Environmental Exposure and Bioaccumulation of Potentially Toxic Elements in Fishery Resources from the Romanian Black Sea and Implications for Seafood Safety

Abstract

Potentially toxic elements (PTE) are persistent contaminants in coastal systems and may accumulate in marine organisms, with relevance for both environmental monitoring and seafood safety assessment. This study provides an exploratory cross-biota assessment of Cd, Cr, Cu, Ni, and Pb in fishery resources from the Romanian Black Sea in 2024. The dataset included 24 composite samples and 120 analyte-level observations across bivalves, gastropods, pelagic fish, and demersal fish. Tissue concentrations were integrated with regulatory maximum levels, bioconcentration factors (BCF), biota–sediment accumulation factors (BSAF), and adult dietary risk indices, including estimated daily intake (EDI), target hazard quotient (THQ), and total target hazard quotient (TTHQ). Within the limits of this single-year dataset, Cd and Pb concentrations were generally higher in bivalves than in fish and gastropods, whereas Cr showed higher values in several fish samples, particularly pelagic fish. Cd was the main element of concern, with regulatory exceedances occurring mainly in bivalves and fewer exceedances in pelagic fish, while Pb exceedance was isolated. BCF and BSAF supported the relevance of Cd as a priority element but were interpreted only as descriptive tissue–water and tissue–sediment ratios, not as evidence of specific uptake pathways. Low abiotic Cd concentrations may have inflated some ratio-based values, and Cr interpretation remains limited by the absence of Cr speciation and dissolved/particulate partitioning data. The adult dietary risk assessment did not indicate substantial non-carcinogenic concern, as all individual THQ values and cumulative TTHQ values remained below 1. Overall, the findings support continued PTE monitoring in the Romanian Black Sea, using sessile bivalves as indicators of local environmental contamination and including gastropods and representative pelagic and demersal fish species of ecological and fisheries relevance to capture contaminant patterns across benthic and mobile fishery resources. Future monitoring should improve species-level replication, integrate metal partitioning in abiotic matrices, and include additional contaminants of seafood safety relevance, particularly Hg and As.

1. Introduction

Potentially toxic elements (PTE) are among the most persistent contaminants in coastal environments because they are not biodegradable, may remain associated with suspended particles and sediments for long periods, and can be translocated into aquatic food webs through multiple exposure pathways [1,2,3]. In coastal areas affected by river discharge, wastewater inputs, industrial activity, harbor operations, maritime transport, tourism, and substrate disturbances, contaminants may circulate repeatedly among seawater, suspended matter, seabed substrate, and aquatic organisms [4,5,6]. Their environmental significance therefore extends beyond their measured amounts in abiotic matrices and includes the efficiency with which they are accumulated by fishery resources and transferred to seafood consumers [7,8].

The Black Sea is a notably relevant basin for this type of assessment. As a semi-enclosed sea with limited water exchange, strong vertical stratification, large riverine inputs, and marked coastal pressure gradients, it is vulnerable to the retention and redistribution of contaminants [9,10,11]. The Romanian sector is influenced especially by the Danube River in the northern area, while the central and southern coastal zones are more directly affected by urban development, wastewater discharge, ports, shipping, tourism, and other shoreline activities [12,13,14,15,16]. These pressures generate heterogeneous environmental conditions that may influence PTE partitioning, bioavailability, uptake, and retention in marine organisms [17,18,19].

Aquatic organisms can provide integrative information on environmental contamination, although the relationship between external pollutant loads, bioavailability, and tissue concentrations is shaped by multiple biological and environmental factors [20]. Different taxa do not record PTE burdens in the same way. Bivalves are relatively sedentary filter feeders and are widely used as bioindicators of local contamination, particularly where waterborne and particle-associated sources are relevant [4,21,22]. However, bivalves should not be treated as a homogeneous ecological group: epifaunal, semi-infaunal, and infaunal species may differ in their contact with suspended matter, porewater, surface sediments, and resuspended particles, as well as in food selection, filtration activity, and physiological regulation [23,24]. Gastropods [25,26,27], pelagic fish, and demersal fish differ in feeding ecology, mobility, habitat use, and trophic position, and may therefore provide complementary information on PTE occurrence in marine biota [28,29,30].

This distinction is important for seafood safety appraisal. Many studies on PTE in marine organisms focus mainly on tissue contents and compare them with regulatory maximum levels. Such information is essential for seafood safety appraisal, especially when accumulated concentrations exceed established limits. In these cases, dietary risk indices such as estimated daily intake (EDI), target hazard quotient (THQ), and total target hazard quotient (TTHQ) provide additional scenario-based exposure context [31,32], but they do not identify the environmental pathways responsible for the observed tissue burdens [33,34,35,36,37,38,39]. The combined use of biological matrix, bioconcentration factors (BCF), biota–sediment accumulation factors (BSAF), and human health hazard indices can therefore provide a more complete picture of both contamination processes and seafood safety implications [40,41,42,43].

Previous studies from the Romanian Black Sea (RoBS) have provided important information on PTE burden in fishery resources, including long-term assessments of mussels and small pelagic fish [19,44,45,46,47]. These studies have indicated that elements such as Cd, Pb, Cu, Ni, and Cr may vary substantially over time and space, and that some elements may occasionally exceed regulatory maximum levels in edible organisms. However, previous studies have focused on individual taxa or single organism groups [48]. As a result, less is known about how different species compare within the same recent monitoring framework and whether shellfish and fish provide complementary information on contaminant occurrence and tissue accumulation patterns. A cross-biota approach can help describe how PTE burdens differ among organism groups within the same monitoring context. Comparing bivalves (BM), gastropods (G), pelagic fish (PF), and demersal fish (DF) within a common dataset allows a descriptive comparison of broad accumulation patterns and may suggest tissue–environment relationships that require confirmation through more detailed replicated studies. This approach is especially useful for coastal monitoring because it links environmental loads with seafood safety and can support more targeted surveillance of both pollution patterns and consumer concern endpoints.

Mollusks are routinely included in the RoBS monitoring activities carried out by the National Institute for Marine Research and Development “Grigore Antipa” (NIMRD) in the context of the Marine Strategy Framework Directive (MSFD) implementation. These activities usually combine biota sampling with seawater and sediment analyses, including measurements of PTE and other contaminants. The results are used in annual national assessments prepared for the environmental authority and also contribute to Romania’s six-year MSFD reporting through the European Environment Agency (EEA), WISE Marine platform: https://water.europa.eu/marine (accessed on 3 March 2026), mainly in relation to Descriptor 8, Contaminants, and Descriptor 9, Contaminants in seafood [49]. The mollusk component of such monitoring is ecologically relevant but not homogeneous. Mytilus galloprovincialis is a sessile epifaunal suspension-feeding bivalve attached to hard substrates by byssal threads, whereas Anadara kagoshimensis is an infaunal to semi-infaunal suspension-feeding bivalve associated with soft sediments. In contrast, Rapana venosa is a benthic predatory gastropod that feeds mainly on bivalves and may reflect contaminant patterns linked to benthic habitat use and trophic exposure [50,51,52]. These contrasting lifestyles may influence contact with suspended matter, surface sediments, porewater, resuspended particles, and prey, and therefore may contribute to differences in PTE accumulation patterns.

Fish, however, are not yet systematically integrated into the same monitoring framework, and available data remain comparatively scarce and catch-dependent. This gap is relevant for seafood safety assessment because fish represent important resources and may integrate exposure pathways that differ from those reflected by relatively sedentary benthic organisms. The fish species included in this study represent different ecological traits, habitat use, mobility patterns, and feeding strategies [53]. The selected taxa include small pelagic schooling species, such as Sprattus sprattus, Engraulis encrasicolus, Atherina boyeri, and Trachurus mediterraneus; demersal or benthic-associated species, such as Mullus barbatus and Neogobius melanostomus; and migratory clupeids, such as Alosa tanaica (

Table A1. Main ecological and trophic characteristics of fish species analyzed in the present study.

