18	0.1	0.05	0.08	0.08	1–2	[59,60]
	Hepato. G.	0.26	0.29	0.23	0.21	0.21
Hg	Muscle. T.	0.04	0.04	0.03	0.06	0.30	0.05–1	[60]
	Hepato. G.	0.15	0.14	0.09	0.18	0.32
Ni	Muscle. T.	0.35	0.14	0.14	0.1	0.01	80	[61]
	Hepato. G.	0.45	0.21	0.82	0.55	0.35
Pb	Muscle. T.	0.43	0.06	0.23	0.07	0.12	1–6	[60]
	Hepato. G.	0.61	0.09	1.00	0.41	0.58
Cr	Muscle. T.	0.32	0.98	0.42	0.34	0.22	13	[62]
	Hepato. G.	0.79	1.29	1.08	1.22	0.65
Cu	Muscle. T.	0.81	0.7	1.03	1.64	2.47	30	[63,64]
	Hepato. G.	2.36	2	2.91	2.33	2.29
As	Muscle. T.	1.82	3.53	2.32	3.87	LLD *	1–3.5	[60]
	Hepato. G.	4.07	9.35	4.94	15.9	LLD *
Zn	Muscle. T.	14.0	13.8	11.7	8.96	11.3	100	[63,64]
	Hepato. G.	17.5	17.9	17.3	18.6	15.0

Note: * LLD, Lower Limit of Detection.

shows the levels of heavy metals in the organs of the mussel. According to the data, Zn has the highest concentrations in the majority of months, while Hg has the lowest concentrations (Figure 2). The greater bioavailability of Zn in bivalves may be the reason for its predominance [17,51], and the intensive waterway and port activities in the Hai Phong coastal area contribute to increased Zn levels in the environment. To minimize the health risks associated with exposure to metals in various foods, countries and international organizations have established guideline levels for seafood. The guideline levels for Cd, Hg, Ni, Pb, Cr, Cu, As, and Zn are 1–2, 0.05–1, 80, 1–6, 13, 30, 1–3.5, and 100 mg/kg, respectively (Table 1). The selected heavy metal concentrations in mussels were recorded as significantly lower than the guideline levels, except for As. Urban development, particularly coal mining activities, has been identified as a major source of arsenic contamination in this area [31], while the Red River Delta is also known for its high natural arsenic mobilization in groundwater (up to 3050 µg/L) [52]. It should be noted that marine organisms can accumulate large amounts of As compounds; however, most of the As in bivalves exists in organic forms such as arsenobetaine, arsenoribosides, and arsenocholine, which are less toxic [11,29,53,54,55]. Previous studies have reported that the inorganic fraction of arsenic accounts for only about 13–22% of the total arsenic in oysters and mussels [56,57,58]. This means that, even at the upper bound of 22%, the level of inorganic As in the hepatopancreas during June (when the highest total As concentration was observed) remains within recommended safety limits.

Table 2. Comparison of heavy metal concentrations in Perna viridis (P. viridis) and Mytilus galloprovincialis (M. galloprovincialis) reported from different countries, including the findings of the present study.

Country	Species	Concentrations (mg/kg Wet Weight)									References
		Cs	Cd	Hg	Ni	Pb	Cr	Cu	As	Zn
Turkey	M. galloprovincialis	-	-	-	12.7	3.16	0.56	2.65	3.16	69.06	[65]
Morocco	M. galloprovincialis	-	1.33–25.3	0.02–2.30	-	0.50–34.2	-	4.35–142.2	-	112.6–612.3	[66]
Spain	M. galloprovincialis	-	0.46–1.40	0.11–0.61	-	0.9–3.0	-	5.33–7.2	-	176–316	[67]
Korea	M. galloprovincialis	-	0.63–9.98	0.029–0.627	-	0.44–18.9	-	6.99–58.8	-	107–279	[68]
Indonesia	P. viridis	-	0.95	1.27	-	1.27	-	-	-	-	[69]
Singapore	P. viridis	-	BLD-0.04	-	1.6	1.2	0.4	5.6	4.2	56	[70]
Malaysia	P. viridis	-	1.1	-	-	38	-	34	-	46	[71]
Hong Kong	P. viridis	-	2.5	-	8.9	3.7	5.1	15.1	-	-	[72]
Venezuela	P. viridis	-	0.16	-	0.65	-	0.12	1.7	-	25	[73]
United Kingdom	M. galloprovincialis	-	0.77	-	1.4	3.9	1.6	7.4	14	88	[74]
Spain	Mussels	-	0.3–3.1	-	5.2–23.8	1.0–30.1	3.1–25.8	12–292	-	190–997	[75]
Indian	P. viridis	-	0.65–2.99	-	-	1.20–4.00	-	3.52–23.21	-	20.16–117.17	[76]
Vietnam	P. viridis (Muscle. T. 1)	0.018	0.098	0.094	0.148	0.182	0.456	1.33	2.31	11.95	This study
	P. viridis (Hepato. G. 2)	0.081	0.240	0.176	0.476	0.538	1.01	2.38	6.85	17.26

Notes: ¹ Muscle tissue. ² Hepatopancreas gland.

summarizes the concentrations of heavy metals in the soft tissues of some bivalve species reported from different locations in the world. When compared to the present findings, it is apparent that the levels of heavy metals in both muscle and hepatopancreatic tissues of P. viridis are lower than those reported in several other regions. For instance, the lead (Pb) concentrations observed here were substantially lower than those recorded in Mytilus galloprovincialis from Morocco (34.2 mg/kg), as well as in samples from Korea (18.9 mg/kg) and Malaysia (38 mg/kg). Similarly, cadmium (Cd) concentrations in the present study were recorded as lower than the values reported in Morocco (up to 25.3 mg/kg) and Korea (9.98 mg/kg). These discrepancies may be attributed to differences in local anthropogenic pressures, particularly industrial discharge, urban runoff, and mining activities, which are more intense in regions such as Morocco and Hong Kong. Species-specific bioaccumulation capacities may also account for observed differences in metal concentrations. For example, M. galloprovincialis is known to accumulate certain metals more efficiently than P. viridis, as evidenced by the elevated copper (Cu) concentration of 142.2 mg/kg reported in Moroccan specimens compared to a maximum of 2.378 mg/kg observed in this study. Furthermore, the arsenic (As) concentration is shown to be lower than the exceptionally high concentrations reported in Spain (up to 292 mg/kg) and United Kingdom (14 mg/kg). The variability in heavy metal concentrations across species and geographic regions likely reflects a combination of biological factors (e.g., species physiology, age, body size, sex, and reproductive status) and environmental conditions (e.g., pH, salinity, and site-specific pollution sources). Taken together, these results suggest that the study area is subject to comparatively lower levels of anthropogenic contamination than many industrialized and urbanized coastal regions globally.

The differences in the relationship between pollutants and binding sites and rates of pollutant accumulation and excretion from tissues lead to differences in concentrations between mussel organs [13]. The concentrations of the chosen heavy metals in the hepatopancreas gland is higher in this study than in the mussels’ muscle tissue (statistically significant at the 0.05 level). Because the mussels’ filter-feeding behavior is linked to food digestion, the hepatopancreas gland generally contains higher concentrations of heavy metals [5,12,77,78]. This study highlights the importance of food digestion for heavy metal accumulation in mussels. Figure 2 shows the distribution of heavy metal concentrations in muscle samples in terms of the month.

Biological factors such as the shell length, weight, and condition index (CI) are known to influence the metal accumulation capacity of mussels [39,79]. In this study, statistically significant relationships between heavy metal concentrations in the muscle tissue and hepatopancreas gland of mussels and the shell length, weight, and CI were analyzed in March (spring, dry season) and July (summer, rainy season) (Figure 3).

In March, the concentrations of Hg, Cr, and Cu in muscle tissue were negatively correlated with the shell length, weight, and CI, indicating that heavy metal concentrations decreased as the shell length, weight, and CI increased. Similar trends were also observed in the hepatopancreas gland, where the concentrations of Pb, Cr, Cu, As, and Zn showed negative correlations with the shell length, weight, and condition index (CI). These results imply that variations in steady-state kinetics as mussels mature may be connected to modifications in heavy metal accumulation in muscle tissue. Due to their increased sensitivity and quicker growth, younger mussels typically show higher rates of metal uptake [39,80]. Smaller mussels tend to accumulate more metals due to their higher surface area-to-volume ratio, which enhances metal adsorption. In contrast, larger and older individuals filter less water per unit body weight and have a lower surface area relative to volume, resulting in reduced metal uptake [39,81]. In addition, other metals showed no significant correlation. Notably, Ni exhibited a positive correlation with shell length and weight in both organs, indicating size-dependent accumulation.