Species	Common Name	Ecological Group	Habitat and Seasonal Occurrence	Main Feeding Traits	Relevance for PTE Exposure Context and Seafood-Safety Assessment
Alosa tanaica (Grimm, 1901)	Azov shad	Migratory clupeid; anadromous/coastal pelagic	Native to the Ponto–Caspian basin, including the Black Sea, Sea of Azov, and adjacent river systems. Adults spend most of the life cycle in marine coastal waters and migrate toward freshwater or slightly brackish areas during the reproductive period, mainly in spring.	Feeds mainly on crustaceans; insect larvae and small fish may also be consumed seasonally.	Relevant as a migratory fish linking coastal marine and estuarine/freshwater-influenced habitats. Its trophic position may provide information on contamination in coastal and estuarine/freshwater-influenced habitats from planktonic and small nektonic prey.
Atherina boyeri (Risso, 1810)	Big-scale sand smelt	Small pelagic/coastal schooling species	Lives in schools near the shore and may enter coastal lakes in spring for feeding and spawning. It is highly tolerant of salinity variation and can occur in marine, brackish, and freshwater environments.	Feeds mainly on small planktonic and benthic organisms, depending on habitat and life stage.	Relevant for assessing contamination in shallow coastal and brackish environments, especially because of its tolerance to variable salinity and frequent occurrence near shore.
Engraulis encrasicolus (Linnaeus, 1758)	European anchovy	Small pelagic schooling species	Common in the Black Sea, forming large schools. It approaches coastal waters mainly from April to September. In autumn, adults migrate offshore and descend to deeper waters, approximately 60–70 m, for overwintering. Spawning occurs from June to September, both offshore and near the coast.	Feeds mainly on planktonic organisms, especially zooplankton, with diet varying according to season and prey availability.	Important commercial small pelagic fish and relevant seafood item. Its plankton-based feeding and seasonal coastal occurrence make it useful for evaluating PTE occurrence in pelagic resources.
Sprattus sprattus (Linnaeus, 1758)	European sprat	Small pelagic schooling species	Lives in large schools and does not enter rivers or coastal lakes. It approaches the shore when water temperature reaches approximately 7–8 °C and remains nearshore until water warms to about 18 °C. Spawning occurs during the cold season, with peak intensity between December and March.	Feeds mainly on zooplankton, especially copepods and cladocerans, but may also consume small larvae, mollusks, and phytoplankton.	High economic and dietary relevance, being among the main small pelagic resources in the Black Sea. Its planktonic feeding and schooling behavior make it suitable for assessing pelagic exposure to bioavailable metals.
Trachurus mediterraneus ponticus (Aleev, 1956)	Mediterranean/Black Sea horse mackerel	Pelagic schooling predator	Pelagic species forming large schools. It appears along the Romanian coast mainly in April–May, when water temperature reaches approximately 13–16 °C. Its arrival is often associated with the presence of anchovy and sand smelt schools near shore.	Feeds mainly on small fish, including anchovy, sand smelt, sprat, and juvenile fish; crustaceans, polychaetes, diatoms, and algae may also occur in the diet.	Relevant from both fishery and PTE exposure-context perspective because it occupies a higher trophic position among the small pelagic species analyzed and may integrate dietary exposure from prey fish.
Mullus barbatus (Linnaeus, 1758)	Red mullet	Demersal/benthic-associated species	Occurs in small schools, mainly over muddy, clayey, or mussel-associated substrates. It appears near the shore in spring when water temperature reaches approximately 7–8 °C, then retreats to deeper bottom habitats as waters warm. It is commonly fished from May to autumn.	Feeds on benthic organisms, including crustaceans, worms, and detritus.	Relevant for assessing sediment-associated and near-bottom exposure pathways, because its feeding ecology and demersal habitat use increase interaction with benthic prey and seabed substrate.
Neogobius melanostomus (Pallas, 1814)	Round goby	Demersal/benthic-associated Ponto–Caspian goby	Ponto–Caspian demersal species adapted to marine and brackish coastal waters. Gobies are common along the Romanian coast, especially in sheltered bays, coastal lakes, and shallow areas with rocky or algae-covered substrates. Spawning generally occurs from late spring to summer, depending on temperature.	Feeds on benthic and nektonic prey, including mollusks, crustaceans, shrimps, crabs, insects, small fish, and other gobies.	Relevant for benthic food-web exposure because of its close association with substrate and consumption of benthic prey, including mollusks and crustaceans. It can help provide information on PTE occurrence in demersal resources.

). These species differ in their seasonal occurrence in coastal waters, trophic position, and interaction with planktonic, benthic, or nektonic prey, which may influence their exposure to PTE and their relevance for environmental monitoring and seafood safety assessment [54,55,56,57].

Aquatic foods are important components of human diets, particularly in coastal regions, where fish and mollusks contribute both to nutrition and to potential dietary exposure to contaminants [58]. Globally, apparent consumption of aquatic animal foods reached approximately 20.7 kg per capita in 2022, confirming the importance of seafood as a dietary exposure pathway [59]. Although apparent fish and seafood consumption in Romania remains below the global average, recent estimates of approximately 8 kg per capita per year [60] may underestimate intake among coastal residents, frequent seafood consumers, local fishing communities, and tourists during the summer season. Seafood therefore remains a relevant, though generally moderate, dietary exposure route for contaminants in specific consumption scenarios. Among the species analyzed here, sprat, anchovy, and horse mackerel are particularly relevant from a fisheries and seafood consumption perspective, as they recorded the highest catches and are frequently requested on local markets [59,60]. Therefore, the inclusion of fish species strengthens mollusk-based monitoring by extending the assessment to mobile fishery resources, with emphasis on measured tissue accumulation and seafood safety relevance rather than on inferring specific uptake pathways. Additional information on the main ecological and trophic characteristics of the fish species included in the present study, with emphasis on traits relevant to PTE exposure and seafood safety is provided in Table A1, together with catch data for the main commercial taxa in Figure A1.

The present study provides an exploratory and descriptive cross-biota assessment of Cd, Cr, Cu, Ni, and Pb in bivalves, gastropods and fish species collected from the RoBS in 2024. The study combines tissue burdens with BCF, BSAF, and dietary risk indices in order to contextualize measured concentrations in relation to local abiotic matrices and seafood safety relevance. Given the limited dataset size, uneven species-level replication, and the presence of several taxa represented by single composite samples, interpretation of ecological pattern is intended as hypothesis-generating rather than mechanistic. Specifically, the study aimed to (i) compare PTE concentrations among bivalves, gastropods, pelagic fish, and demersal fish species at a broad descriptive level; (ii) examine whether BCF and BSAF provide complementary contextual information on tissue–environment relationships; and (iii) evaluate whether measured PTE concentrations in edible tissues indicate potential consumer health risk under the considered exposure scenario. By integrating descriptive tissue–environment indicators with human health risk indices, this work supports a preliminary framework for marine pollution assessment, monitoring prioritization, and seafood safety evaluation in the RoBS.

2. Materials and Methods

2.1. Study Area

The study was conducted along the RoBS, a coastal zone shaped by pronounced gradients in freshwater influence, human pressure, and water masses circulation (Figure 1). This sector occupies the western margin of the basin and includes areas affected to different degrees by river discharge, coastal urbanization, port activity, maritime transport, tourism, and other shoreline uses. Because these pressures are unevenly distributed, the Romanian coast provides a suitable framework for examining spatial differences in contaminant availability and biological interactions [12,13,14]. A defining feature of the area is the influence of the Danube, which affects especially the northern part of the coast through the delivery of freshwater, suspended solids, nutrients, and associated contaminants [15,61,62,63]. These inputs can alter local salinity, turbidity, sediment dynamics, and the partitioning of PTE between dissolved and particulate phases [5,6]. Farther south, the relative importance of direct coastal pressures increases, including wastewater discharge, harbor-related activities, shipping, and urban development [64,65]. From the perspective of PTE behavior, this coastal system is relevant because contaminants may circulate through several interconnected compartments. Depending on local conditions, they may remain in the water column, bind to suspended matter, accumulate in surface sediments, or become available again through substrate disturbance and other environmental processes [5,6,66]. Organisms are therefore exposed through multiple routes, including direct uptake from water, contact with or ingestion of particle-bound material, benthic interaction, and food web passage [4,18,67]. This environmental complexity is notably important for a comparative study of molluscan and fish taxa. Bivalves, gastropods, pelagic fish, and demersal fish differ in feeding ecology, mobility, and habitat use, and may therefore provide complementary information on PTE occurrence in marine biota.

2.2. Sampling Design

Biological specimens were collected in 2024 from monitoring stations distributed along the RoBS coast. To support the comparative design of the study, stations were ordered geographically from north to south and classified into three station types according to their environmental setting and bathymetric position: transitional (T), corresponding to stations located in front of the Danube-influenced sector; coastal (C), corresponding to shallower stations, situated at depths up to 20 m; and marine (M), corresponding to stations located at depths greater than 20 m [49].

The biological material comprised marine organisms belonging to four major categories: bivalves (BM), gastropods (G), pelagic fish (PF), and demersal fish (DF). The BM group included Mytilus galloprovincialis and Anadara kagoshimensis, and the G group was represented by Rapana venosa. The fish taxa component included pelagic species (PF) (Alosa tanaica, Atherina boyeri, Engraulis encrasicolus, Sprattus sprattus, and Trachurus mediterraneus ponticus) and demersal species (DF) (Mullus barbatus and Neogobius melanostomus). These taxa were selected because they are relevant for environmental and seafood safety assessment and differ in feeding ecology, mobility, habitat use, and association with benthic or pelagic compartments.

Mollusk samples were collected during regular monitoring campaigns, together with seawater and sediment from the corresponding stations. Bivalves and gastropods were sampled using a biological dredge, allowing the collection of benthic organisms directly associated with the seabed substrate. This sampling strategy provided paired biological and abiotic data suitable for evaluating relationships between tissue burdens and local environmental conditions.