In July, Cu concentrations in muscle were negatively correlated with the condition index (CI), whereas positive correlations were observed for Cd and Zn concentrations with shell length, Hg concentrations with the CI, and Ni concentrations with shell length and body weight. In the hepatopancreas gland, Cs, Cd, Pb, Cr, Cu, and Zn concentrations were negatively correlated with shell length, body weight, and CI. These seasonal differences in metal accumulation may be linked to variations in water metal concentrations and the physiological state of mussels over time. Additionally, factors such as sex and reproductive status may also influence metal accumulation in mussels [6,17,37,40]. The sex, sexual maturity, reproductive stages, and seasonal growth cycles might need to be considered when interpreting the results, and pollution itself might shift sex ratios, potentially influencing the biomonitoring response [82]. Mussel age is positively correlated with size, with environmental and genetic factors influencing this relationship [83], and selecting the sampling time and evaluating the sex of the sampled mussels might reduce the uncertainty of the method.

PCA reveals how seasonal variations influence heavy metal accumulation in Perna viridis, specifically in muscle and hepatopancreas. The PCA clusters include data from March, April, May, June, and July, representing seasonal variations. Group ellipses were used to visualize these clusters. In muscle tissue (Figure 4), PCA explained 82.59% of the total variance, with Component 1 (57.69%) grouping Cd, Pb, Ni, As, and Zn, likely originating from industrial and anthropogenic sources, and Component 2 (24.90%) grouping Hg, Cr, and Cu, possibly influenced by seasonal water chemistry changes. The corresponding component matrix is presented in supplementary Table S1. Metal accumulation trends are affected by monsoonal variations, salinity, and sediment resuspension, altering metal bioavailability. Feeding behavior and metabolic activity, which fluctuate with seasonal changes, also influence uptake rates, as some metals preferentially bind to muscle proteins.

In the hepatopancreas (Figure 5), PCA explained 97.55% of the total variance, with Component 1 (42.78%) grouping Cu, Ni, and Pb, Component 2 (38.21%) grouping Cd, Hg, Cr, and Zn, and Component 3 (16.56%) grouping As. PC1 (Cu, Ni, and Pb) in the PCA results most likely represents man-made sources such as industrial discharge, urban runoff, and marine activities; these are frequently connected to metal plating, petroleum refining, and antifouling coatings, with Hai Phong having a river and sea port. The corresponding component matrix is presented in supplementary Table S2. Anthropogenic influences from agricultural runoff, electroplating, and mixed industrial inputs are also suggested by PC2 (Cd, Hg, Cr, and Zn), with a partial contribution from natural geochemical processes, especially for Zn and Cr. As predominates in PC3, which may have a geogenic or environmental origin, perhaps from mineral weathering or background levels, while aquaculture or legacy contamination contributions cannot be completely ruled out. Urban development, particularly coal mining activities, has been identified as a major source of arsenic contamination in the study area [31], while the Red River Delta is also known for its high natural arsenic mobilization in groundwater (up to 3050 µg/L) [52]. The hepatopancreas, being a major detoxification organ, shows seasonal fluctuations in the metal burden due to enzymatic detoxification, metallothionein binding, and lipid content variations. Increased metal accumulation during monsoon seasons may be linked to higher runoff and pollution influx, affecting metal bioavailability and uptake. The PCA findings confirm that the ways heavy metals are absorbed, retained, and detoxified are much influenced by seasonal changes in water quality and biological activities.