Fish samples were obtained from fixed coastal fishing points and from research fishing operations. Specimens collected from fishing points were taken from pound nets, representing passive trap-type fishing gear, whereas other samples were obtained using pelagic and demersal trawls, representing active fishing gears. Fish sampling was performed randomly from the available catch, so that the analyzed material would reflect the structure and composition of the catch as accurately as possible. Because fish were not collected as part of the regular abiotic monitoring design, their sampling locations were assigned to the nearest monitoring station or transect to allow integration with the corresponding seawater and sediment data used for transfer factor calculations. Adult specimens were selected for analysis in order to reduce variability related to ontogenetic stage and to ensure that the material was representative of commercial fish commonly entering the local seafood chain.

In total, 24 samples were included in the analytical dataset. For each of them, a composite of 5–10 adult individuals of the same species were selected to provide sufficient material for analysis while minimizing the effect of individual variability [68]. Sample identity, species composition, station code, station type, sampling date, and bottom depth are summarized in

Table 1. Sampling design and biological material analyzed from the RoBS in 2024.

No.	Species	Transect	Station Code	Station Type	Biota Type	Sampling Date	Bottom Depth (m)
1	Mullus barbatus (Linnaeus, 1758)	Portița	P10	T	DF	23/7/2024	10
2	Alosa tanaica (Grimm, 1901)	Portița	P10	T	PF	23/7/2024	10
3	Engraulis encrasicolus (Linnaeus, 1758)	Portița	P10	T	PF	23/7/2024	10
4	Sprattus sprattus (Linnaeus, 1758)	Portița	P10	T	PF	23/7/2024	10
5	Sprattus sprattus (Linnaeus, 1758)	Portița	P20	T	PF	22/7/2024	20
6	Trachurus mediterraneus ponticus (Aleev, 1956)	Gura Buhaz	GB10	C	PF	8/8/2024	10
7	Trachurus mediterraneus ponticus (Aleev, 1956)	Gura Buhaz	GB10	C	PF	27/9/2024	10
8	Mytilus galloprovincialis (Lamarck, 1819)	Gura Buhaz	GB20	C	BM	1/6/2024	20
9	Rapana venosa (Valenciennes, 1846)	Gura Buhaz	GB20	C	G	1/6/2024	20
10	Rapana venosa (Valenciennes, 1846)	Gura Buhaz	GB20	C	G	28/6/2024	20
11	Mullus barbatus (Linnaeus, 1758)	Cazino Mamaia	CM10	C	DF	10/10/2024	10
12	Neogobius melanostomus (Pallas, 1814)	Cazino Mamaia	CM10	C	DF	12/7/2024	10
13	Atherina boyeri (Risso, 1810)	Costinești	COS10	C	PF	28/6/2024	10
14	Engraulis encrasicolus (Linnaeus, 1758)	Costinești	COS10	C	PF	7/8/2024	10
15	Mytilus galloprovincialis (Lamarck, 1819)	Mangalia	MAN10	C	BM	25/7/2024	10
16	Rapana venosa (Valenciennes, 1846)	Mangalia	MAN20	C	G	1/6/2024	20
17	Anadara kagoshimensis (Tokunaga, 1906)	Mangalia	MAN20	C	BM	1/6/2024	20
18	Anadara kagoshimensis (Tokunaga, 1906)	Portița	P30	M	BM	1/6/2024	30
19	Mytilus galloprovincialis (Lamarck, 1819)	Portița	P30	M	BM	24/7/2024	30
20	Rapana venosa (Valenciennes, 1846)	Portița	P30	M	G	1/6/2024	30
21	Anadara kagoshimensis (Tokunaga, 1906)	Cazino Mamaia	CM30	M	BM	1/6/2024	30
22	Rapana venosa (Valenciennes, 1846)	Cazino Mamaia	CM30	M	G	1/6/2024	30
23	Anadara kagoshimensis (Tokunaga, 1906)	Costinești	COS30	M	BM	1/6/2024	30
24	Rapana venosa (Valenciennes, 1846)	Costinești	COS30	M	G	1/6/2024	30

Note: T, transitional stations located in front of the Danube-influenced sector; C, coastal stations up to 20 m depth; M, marine stations deeper than 20 m; BM, bivalves; G, gastropods; PF, pelagic fish; DF, demersal fish.

.

2.3. Sample Processing and Preparation

After collection, all biological material was placed in clean containers, kept under frozen conditions, and transported to the laboratory for preparation and analysis. Once in the laboratory, each composite sample was processed as pooled biological material. Each composite sample represented one biological analytical unit, corresponding to a single species–station combination. Although each composite included 5–10 adult individuals, individuals within a composite were pooled before analysis and were not treated as independent biological replicates. Therefore, the effective biological sample size of the study was n = 24 composite samples. Consequently, individual-level variability was not evaluated. For shellfish, external debris and adhering material were first removed by careful rinsing. The shells were then opened and discarded, and the entire soft body was retained for analysis. For fish specimens, the analytical material consisted of dorsal muscle, which was dissected using clean instruments and separated from non-edible parts prior to further preparation. Prepared biological matrices were stored frozen and later were lyophilized to remove moisture. The dried material was then homogenized thoroughly prior to digestion and metal analysis [68].

To support transfer factor calculations, abiotic data were obtained from the corresponding monitoring stations [69]. Surface seawater was collected from the 0–1 m layer using Niskin bottles and transferred into acid-cleaned polyethylene containers; approximately 1 L of seawater was retained per station for PTE analysis. Sediments were collected with a Van Veen grab, and the upper 0–5 cm layer was subsampled to represent recently deposited surface material; approximately 250 g of wet sediment was retained per station. For each station, one representative seawater sample and one representative surface sediment sample were used as the corresponding abiotic matrices. Seawater samples were acidified with HNO₃ (Suprapur, Merck KGaA, Darmstadt, Germany), whereas sediment samples were lyophilized, homogenized, sieved through a 2 mm mesh to remove the coarse fraction, and digested with HNO₃ (Suprapur, Merck KGaA, Darmstadt, Germany) prior to PTE analysis [65,70,71,72]. When the corresponding water or sediment measurement was below the analytical limit of detection, the respective factors were not calculated.

2.4. PTE Determination

Cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), and lead (Pb) were determined in homogenized material after acid digestion. Approximately 0.5 g of lyophilized material was digested with 10 mL HNO₃ (Suprapur, Merck KGaA, Darmstadt, Germany) in sealed Teflon vessels at 120 °C. After mineralization, the digests were quantitatively transferred and diluted to 100 mL with deionized water (Tracepur, Merck KGaA, Darmstadt, Germany). Element concentrations were measured by graphite furnace atomic absorption spectrometry (GF AAS) using a high-resolution continuum-source instrument, HR-CS ContrAA 800 G, Analytik Jena GmbH + Co. KG, Jena, Germany. Final concentrations in biota were expressed as µg/g wet weight after conversion from dry-weight values using the sample-specific wet-to-dry mass ratio determined for each composite sample [70]. Analytical quality assurance and quality control were applied throughout the procedure. External calibration was performed using freshly prepared working standards obtained by serial dilution of certified multi-element standard solution (Certipur, Merck KGaA, Darmstadt, Germany). The calibration ranges were 0–10 µg/L for Cd, 0–25 µg/L for Pb, and 0–50 µg/L for Cu, Ni, and Cr. Calibration curves included a blank and at least three concentration levels and were accepted when the correlation coefficient was ≥0.995. Procedural blanks were processed together with the samples to check possible contamination during digestion and instrumental analysis. Each final analytical solution was measured through three instrumental readings, and the mean value was used for data interpretation. These repeated readings were treated as analytical/technical replicates only and were not considered independent observations. Replicate readings were accepted when the relative standard deviation (RSD) was ≤10%; otherwise, the solution was remeasured [70].

Instrumental stability was checked during the analytical sequence using a continuing calibration verification standard prepared at a concentration close to the middle of the calibration range. The sequence was accepted when the measured value of this verification standard remained within ±10% of the expected concentration. Instrumental limits of detection (LOD) and limits of quantification/reporting limits (LOQ/RL) were established during routine method/instrument validation procedure. LOD and LOQ were derived from repeated blank measurements using the standard-deviation approach, with LOD estimated as 3σ and LOQ as 10σ of the blank response after conversion through the calibration curve. The validated LODs were 0.001 µg/L for Cd and Pb and 0.01 µg/L for Cu, Ni, and Cr. Values below the LOQ were retained for descriptive screening but were not used as denominator values in transfer factor calculations, because very low abiotic concentrations may artificially inflate estimates. Calibration and analytical performance criteria are provided in Table S1.

2.5. Bioconcentration Factor (BCF)

The BCF was used as a descriptive ratio between tissue concentrations and corresponding seawater concentrations, providing a simplified indicator of tissue–water association [73,74]. For each analyte, BCF was calculated as the ratio between its tissular concentration and its corresponding concentration in seawater:

BCF = C_biota/C_water

(1)

where C_biota is the metal concentration in tissue expressed on a wet-weight basis (µg/kg ww), and C_water is the concentration of the same metal in seawater (µg/L).

This metric was used to support contextual interpretation of relative tissue–water relationships for each element and to complement the sediment-normalized BSAF perspective.

2.6. Biota–Sediment Accumulation Factor (BSAF)

The BSAF was used to evaluate the relationship between metal loads in tissues and those measured in the corresponding seabed substrate, thereby providing a descriptive tissue–sediment ratio [69]. For each analyte, BSAF was calculated as the ratio between its tissular concentration and its concentration in sediment:

BSAF = C_biota/C_sediment

(2)

where C_biota is the metal concentration in tissue expressed on a dry-weight basis (µg/g dw), and C_sediment is the concentration of the same metal in sediment (µg/g dw).

This metric was included to complement the water-based perspective provided by BCF and to support contextual interpretation of tissue concentrations relative to seabed substrate concentrations. In the present study, BSAF was used as a comparative indicator of relative tissue–sediment relationships across PTE and broad biota categories, rather than as evidence of direct sediment uptake.

2.7. Human Health Risk Assessment

Potential effects associated with seafood consumption were evaluated using three standard indices: estimated daily intake (EDI), target hazard quotient (THQ), and total target hazard quotient (TTHQ) [32,37,39,75].

Estimated daily intake was calculated as:

EDI = (C_metal × DIR)/BW

(3)

where C_metal is the concentration of the metal in tissue (mg/kg wet weight), DIR is the daily ingestion rate of seafood (kg/day), calculated from the annual per capita fish and shellfish consumption data for Romania reported by FAOSTAT (the food ingestion rate, FIR, kg/capita/year) [76], and BW is the average adult body weight (70 kg). EDI was used as the basic measure of dietary hazard for each element [75,77].

Non-carcinogenic risk was evaluated through the target hazard quotient:

THQ = EDI/RfD

(4)

where RfD is the chronic oral reference dose (mg/kg/day). In the present study, RfD values of 0.0001 for Cd, 0.04 for Cu, 0.02 for Ni, 0.003 mg/kg/day for Cr and 0.004 mg/kg/day for Pb were applied [78,79]. Although a formal USEPA oral RfD is not provided for Pb, the value of 0.004 mg/kg/day has been widely used in previous THQ-based dietary exposure assessments and was therefore adopted here for consistency with comparable seafood risk studies [80,81,82].

To estimate the cumulative non-carcinogenic risk associated with simultaneous exposure to multiple metals, individual THQ estimates were summed:

TTHQ = ΣTHQ_i

(5)

This cumulative index was used to integrate the combined contribution of Cd, Cr, Cu, Ni, and Pb to overall non-carcinogenic risk [39,83,84,85,86].

2.8. Data Structure and Grouping Strategy

For interpretation and statistical analysis, specimens were grouped into four broad biota categories: bivalves (BM), gastropods (G), pelagic fish (PF), and demersal fish (DF). This grouping was used as a practical descriptive and statistical framework because species-level replication was limited and uneven, with some taxa represented by only one composite sample. Therefore, these categories should not be interpreted as homogeneous ecological units. Species included within the same broad group may differ in mobility, feeding ecology, habitat use, physiology, uptake efficiency, and detoxification capacity. Accordingly, group-level comparisons were used to identify broad exploratory patterns in the dataset, while species-level observations were retained for descriptive interpretation only. No population level or species-wide inference was intended. Station type was also retained as a secondary grouping variable. Each sample was assigned to one of three station classes: transitional (T), coastal (C), or marine (M), allowing additional examination of broad spatial structure in the dataset. Derived datasets used for BCF, BSAF, and health risk calculations were constructed from the same framework.

2.9. Statistical Analysis

The distribution of each variable was evaluated using the Shapiro–Wilk test. Because tissue concentrations, transfer factors, and most abiotic variables did not meet normality assumptions (Tables S2, S5 and S8), nonparametric methods were used throughout the study. Differences among groups were tested using the Kruskal–Wallis test. This approach was applied primarily to compare major biota categories and, where relevant, station types and species. The Kruskal–Wallis test statistic was reported as H, together with the associated p-value, which indicates the statistical significance of overall differences among groups. The magnitude of group effects was expressed as epsilon-squared (ε²), which provides an estimate of the proportion of variability in ranked data attributable to group membership. When the overall Kruskal–Wallis test was significant at p < 0.05, pairwise comparisons were performed using Dunn’s post hoc test with Holm correction for multiple testing. Species-level analyses were treated as exploratory because replication was uneven and limited for several taxa. Species represented by a single sample were excluded from inferential testing but were retained in descriptive summaries and graphical interpretation. Because several independent Kruskal–Wallis tests were performed across elements, station types, transfer factors, dietary risk indices, and exploratory species-level comparisons, the possibility of inflated type I error at the manuscript level should be acknowledged. Holm correction was applied to Dunn’s post hoc pairwise comparisons following significant omnibus tests, but no global correction was applied across all independent Kruskal–Wallis tests. Therefore, statistically significant results were interpreted cautiously, with emphasis on consistency across endpoints, effect sizes, biological plausibility, and descriptive patterns. To further explore the multivariate structure of the dataset, principal component analysis (PCA) was applied as an ordination tool. In addition, a shade plot based on square-root-transformed data and boxplots were used to visualize cross-biota patterns and within-group heterogeneity. Data preparation was carried out in Microsoft Excel 365; univariate and nonparametric analyses were performed in TIBCO Statistica 14.0.1.25 [87]; multivariate analyses, shade plot and boxplots visualization were performed in PRIMER v7 [88]; and the station map was prepared in Ocean Data View (ODV), version 5.1.7 [89].

3. Results

3.1. PTE Concentrations and Environmental Transfer Factors in Marine Biota

3.1.1. Tissue Burdens in Seafood Taxa

To highlight species-level variability, tissue concentrations were examined descriptively by species (Figure 2). This visualization showed that PTE concentrations were not uniform within broad biota categories, reflecting differences in feeding ecology, habitat use, mobility, uptake efficiency, and physiological regulation. Cd and Pb signals were generally higher in bivalve species, particularly Anadara kagoshimensis and Mytilus galloprovincialis. In contrast, Cr was elevated in fish, with the highest individual value recorded in Trachurus mediterraneus ponticus and relatively consistent values also observed in other fish species. Cu showed a broader species-level range, with elevated values in Rapana venosa and selected fish samples, particularly E. encrasicolus, S. sprattus, and T. m. ponticus. Ni variability was influenced mainly by a limited number of elevated observations, especially in E. encrasicolus. Because several taxa were represented by few composite samples, species-level patterns were interpreted descriptively, while formal comparisons focused on broader biota-type groups. These species-level observations illustrate why differences in feeding ecology, habitat use, mobility, and physiological regulation must be considered when contextualizing PTE patterns. For the fish component, relevant ecological and trophic traits are summarized in Table A1.

Because all analytes departed from normality according to the Shapiro–Wilk test (Table S2), nonparametric methods were used for inferential analyses throughout. Within the limits of this exploratory dataset, broad biota category provided the clearest descriptive structure for the observed PTE patterns. Because all analytes departed from normality according to the Shapiro–Wilk test (Table S2), nonparametric methods were used for inferential analyses throughout. Within the limits of this exploratory dataset, broad biota category provided the clearest descriptive structure for the observed PTE patterns.

Significant overall effects were detected for Cd (H = 15.638, p = 0.0013, ε² = 0.632), Cr (H = 9.490, p = 0.0234, ε² = 0.324), and Pb (H = 9.898, p = 0.0194, ε² = 0.345). Cu did not show a statistically significant difference among broad biota categories (H = 7.780, p = 0.0508, ε² = 0.239), and no pairwise post hoc comparisons were performed. Also Ni did not exhibit a statistically significant difference among groups. For Cd, the highest median was observed in BM (1.046 µg/g ww), and Dunn–Holm post hoc testing indicated that BM loads were more elevated than those in PF (0.048 µg/g ww; pHolm = 0.0031) and DF (0.043 µg/g ww; pHolm = 0.0090). For Cr, median values were higher in the fish categories, although only the PF versus G contrast remained significant after multiple-comparison correction (0.439 vs. 0.006 µg/g ww; pHolm = 0.0220). For Pb, the strongest supported difference was between BM and G, with elevated Pb in BM (0.232 vs. 0.002 µg/g ww; pHolm = 0.0103) (

Table 2. Median metal concentrations (µg/g ww) by biota type, Kruskal–Wallis results, effect sizes, and significant Dunn–Holm post hoc comparisons.

Metal	BM	G	PF	DF	H	p	ε2	Significant Dunn–Holm Pairs
Cd	1.046 [0.655]	0.171 [0.252]	0.048 [0.028]	0.043 [0.014]	15.638	0.0013	0.632	BM vs. PF (BM higher) (pHolm = 0.0031); BM vs. DF (BM higher) (pHolm = 0.0090)
Cr	0.205 [0.269]	0.006 [0.026]	0.439 [0.030]	0.432 [0.096]	9.490	0.0234	0.324	PF vs. G (PF higher) (pHolm = 0.0220)
Cu	1.619 [0.638]	3.645 [0.666]	1.394 [2.317]	2.276 [1.693]	7.780	0.0508	0.239	Not performed (overall p > 0.05)
Ni	0.631 [0.332]	0.061 [0.139]	0.527 [0.790]	0.248 [0.628]	6.371	0.0949	0.169	Not performed (overall p > 0.05)
Pb	0.232 [0.220]	0.001 [0.002]	0.052 [0.112]	0.039 [0.043]	9.898	0.0194	0.345	BM vs. G (BM higher) (pHolm = 0.0103)

BM, bivalves; G, gastropods; PF, pelagic fish; DF, demersal fish. Group values are presented as median [IQR], where IQR = interquartile range (P25th–P75th). H, Kruskal–Wallis test statistic; p, significance level of the Kruskal–Wallis test; ε², epsilon-squared effect size. Pairwise Dunn–Holm comparisons were conducted only for analytes with significant overall Kruskal–Wallis results.