3.2. Seasonal and Monthly Variations in Heavy Metal Concentrations

Figure 6 shows the temporal changes in heavy metal concentrations based on the monthly measurements of Cs, Cd, Hg, Ni, Pb, Cr, Cu, As, and Zn in the muscle tissue and hepatopancreas gland of mussels. In general, statistically significant changes (p < 0.05) in heavy metal concentrations in the two organs were observed across seasons and months. The Pb (muscle), Cr (muscle) and As (both organs) concentrations showed a decreasing trend from March (dry season) to July (rainy season), while Cs (both organs) and Cu (muscle) concentrations increased during this period. The dispersion of heavy metal concentrations is linked to significant rainfall and river flow. This may be the cause of the drop in heavy metal concentrations from the dry season to the wet season. It is impossible to rule out the possibility that river flow could also carry significant concentrations of heavy metals, such as Cu and Hg, into the marine environment. In addition, biological factors may also influence seasonal changes in heavy metal concentrations in mussels [37]. Typically, the metal burden of mussels is highest in winter and spring [37,40,84,85], as metal concentrations are closely tied to the mussels’ weight and nutritional status, which may be suboptimal during these times [37]. For example, the highest concentrations of Zn, Cu, and Cd in M. galloprovincialis on the west coast of Algeria were observed in winter, likely due to the spawning period [17]. Similar trends have been noted in other studies [6,40,41,86,87], although some differences exist. For instance, the highest concentrations of Pb were found in summer [17], while Rainbow et al. (2004) reported limited seasonal variation in Zn and Cu concentrations in Mytilus trossulus in Gdansk Bay (Baltic Sea) [88]. Additionally, Scudiero et al. (2014) found that Cd levels did not correlate closely with seasonal changes in M. galloprovincialis on the Campania coast (Italy) [8]. In this study, there was no clear uniformity in the seasonal trends of changes in the concentrations of the studied heavy metals. This may result from a combination of factors directly correlated with mussel weight, such as food availability, reproductive cycles, and metabolism. Furthermore, the bioavailability of metals is influenced by location and abiotic factors, such as temperature and salinity [17,89,90,91].

Monthly variations in metal concentrations revealed significant differences among the analyzed metals, with no consistent trends observed except for similar patterns in the hepatopancreas gland for Cr and As. Previous studies have shown that local industrial activities and urban wastewater contribute to heavy metal pollution in the area [29,30]. These variations may be linked to fluctuations in heavy metal concentrations in seawater and mussel food sources resulting from discharge activities. Interestingly, significant monthly variations in concentrations were also observed for Pb, Hg, Ni, Cr, and As in both organs, and for Cs and Cu in the muscle tissue (statistically significant at the 0.05 level). In contrast, no significant differences were detected for Cd and Zn concentrations in both organs, as well as for Cu and Cs concentrations in the hepatopancreas gland. These findings may reflect fluctuations in background metal levels and seasonal biological changes in mussels. Additionally, this could be a result of variations in the hepatopancreatic gland’s and muscle tissue’s capacities for metal accumulation. In contrast to muscle tissue, the hepatopancreas gland exhibited more consistent variations and higher metal concentrations. These results imply that the hepatopancreas is more susceptible to variations in local discharge activity and pollution levels. This investigation reveals significant evidence in support of using mussels (P. viridis) as biological indicators for heavy metal monitoring in the Hai Phong coastal area, even if more research is required for a more thorough understanding. We consider that assessing the baseline concentrations of heavy metals in seawater and marine sediments is essential in future studies to elucidate the relationship between bioaccumulation and environmental exposure. A more comprehensive study covering all months of the year would allow for a more robust generalization of monthly and seasonal trends. Further, more complex studies may also incorporate the sex, sexual maturity, reproductive stages, and seasonal growth cycles and multiple methods of age determination in addition to size and CI.

3.3. Health Risk Assessment Through Target Hazard Quotients and the Hazard Index

To assess potential health risks from consuming mussels from Hai Phong Province, the target hazard quotient (THQ) and total hazard index (HI) were calculated (

Table 3. Target hazard quotient (THQs) and hazard indices (HIs) of heavy metals for consumers based on the organs—muscle tissue (Muscle. T.) and hepatopancreas gland (Hepato. G.)—of the Asian green mussel.