; Figure 3; Table S3).

Species-level analyses were treated as exploratory because of uneven group sizes, and taxa represented by a single sample were excluded from inferential testing. Significant overall species effects were detected for Cd (H = 14.310, p = 0.0264, ε² = 0.594) and Pb (H = 12.900, p = 0.0447, ε² = 0.493), whereas Cr, Cu, and Ni did not present clear species differences (Table S4). Within the replicated subset, the highest median Cd amounts were recorded for A. kagoshimensis and M. galloprovincialis, while greater median Pb concentrations were recorded for A. kagoshimensis and S. sprattus.

To further visualize sample-level multivariate variation in PTE burdens, PCA was applied as an exploratory ordination tool. Given the limited number of composite samples relative to the number of variables and the uneven species-level replication, the ordination was used only as a descriptive visualization. Several bivalve samples were positioned in the direction of the Cd and Pb vectors, whereas several fish samples, particularly PF, extended toward the Ni- and Cr-related direction. Gastropod samples showed comparatively lower Cd and Pb values, while DF occupied an intermediate ordination space. Overall, the PCA should not be interpreted as evidence of stable ecological clustering, robust separation among groups, or homogeneous group-level behavior. (Figure 4).

The shade plot of square-root transformed PTE concentrations provided an additional exploratory visualization of sample-level variation. The heatmap broadly illustrated higher Cd/Pb values in several bivalve samples and higher Cr/Ni values in some fish samples, while also showing variability within broad biota categories. Therefore, the shade plot was used as a descriptive complement to the univariate analyses and PCA, not as evidence of distinct ecological clusters (Figure S1).

When interpreted against the maximum levels established by Commission Regulation (EU) 2023/915 [90], Cd exceedances were concentrated mainly in BM. Four of seven BM samples exceeded the Cd maximum level of 1.0 mg/kg ww, including three specimens of A. kagoshimensis and one of M. galloprovincialis. Among fish species, exceedances were limited to three pelagic species, namely A. tanaica, S. sprattus, and T. m. ponticus, while no DF exceeded the applicable Cd limit of 0.05 mg/kg ww. Notably, E. encrasicolus remained below its species-specific Cd maximum level of 0.25 mg/kg ww. For Pb, exceedances were much less frequent: no BM sample exceeded the maximum level of 1.50 mg/kg ww, and only one PF, S. sprattus from Portița, exceeded the maximum level of 0.30 mg/kg ww. Rapana venosa remained below these BM-based reference thresholds for both toxic metals; however, because the regulation does not explicitly define corresponding Cd and Pb maximum levels for marine gastropods, this comparison should be regarded as contextual rather than as a formal compliance examination. These regulatory comparisons provide the primary benchmark for seafood compliance. The complementary exposure-based dietary-risk assessment is presented separately in Section 3.2.

3.1.2. Bioconcentration Factors (BCF)

To contextualize tissue concentrations in relation to measured seawater concentrations, BCF values were calculated as descriptive tissue–water ratios. BCF were not calculated in a few cases where water measurements of Cd and Pb were below the analytical limit of detection. Distributions of BCF and seawater values were non-normal (Table S5). Seawater PTE concentrations showed station-level variability (

Table 3. Seawater PTE concentrations at the monitoring stations used for transfer factor calculations.

Transect	Station Code	Bottom Depth [m]	Cu (µg/L)	Cd (µg/L)	Pb (µg/L)	Ni (µg/L)	Cr (µg/L)
Portita	P10	10	13.159	<LOQ *	2.135	2.873	2.491
Portita	P20	20	12.498	<LOQ *	2.084	7.446	2.303
Portita	P30	30	28.674	0.010	4.491	30.235	2.844
Gura Buhaz	GB10	10	10.305	<LOQ *	<LOD *	5.202	0.970
Gura Buhaz	GB20	20	16.472	0.023	6.322	5.903	0.893
Cazino Mamaia	CM10	10	46.908	0.010	0.942	27.658	37.678
Cazino Mamaia	CM30	30	5.335	0.241	25.067	1.907	7.955
Costinesti	COS10	10	9.883	<LOQ *	1.573	16.204	0.979
Costinesti	COS30	30	27.371	<LOQ *	<LOD *	19.235	16.506
Mangalia	MAN10	10	9.059	<LOQ *	<LOD *	1.055	1.822
Mangalia	MAN20	20	7.479	<LOD *	<LOD *	3.086	1.138

* For Cd and Pb, LOD = 0.001; LOQ = 0.005.

). Cu was detected at all stations, with the highest value recorded at Cazino Mamaia CM10, while Ni and Cr also showed elevated values at selected stations, particularly Cazino Mamaia CM10 and Costinești COS30. Cd was generally low in seawater, with several measurements below the quantification limit (7 stations) and the highest value recorded at Cazino Mamaia CM30. Pb was also variable, with few values below the detection limit (4 stations) and the highest concentration observed at Cazino Mamaia CM30 (Table 3). No analyte presented a significant station-type effect (T, C, M) in seawater. Kruskal–Wallis tests were non-significant for Cd (H = 0.167, p = 0.6831), Pb (H = 2.464, p = 0.2917), Cu (H = 0.091, p = 0.9556), Ni (H = 0.240, p = 0.8868), and Cr (H = 1.649, p = 0.4384) (Table S6).

BCF values were used as relative indicators of the relationship between tissue concentrations and measured seawater concentrations, rather than as direct evidence of uptake exclusively from seawater. Overall, Cd showed the highest apparent BCF values, particularly in bivalves. However, this pattern should be interpreted with caution because Cd concentrations in seawater were very low and, in several cases, close to the quantification limit. Consequently, elevated Cd BCF values may partly result from the low denominator used in the calculation, rather than indicating direct uptake from seawater alone. At the same time, the pattern is consistent with the capacity of bivalves to retain Cd through combined dissolved and particle-associated exposure pathways. Cu, Ni, Cr, and Pb generally showed lower or more variable BCF patterns.

Significant overall biota-type differences were detected only for Cr (H = 15.638, p = 0.0013, ε² = 0.6319), whereas Cu, Cd, Ni, and Pb did not differ significantly among groups. For Cr, PF reached the highest median BCF (234.97), followed by BM (55.06), DF (13.96), and G (5.60). Dunn–Holm testing indicated that only the G versus PF contrast remained significant after correction, with elevated Cr BCF estimates in PF (pHolm = 0.0009) (

Table 4. Median BCF values by biota type, Kruskal–Wallis results, and significant Dunn–Holm comparisons.

Metal	BM	G	PF	DF	H	p	ε2	Significant Dunn–Holm Pairs
Cu	109.56 [61.52–143.94]	234.27 [196.45–341.01]	121.00 [81.24–248.65]	48.53 [42.02–216.24]	5.352	0.1478	0.118	None
Cd *	45,166.09 [11,437.89–92,473.50]	2056.52 [1449.15–11,396.09]	—	3031.25 [2403.12–3659.38]	4.555	0.1026	0.365	None
Pb	30.02 [6.94–53.63]	0.37 [0.03–1.44]	44.92 [17.40–77.87]	40.95 [24.56–42.54]	7.071	0.0697	0.313	None
Ni	33.75 [23.68–243.48]	4.51 [1.01–21.61]	78.97 [18.21–132.33]	8.98 [8.54–261.97]	6.408	0.0934	0.170	None
Cr	55.06 [36.32–145.74]	5.60 [0.19–12.32]	234.97 [188.28–443.14]	13.96 [11.41–93.64]	15.638	0.001345	0.632	G vs. PF (PF higher), pHolm = 0.000879

BM, bivalves; G, gastropods; PF, pelagic fish; DF, demersal fish. Group values are presented as median [(P25th–P75th)]. H, Kruskal–Wallis test statistic; p, significance level of the Kruskal–Wallis test; ε², epsilon-squared effect size. Pairwise Dunn–Holm comparisons were conducted only for analytes with significant overall Kruskal–Wallis results. * PF had no analyzable Cd BCF values because corresponding seawater concentrations were below LOD.

; Figure 5).

Station type was less informative overall for BCF, although Pb displayed a significant secondary station-type effect (H = 8.346, p = 0.0154, ε² = 0.4533). Descriptively, Pb BCF increased from coastal (C) stations to marine (M) stations and was maximum in transitional (T) stations, suggesting a possible station-type pattern, although this should be interpreted cautiously because of the limited number of observations and the ratio-based nature of the metric (Table S7).

Species-level BCF analyses were treated as exploratory because taxa represented by a single sample were excluded from inferential testing. Only Cr presented an overall species effect (H = 15.494, p = 0.01675, ε² = 0.6781). Considered together with the biota-type BCF medians, Cr-related BCF estimates were highest in PF, namely E. encrasicolus, S. sprattus, and T. m. ponticus, with secondary elevation in the BM subset (A. kagoshimensis and M. galloprovincialis). Rapana venosa was characterized by the lowest Cr-related BCF observations, while M. barbatus appeared intermediate.

To further contextualize the observed BCF trends, the data were interpreted against the REACH/ECHA screening thresholds for bioaccumulative potential [73,74]. Within this framework, Cu and Pb remained in the non-bioaccumulative range (BCF < 1000), whereas Cd showed the highest apparent BCF values in the dataset. Cd exceeded the bioaccumulative threshold in non-pelagic biota groups, placing BM in the very bioaccumulative range (BCF > 5000) and G and DF in the bioaccumulative range (BCF > 1000). This was aligned with the broader Cd profile in biological specimens, especially in A. kagoshimensis and M. galloprovincialis.

By contrast, Ni and Cr were generally below the bioaccumulative threshold, although elevated Ni-related BCF patterns were associated mainly with PF and BM, and Cr-related BCF patterns were most evident in PF, namely E. encrasicolus, S. sprattus, and T. m. ponticus, with R. venosa remaining lowest. Across the dataset, these threshold-based comparisons indicate that Cd was the only element consistently exceeding the applied BCF screening thresholds in the analyzable samples (Figure 6).

3.1.3. Biota–Sediment Accumulation Factors (BSAF)

To complement the water-based transfer perspective, tissue burdens were also interpreted relative to sediment amounts using BSAF. Few Cd and Ni BSAF observations were excluded because corresponding measurements were below LOD. Shapiro–Wilk testing showed that all BSAF variables were non-normal, while sediment distributions were non-normal for Cd, Ni, and Cr, with Cu and Pb closer to normality (Table S8); however, nonparametric methods were retained consistently throughout because of the small and uneven group sizes.

Sediment PTE concentrations showed station-level variability (

Table 5. Sediment PTE concentrations at the stations used for transfer factor calculations.

Transect	Station Code	Bottom Depth [m]	Cu (µg/g dw)	Cd (µg/g dw)	Pb (µg/g dw)	Ni (µg/g dw)	Cr (µg/g dw)
Portita	P10	10	4.488	0.018	3.674	10.662	16.699
Portita	P20	20	18.044	0.088	14.027	23.594	27.815
Portita	P30	30	29.587	0.093	22.463	46.232	42.317
Gura Buhaz	GB10	10	12.736	0.033	4.934	10.226	14.966
Gura Buhaz	GB20	20	16.490	0.051	12.024	16.454	20.973
Cazino Mamaia	CM10	10	8.310	0.214	38.673	11.544	18.030
Cazino Mamaia	CM30	30	26.996	0.100	22.296	42.967	163.854
Costinesti	COS10	10	6.738	0.021	5.273	<LOD **	13.682
Costinesti	COS30	30	4.066	<LOD *	2.321	3.750	11.622
Mangalia	MAN10	10	6.488	0.015	4.397	10.150	13.773
Mangalia	MAN20	20	7.383	0.077	14.863	12.448	19.758

* Cd LOD = 0.01 µg/g; ** Ni LOD = 0.06 µg/g.

). Cr generally presented the highest concentrations, with a pronounced maximum at Cazino Mamaia CM30, while Pb and Cd showed their highest values at Cazino Mamaia CM10. Cu and Ni were also variable among stations, with higher values recorded mainly at Portița P30 and Cazino Mamaia CM30. Cd remained low across most sediments, with one value below the detection limit, while Ni was below the detection limit at Costinești COS10.

Despite these descriptive differences, no significant station-type effects were detected for sediment. Kruskal–Wallis tests were non-significant for Cd (H = 2.473, p = 0.2904, ε² = 0.068), Pb (H = 2.844, p = 0.2412, ε² = 0.106), Cu (H = 4.558, p = 0.1024, ε² = 0.320), Ni (H = 5.018, p = 0.0813, ε² = 0.431), and Cr (H = 5.435, p = 0.0660, ε² = 0.429) (Table S9).

BSAF values were used as relative indicators of the relationship between tissue and sediment concentrations, rather than as direct evidence of uptake from sediment alone. Overall, Cd showed the highest apparent BSAF values, but this pattern should be interpreted cautiously because Cd concentrations in sediments were low. Consequently, elevated Cd BSAF values may partly reflect the low denominator used in the calculation, rather than indicating direct sediment-derived accumulation. Nevertheless, the higher Cd BSAF values observed mainly in bivalves remain ecologically relevant, as they suggest that Cd can be efficiently retained in biological tissues even when sediment concentrations are low.

A similar descriptive pattern was observed when samples were grouped by broad biota category. Significant overall differences were detected for Cd (H = 13.641, p = 0.0034, ε² = 0.5912), Cr (H = 13.170, p = 0.0043, ε² = 0.5085), and Pb (H = 9.562, p = 0.0227, ε² = 0.3281), whereas Cu and Ni did not differ among groups (

Table 6. Median BSAF values by biota type, Kruskal–Wallis results, and significant Dunn–Holm contrasts.

Metal	BM	G	PF	DF	H	p	ε2	Significant Dunn–Holm Pairs
Cu	0.5309 [0.3044–0.8512]	1.1788 [0.9812–1.6661]	0.6857 [0.5621–1.3706]	1.0957 [0.9489–2.7994]	5.446	0.1419	0.122	None
Cd	66.7463 [45.5604–94.5340]	9.1984 [5.4902–10.2474]	8.0252 [7.0689–9.7063]	0.8014 [0.5666–5.3734]	13.641	0.0034	0.591	BM vs. DF (BM higher), pHolm = 0.0095; BM vs. PF (BM higher), pHolm = 0.0138
Pb	0.0516 [0.0405–0.0818]	0.000257 [0.000083–0.000642]	0.0545 [0.00927–0.1036]	0.00399 [0.00239–0.0533]	9.562	0.0227	0.328	BM vs. G (BM higher), pHolm = 0.0252
Ni	0.1104 [0.0560–0.3405]	0.00886 [0.00127–0.0156]	0.1357 [0.0583–0.3021]	0.0860 [0.0818–0.3205]	5.950	0.1141	0.164	None
Cr	0.0185 [0.0126–0.1114]	0.00140 [0.000059–0.00344]	0.1145 [0.0948–0.1268]	0.1034 [0.0887–0.1100]	13.170	0.0043	0.508	G vs. PF (PF higher), pHolm = 0.0024

BM, bivalves; G, gastropods; PF, pelagic fish; DF, demersal fish. Group values are presented as median [(P25th–P75th)]. H, Kruskal–Wallis test statistic; p, significance level of the Kruskal–Wallis test; ε², epsilon-squared effect size. Pairwise Dunn–Holm comparisons were conducted only for analytes with significant overall Kruskal–Wallis results.

; Figure 7). For Cd, BM displayed by far the highest median BSAF (66.75), and Dunn–Holm testing confirmed greater results in BM than in DF (pHolm = 0.0095) and PF (pHolm = 0.0138). The PF category had the highest median Cr BSAF (0.1145), while G showed the lowest (0.00140); only the G versus PF contrast remained significant after correction (pHolm = 0.0024). For Pb, the only supported pairwise difference was observed between the BM and G categories, with higher BSAF ratios in BM (0.0516 vs. 0.000257; pHolm = 0.0252).

Station type was less informative overall, although Pb indicated a significant secondary station-type effect (H = 10.490, p = 0.0053, ε² = 0.4043), whereas Cu, Cd, Ni, and Cr did not differ significantly among station types (Table S10). Descriptively, Pb BSAF increased from C stations (median 0.00528) to M stations (0.0516) and was maximum in T stations (0.1026), suggesting a possible spatial gradient that merits cautious explanation.

Species-level BSAF analyses were treated as exploratory because taxa represented by a single sample were excluded from inferential testing. Pb indicated an overall species effect (H = 13.099, p = 0.0415, ε² = 0.5071), while Cd and Cr were borderline (p = 0.0523 and p = 0.0503, respectively) (Table S11). In exploratory terms, and considered alongside the biota-type BSAF medians, Cd-related BSAF appeared highest in the BM subset, particularly A. kagoshimensis and M. galloprovincialis. For Pb, elevated BSAF ratios appeared to be shared between BM and PF, while R. venosa (G) remained low. For Cr, increased BSAF estimates were associated mainly with fish taxa, especially the pelagic subset, while R. venosa (G) had the lowest levels.

To further contextualize the observed BSAF trends, the data were interpreted against the general USEPA benchmark whereby values > 1 suggest a meaningful sediment contribution, while recognizing that contextualization depends on species traits and local seabed substrate characteristics [69]. Under this framework, Cd indicated the clearest signal in the dataset, with BM and G above the benchmark. This was aligned with the stronger Cd profile observed in A. kagoshimensis and M. galloprovincialis.