Metal	Organs	THQ March (n = 10)			THQ July (n = 10)
		Average	Min	Max	Average	Min	Max
Cd	Muscle. T.	0.01	<0.01	0.02	<0.01	<0.01	<0.01
	Hepato. G.	0.01	<0.01	0.03	0.01	<0.01	0.01
Pb	Muscle. T.	0.65	0.50	0.80	0.19	0.11	0.25
	Hepato. G.	0.91	0.65	1.29	0.86	0.45	1.84
Hg	Muscle. T.	<0.01	<0.01	<0.01	<0.01	<0.01	0.01
	Hepato. G.	<0.01	<0.01	<0.01	<0.01	<0.01	0.01
Ni	Muscle. T.	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
	Hepato. G.	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
Cr	Muscle. T.	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
	Hepato. G.	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
Cu	Muscle. T.	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
	Hepato. G.	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
As	Muscle. T.	0.20	0.16	0.24	<0.01	<0.01	<0.01
	Hepato. G.	0.45	0.38	0.55	<0.01	<0.01	<0.01
Zn	Muscle. T.	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
	Hepato. G.	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
HI		2.25	1.70	2.93	1.08	0.58	2.14

). Data from two months, March and July, were used in the analysis to symbolize the dry and wet seasons, respectively. The THQs for the majority of studied heavy metals were less than one in both months, suggesting no expected health hazards. Nonetheless, in both months, the THQs for Pb in the hepatopancreas were greater than one, indicating possible health hazards for consumers who may be exposed to Pb.

The HI was recorded to range from 1.7 to 2.93 with a mean of 2.25 in March (dry season) and from 0.58 to 2.14 with a mean of 1.08 in July (rainy season). These findings indicate that HI values were higher during the dry season compared to the rainy season. Notably, the THQ for Pb contributed the most to the HI values. Pb, a non-essential element, is commonly used in industries such as pigment, alloy, battery, and gasoline production [13]. The major sources of lead (Pb) in the marine environment may be associated with runoff from inland areas, industrial wastewater discharge, and leakage from coal mining activities [31,92]. Hai Phong is a heavily industrialized city, with numerous factories and manufacturing facilities that contribute to the release of toxic heavy metals into the environment [27,29,93]. Notably, this area borders Quang Ninh Province, which possesses the largest coal reserves in the country and hosts extensive mining operations. These factors may significantly elevate Pb levels in the local marine environment. The results of this study highlight the potential health risks from Pb exposure associated with the consumption of Asian green mussels from the study area. Beyond the dangers to human health, the bioaccumulation of heavy metals or trace elements (TE) in the mussel hepatopancreas has important ecological ramifications. Fish, crustaceans, and seabirds may be impacted by sub-lethal toxicity or reproductive disruption from metals that are stored in this important detoxifying organ and can be passed to predators through trophic interactions [94,95]. Furthermore, as sentinel species, mussels’ higher TE levels suggest widespread pollution that could affect ecosystem health and food web dynamics.

4. Conclusions

Various concentrations of the studied heavy metals were observed in the two mussel organs, with Zn showing the highest concentration and Hg having the lowest concentration. Additionally, most of the studied heavy metals showed higher concentrations in the hepatopancreas compared to the muscle tissue, possibly due to a preferential accumulation in digestive organs. This suggests that food digestion plays a crucial role in the accumulation of heavy metals in mussels. The seasonal variation in these correlations may reflect changes in metal concentrations in the seawater and the physiological states of the mussels. The studied heavy metal concentrations varied both seasonally and monthly. The highest levels were recorded in spring, while the lowest levels were found in summer for Pb, Cr and As. Cs and Cu showed the opposite trend. This suggests a lack of uniformity in the seasonal concentration trends across different metals. The authors propose that the observed variations may be due to a combination of biotic factors, abiotic factors, fluctuating heavy metal concentrations in the seawater, and site-specific metal bioavailability. According to our observations, the hepatopancreas gland exhibited higher metal concentrations and stronger fluctuations compared to muscle tissue, making it more sensitive to changes in regional pollution levels and anthropogenic sources. The seasons/monthly fluctuations in heavy metal levels are valuable data for using mussels (Perna viridis) as bioindicators for heavy metal monitoring in the coastal areas of Hai Phong. The target hazard quotient (THQ) and total hazard index (HI) were calculated to assess potential health risks from consuming mussels from Hai Phong Province. Results showed that the THQ for Pb in the hepatopancreas exceeded one in both months, indicating potential health risks, while no significant risks were associated with other studied heavy metals. The HI in March (dry season) was recorded as higher than that in July (rainy season) with the major contribution coming from the THQ of Pb. These findings suggest that the hepatopancreas is more sensitive to changes in pollution levels and anthropogenic activities in the area.