Cu BSAF values were not interpreted further at group level because Cu did not show significant broad biota category differentiation. Lead, Ni and Cr generally remained below 1, although Cr still presented a relative increase in fish, especially pelagic taxa, while R. venosa remained lowest (Figure 8). In general, these threshold-based comparisons indicate that Cd produced the clearest apparent tissue–sediment ratio in the present dataset, especially in bivalves. However, this result should be interpreted as a relative BSAF pattern rather than as direct evidence of sediment-derived uptake. Because Cd concentrations in sediments were low, elevated BSAF values may partly reflect the low sediment denominator, together with the efficient biological retention of Cd by bivalves.

3.2. Human Health Risk Indices

To complement the regulatory comparison, dietary exposure was evaluated for the adult consumption scenario using EDI, THQ, and TTHQ. This assessment was not intended to replace regulatory maximum levels, which remain the primary benchmark for seafood compliance, but to provide an exposure-based interpretation that integrates measured PTE concentrations with daily ingestion rate, body weight, and oral reference values.

Within the risk index dataset, broad biota category provided the clearest descriptive structure, although this pattern should be interpreted cautiously given the limited and uneven species-level replication. Significant Kruskal–Wallis effects for all analyzable variables (ε² = 0.325–0.803) (Table S12).

Overall, Cd-related EDI and THQ were highest in BM relative to G, whereas Cr-, Ni-, and Pb-related indices were generally greater in PF, with DF also contributing to the Cr and Ni patterns. Cu-related EDI and THQ were higher in fish groups than in BM. Cumulative TTHQ also differed significantly by organism group, with higher medians in BM and PF than in G (Figure 9). Together, these outcomes reveal that the dietary risk profile varied by analyte, with BM driving the Cd-related signal and PF contributing more strongly to Cr-, Ni-, and Pb-related hazard profiles, corroborating with the bioaccumulation and transfer factor findings described in previous section.

Risk index evaluation nevertheless indicated generally low health concern in the dataset studied. Estimated daily intake (EDI) for Cd, Pb, and Ni remained below the corresponding health-based guidance thresholds throughout the dataset. For Cd, all EDI estimates were below the EFSA tolerable daily intake of 0.00036 mg/kg/day [91]; Pb-related EDI were likewise below the health-based guidance of 0.00063 mg/kg/day [92]. For Ni, all values also remained below the EFSA tolerable daily intake of 0.01300 mg/kg/day [93]. Thus, although EDI trends differed among groups, none of the evaluated Cd-, Pb-, or Ni-related dietary intake exceeded the corresponding health-based guidance level. None of the individual THQ exceeded 1.0, indicating no evident non-carcinogenic hazard from exposure to any single metal. Among the analyzed elements, Cd contributed most strongly to THQ, although values remained below the critical threshold (Table S13).

Likewise, all cumulative TTHQ estimates were below 1.0, suggesting no detectable chronic non-carcinogenic concern from combined PTE interaction. (Table S14; Figure 10). In general, the dataset supports low current health concern, while showing that Cd-driven cumulative exposure represented the most sensitive part of the hazard profile.

4. Discussion

4.1. Tissue Concentrations and Regulatory Relevance

Despite heterogeneity in species composition, sampling sites, and depths, the 2024 dataset showed a clear descriptive pattern: Cd and Pb concentrations were generally higher in bivalves than in fish and gastropods, whereas Cr values were higher in several fish samples, particularly pelagic fish. Cu and Ni did not show statistically significant differences among broad biota categories and were therefore not interpreted as group-level ecological signals. These patterns should be considered exploratory, because the dataset included 24 composite samples collected during a single year and species-level replication was uneven. The higher Cd and Pb values recorded in bivalves are consistent with their relatively sedentary feeding ecology and their frequent use as indicators of local contamination, particularly where waterborne and particle-associated exposure is relevant [22]. However, this pattern should not be interpreted as evidence of a single uptake pathway. Bivalves differ among themselves in lifestyle, habitat position, feeding behavior, and physiological regulation, and these differences may influence PTE uptake and retention. Therefore, the present bivalve pattern is best understood as a descriptive tissue burden signal rather than as a mechanistic explanation of exposure [94,95,96,97].

Comparable findings have been reported in previous Black Sea studies using Mytilus galloprovincialis as a biomonitor. Romanian investigations have demonstrated the usefulness of mussels for tracing PTE contamination along the Romanian coast [19,44,46,47,98,99], while studies from the Turkish and Bulgarian Black Sea have also reported spatial variability in Cd, Pb, and other metals in mussels, often with elevated values near anthropogenic pressure zones [39,100,101,102,103]. Cd deserves particular attention because elevated Cd in bivalves may occur even when measured Cd concentrations in seawater or bulk sediment are low. This apparent contrast reflects both Cd environmental behavior and biological retention. Unlike more particle-reactive elements such as Pb, Cd may remain relatively mobile in marine systems and can show partly nutrient-like behavior in seawater [104,105,106,107]. A single measurement of dissolved Cd in seawater or total Cd in sediment may therefore not fully represent the labile, particulate, organic-bound, or food-associated Cd fractions available to filter-feeding organisms [108,109,110]. Bivalves integrate exposure over time through continuous filtration of seawater, suspended particles, phytoplankton, and organic detritus; consequently, tissue Cd concentrations may reflect cumulative retention rather than the instantaneous concentration measured in paired abiotic matrices [108,109]. Gastropods displayed generally lower Cd and Pb concentrations than bivalves. This difference may reflect their distinct feeding ecology and habitat use, but the limited replication does not allow firm inference on uptake processes [111]. The gastropod pattern should therefore be interpreted descriptively. Previous observations indicate that Rapana venosa can act as a useful bioindicator, but its assimilation profile differs from that of filter-feeding bivalves because intake is partly mediated through benthic predation and trophic mobilization [25,99,102].

Fish samples, particularly pelagic fish, showed higher Cr values than gastropods and several bivalve samples. This pattern should also be interpreted cautiously. Fish are mobile organisms, and their tissue concentrations may integrate broader environmental and dietary influences rather than local exposure at the nearest monitoring station [112,113,114]. In addition, Cr speciation and dissolved/particulate partitioning were not determined. The relative occurrence of Cr(VI) and Cr(III), as well as dissolved versus particulate Cr partitioning, may strongly influence Cr mobility, bioavailability, toxicity, and biological uptake [114,115,116,117]. Therefore, Cr-related patterns in fish should be treated as descriptive tissue burden signals, not as evidence of a specific water column or trophic pathway.

The comparison with regulatory maximum levels confirmed that the main compliance issue in the 2024 dataset was Cd. Cd exceedances occurred mainly in bivalves, including some Anadara kagoshimensis samples and one M. galloprovincialis sample, while fewer exceedances were observed in pelagic fish. Pb exceedance was isolated and limited to one pelagic fish sample, whereas bivalves remained below the regulatory maximum level for Pb [90]. These results indicate that regulatory screening remains important, particularly for Cd in bivalves. From a seafood safety perspective, regulatory maximum levels remain the primary benchmark for compliance and consumer protection; dietary risk indices provide additional exposure-based context only after measured concentrations are interpreted against these limits [17,118].

4.2. BCF as a Descriptive Tissue–Water Ratio

BCF values provided additional context by relating tissue concentrations to measured seawater concentrations, but they should be interpreted only as descriptive tissue–water ratios [40,119,120]. In this study, BCF was not used to identify direct uptake routes or to demonstrate causal transfer from seawater to biota.

Cd showed the highest apparent tissue–water ratios, especially in bivalves. However, because Cd concentrations in seawater were very low, elevated BCF values may partly reflect low denominator values rather than direct dissolved-phase uptake [104,121,122]. The high Cd BCF values are therefore consistent with the high Cd tissue burdens observed in bivalves, but they should not be interpreted independently from Cd geochemical behavior and bivalve filtration ecology. Similar patterns have been reported in Chinese coastal waters, where bivalves accumulated Cd efficiently despite relatively low dissolved concentrations, and Cd was identified as an important contributor to health hazard in farmed scallops and other bivalves [123,124].

The Cr-related BCF pattern differed from Cd. Pelagic fish showed higher Cr tissue–water ratios than gastropods, but this result should not be interpreted as evidence of a specific water column exposure pathway. As noted above, Cr speciation was not determined, and fish mobility makes station-matched seawater data only an approximate environmental reference. Previous Black Sea studies also indicate that BCF and BSAF patterns in fish are strongly influenced by habitat, mobility, and diet, supporting their cautious use as integrated descriptive indicators rather than proof of uptake from a single compartment [98,125,126].

4.3. BSAF as a Descriptive Tissue–Sediment Ratio

BSAF outcomes contextualized tissue concentrations relative to surface sediment concentrations, but they also require cautious interpretation. Cd showed the highest apparent tissue–sediment ratios, especially in bivalves. However, low sediment Cd concentrations can inflate BSAF values; therefore, these results indicate a descriptive tissue–sediment relationship rather than direct evidence of sediment-derived uptake [127]. The elevated Cd BSAF values observed here are consistent with the high Cd tissue burdens measured in bivalves, but they do not identify the relative contribution of dissolved, particulate, or resuspended material. They are also consistent with previous RoBS studies in which Cd presented high ratio-based values in shellfish and was identified as one of the more bioavailable PTE in benthic-related matrices [65,99]. Broader experimental and modeling evidence also shows that sediment-bound PTE, including Cd, may be assimilated by suspension- and deposit-feeding bivalves, with uptake depending on element identity, sediment properties, and feeding mode [127,128].

For Pb, BSAF values were also greater in bivalves than in gastropods, although the apparent tissue–sediment ratios were lower than those observed for Cd. This pattern agrees with the tissue concentration results, but it should not be interpreted as direct evidence of a specific particle-associated uptake process [129,130].

Previous BCF/BSAF studies support this cautious contextualization. In the RoBS, earlier work on M. galloprovincialis reported bioaccumulative or very bioaccumulative BCF values for Cd, Cu, Cr, and Ni in some cases, especially in port-influenced areas, while BSAF profiles supported Cd and Ni accumulation and indicated seasonal variability [131]. Similar research on M. galloprovincialis, A. inaequivalvis, and R. venosa reported higher ratio-based values for Cd and Ni and interspecific differences in tissue–environment relationships [99]. Comparable evidence from other marine regions also confirms that BCF and BSAF values are strongly influenced by PTE identity, organism physiology, uptake route, and ambient environmental concentrations [132,133,134,135].

For fish, BSAF must be interpreted with particular caution. Fish do not accumulate PTE directly from seabed substrate in the same way as benthic invertebrates. In pelagic and demersal fish, a sediment-related signal may reflect indirect processes such as benthic–pelagic coupling, resuspension, prey contamination, near-bottom contact, mobility, or broader station-level environmental conditions. BSAF is therefore more biologically plausible for relatively sedentary bivalves than for mobile fish [136]. For gastropods, BSAF may reflect benthic contact and trophic exposure, but direct sediment uptake should still not be assumed. For fish, particularly pelagic species, BCF and BSAF should be treated only as exploratory, station-matched contextual indicators, because tissue concentrations may integrate exposure over broader spatial scales than the paired water or sediment sample [137,138]. Similar regional and international studies have shown that transfer factor values vary according to element identity, organism physiology, organ or tissue analyzed, feeding ecology, and ambient environmental levels [125,139,140,141].

4.4. Dietary Exposure and Seafood Safety Interpretation

The dietary risk assessment did not indicate substantial non-carcinogenic concern under the evaluated adult exposure scenario. All individual THQ values remained below 1, and cumulative TTHQ values also remained below 1. This indicates that, although detectable PTE burdens and some regulatory exceedances were observed, the calculated non-carcinogenic risk from combined exposure to Cd, Cr, Cu, Ni, and Pb was below the level of concern for the consumption scenario considered.

This result is consistent with Black Sea and broader marine studies. Studies of commercial fish species from Georgian and Turkish Black Sea waters have generally reported PTE concentrations and dietary risk indices within acceptable limits, while also emphasizing species-specific enrichment, spatial heterogeneity, and the need for continued monitoring, especially for frequent seafood consumers [83,142,143]. Similar hazard assessments from Mediterranean, Baltic, and Asian coastal areas commonly report detectable PTE burdens but THQ values below concern thresholds under average consumption assumptions, while noting that concern may increase for high-consumption groups or polluted local hotspots [31,144,145,146,147,148]. Therefore, the balanced interpretation of the present dataset is that Cd remains relevant for seafood compliance monitoring, especially in bivalves, but the calculated adult dietary risk indices did not indicate substantial non-carcinogenic concern under the evaluated exposure scenario.

The distinction between regulatory exceedance and dietary risk indices is important. Regulatory maximum levels evaluate whether seafood complies with legal safety thresholds, whereas EDI, THQ, and TTHQ estimate exposure under a defined consumption scenario. Thus, regulatory exceedances, particularly for Cd in bivalves, justify continued surveillance even when calculated THQ and TTHQ values remain below 1.

4.5. Monitoring Implications and Study Limitations

The present findings support a focused multi-species monitoring framework for the Romanian Black Sea. Bivalves, particularly M. galloprovincialis and A. kagoshimensis, should remain priority matrices for Cd and Pb surveillance because they captured the clearest Cd/Pb tissue signal in this dataset. Representative pelagic and demersal fish species of ecological and fisheries relevance should also be included because they provide information on mobile fishery resources and broader tissue burden patterns. Monitoring only bivalves could miss patterns in mobile fish, whereas monitoring only fish could miss localized Cd/Pb accumulation in bivalves. Therefore, both organism groups should be regarded as complementary monitoring matrices, while future monitoring should retain species-level resolution rather than relying only on broad pooled categories. Cd should remain a priority analyte in future seafood safety and environmental contamination assessments because it was the most recurrent element of concern across tissue burdens, BCF, BSAF, regulatory comparison, and dietary risk evaluation.

This is particularly relevant for the RoBS, where contaminant availability may be shaped by interacting pressures, including Danube influence, coastal wastewater inputs, port activities, maritime transport, tourism, sediment resuspension, and local hydrodynamics. Under such conditions, no single organism group can fully represent contaminant movement from the environment to seafood. Bivalves are useful for detecting local and particle-associated loads, while fish may integrate broader water column, trophic, and habitat-related influences [7,149]. This interpretation is supported by regional [10,11,65,150,151,152,153] and broader [154,155,156] marine evidence showing that riverine inputs, sediment texture, port activity, atmospheric inputs, and resuspension influence PTE retention and redistribution in coastal systems.

Several limitations should be considered. The study was based on 24 composite samples collected during a single year, and the design does not allow robust temporal, seasonal, population-level, or mechanistic inference. Seasonal variation, interannual variability, and episodic pollution events could not be evaluated. Composite samples also limited the evaluation of individual-level variability; therefore, the results should be interpreted as broad exploratory patterns, not as population-level estimates. Species-level inference was constrained by uneven replication, and some taxa were represented by single observations. Accordingly, broad biota categories should not be interpreted as homogeneous ecological units. In addition, multiple independent Kruskal–Wallis tests were performed across endpoints; although Holm correction was applied to post hoc pairwise comparisons, no global correction was applied across all omnibus tests. Therefore, statistically significant results should be interpreted cautiously and in relation to effect size, consistency among endpoints, and biological plausibility.

BCF and BSAF interpretation was also constrained by <LOD values in some abiotic measurements and by low Cd concentrations in seawater and sediments, which can inflate ratio-based values and reduce the number of analyzable transfer factor observations. These indices cannot separate dissolved, particulate, sediment-associated, and dietary exposure pathways. This limitation is especially relevant for Cr because Cr speciation was not determined [114,115], and for fish because samples were assigned to the nearest monitoring station rather than being spatially paired with abiotic matrices at the same resolution as bivalves. The dietary risk assessment was also based on standard exposure assumptions and available consumption data; individual risk may vary with seafood consumption frequency, portion size, age, body weight, health status, and exposure to contaminants from other dietary sources.

Future assessments should expand spatial and temporal coverage, include repeated seasonal and multiannual sampling, strengthen species-level replication, and better integrate abiotic variables such as sediment texture, particulate matter, salinity, pH, hydrodynamic conditions, and resuspension processes. Additional contaminants of high seafood safety relevance, especially Hg and As, should also be included, together with persistent organic pollutants, polycyclic aromatic hydrocarbons, microplastics, and possible additive or synergistic effects. Such a framework would better support environmental status assessment, seafood safety surveillance, and management decisions in the Romanian Black Sea.

5. Conclusions

This study provides an exploratory cross-biota assessment of Cd, Cr, Cu, Ni, and Pb in fishery resources from the Romanian Black Sea, integrating tissue concentrations, regulatory comparison, BCF, BSAF, and adult dietary risk indices. Within the limits of a single-year dataset based on 24 composite samples, bivalves showed the clearest Cd and Pb tissue signal, whereas several fish samples, particularly pelagic fish, showed higher Cr values. Cu and Ni did not support clear group-level interpretation.

Cd was the main element of concern in the dataset. Cd exceedances of regulatory maximum levels occurred mainly in bivalves, while Pb exceedance was isolated. BCF and BSAF results supported the relevance of Cd as a priority element, but these ratio-based metrics should be interpreted as descriptive tissue–environment indicators, not as evidence of specific uptake pathways.

The adult dietary risk assessment did not indicate substantial non-carcinogenic concern under the evaluated exposure scenario, as all individual THQ values and cumulative TTHQ values remained below 1. Nevertheless, regulatory exceedances for Cd justify continued seafood safety surveillance, particularly in bivalves.

Overall, the findings support a cross-biota monitoring approach including bivalves, gastropods, and representative pelagic and demersal fish species, while retaining species-level resolution. Bivalves remain especially useful for Cd and Pb surveillance, while selected pelagic fish can provide additional information on mobile fishery resources. Cd should remain a priority analyte in future seafood safety and environmental contamination assessments, together with improved species-level replication, repeated seasonal and multiannual sampling, and inclusion of additional contaminants such as Hg, As, persistent organic pollutants, polycyclic aromatic hydrocarbons, and microplastics.