Contamination Assessment of Sediments and Bivalves in Estuaries of the Southern Iberian Peninsula

Abstract

Human activities directly impact estuaries, where the biota is exposed to sediment contamination. A contamination assessment was carried out in several estuaries in the Southern Iberian Peninsula. Sediment samples were analyzed for the presence of metals/metalloids, and bioaccumulation tests were conducted with clams (Ruditapes philippinarum). Huelva Estuary had the highest contamination levels, while the inner bay of Cádiz and the outer stations of the estuaries from Guadiana, Guadalquivir, Palmones, and Guadarranque were the lesser contaminated. All sampling points (except Huelva) had low contamination levels of As and Cd, but they had high concentrations of Cu. The elements Pb, Cd, Zn, Cu, As, and Hg displayed correlations between the concentrations in sediments and the biota. High bioaccumulation of Zn, Cu, and As was observed in Huelva and Barbate. Important insights into the sediment contamination in Southern Iberian Peninsula estuaries suggest greater management and conservation efforts in these critical ecosystems.

1. Introduction

Marine sediments are a vital component of coastal ecosystem, serving as the ultimate sink for a diverse array of contaminants, including metals and metalloids [1,2,3]. These contaminants enter the marine environment through various pathways, such as runoff from terrestrial sources, industrial discharges, atmospheric deposition, and erosion of coastal land. Once introduced into the aquatic ecosystem, they have the potential to accumulate in the tissues of marine organisms, potentially leading to adverse effects on their health and disrupting the delicate balance of the ecosystem [4,5].

Estuaries, the transitional zones where rivers meet the sea, are highly complex and biodiverse ecosystems that are particularly vulnerable to environmental stressors [6,7]. Their intricate physical, chemical, and biological characteristics make them especially sensitive to contamination [8,9,10]. Therefore, it is imperative to conduct comprehensive assessments of contaminant levels in estuarine sediments. Such assessments are essential for developing a nuanced understanding of the extent and intensity of contamination, which is crucial for devising targeted and effective strategies to mitigate its adverse effects on both the ecosystem and human health [11,12].

The bioaccumulation of contaminants in marine organisms has been used as a reliable tool for assessing the potential for trophic transfer of pollutants in the food chain [11]. Marine bivalves, such as clams and mussels, are particularly useful for this purpose, as they are filter feeders that can accumulate trace elements and other substances from the surrounding water and sediments [13]. In addition to serving as bioindicator organisms of coastal contamination, bivalves are also important links in the transfer of contaminants from sediments to higher organisms, including humans who consume them [14].

The Manila clam Ruditapes philippinarum is a valuable species native to the Western Pacific and has been introduced to various regions worldwide, including the Pacific coasts of North America and European waters. Its cultivation began in areas with traditional fishing practices, and it has become a significant contributor to clam landings in Europe [15]. The species R. phillipinarum has also been used in previous studies to monitor contamination in other studies from the South of the Peninsula Iberian [16,17,18,19]. As filter feeders, clams play a crucial role in the marine ecosystem by filtering water and ingesting particles, including contaminants, from the sediment. Due to their feeding behavior, clams come into direct contact with sediment, making them susceptible to bioavailable contaminants. This direct interaction with the sediment increases the risk of contaminant accumulation in clams, which can have implications not only for clam populations but also for the broader ecosystem.

In the southern Iberian Peninsula, elevated levels of contaminants have been identified in ecosystems. This region has a long history of being affected by mining discharges (both natural contamination and accidental spillages), the development of ports and chemical industries, as well as the presence of major cities, tourism, recreational areas, and marinas, all of which have contributed to the release of contaminants into the environment [20,21,22,23].

In light of these concerns, the main objective of this study is to investigate the relationships between different sources of sediment contamination and the bioavailability of metals in the clam Ruditapes philippinarum on the southern coast of Spain. To achieve this objective, we conducted a comprehensive assessment of sediment quality and metal bioaccumulation in clams collected from various stations along the coast.

2. Materials and Methods

2.1. Sample Collection

Surface sediment samples were gathered from various estuaries in the south of Spain, stretching between the provinces of Huelva and Cádiz (Figure 1). The sampling was conducted at twelve stations, including two in the Guadiana River Estuary (1, 2), bordering Portugal and Spain. Three stations were sampled from the Ria of Huelva (3, 4, 5), an area that has been historically affected by mining activities, but now also has several industrial activities [7]. Two stations were situated in the Guadalquivir River Estuary (6, 7), which is affected by maritime traffic and urban waste. Three stations were sampled from the Bay of Cádiz, which is also impacted by intense maritime traffic (8, 9, 10). Station 11 was located at the port of Barbate, a large harbor used for recreational and commercial fishing. Finally, the remaining three stations were located in the Bay of Algeciras and were influenced by industrial discharges (12—Palmones River Estuary; 13, 14—Guadarranque River Estuary).

At the stations identified in Figure 1, sediment samples were gathered using a 0.025 m² Van Veen grab. These samples were then stored in a cooler and transported to the laboratory. In the lab, the samples were kept at 4 °C in dark conditions until a sediment bioaccumulation test could be conducted. The methodology for this test was based on the procedures outlined in [21,22,23,24,25].

2.2. Bioaccumulation Test

To acclimate the adult clams (Ruditapes philippinarum) obtained from an aquaculture farm, they were kept under controlled laboratory conditions with continuous flow for 10 days before conducting the tests.

For the tests, sediment samples from each station were added in replicates to aquariums, with a sediment and clean seawater (1:4). The mixture was then left for 24 h to allow particle settling, and aeration was pumped for 12 h before introducing the organisms [24]. No additional food was added, and the water was renewed every three days. The physicochemical parameters, including pH, temperature, oxygen, and salinity, were regularly monitored and controlled to ensure quality control. Mortality was recorded daily, and samples of organisms were collected on days 7, 14, and 28. After a 12 h gut depuration period, the clams were individually dissected, frozen at −80 °C, and lyophilized.

2.3. Analytical Procedure

To determine the grain size, a portion of the wet sediment was analyzed using a laser particle size Fritsch analyzer (model Analysette 22, Laval lab, Laval, PQ, Canada), following the method described by [21]. In addition, dried and homogenized sediment samples were used to analyze the organic carbon content (OC) and metal(loid) concentrations. The OC was determined using the method outlined by [24], while elemental analysis in sediment samples was carried out after digestion with HF and aqua regia in closed Teflon bombs, following the methodology described by [13,14]. Freeze-dried and homogenized organisms were acid-digested using the method described by [24].

Sediment and soft tissue concentrations of Zn were determined using flame atomic absorption spectrometry (Perkin Elmer AAnalyst 100, USA). The concentrations of As, Cd, Co, Cu, Ni, and Pb were analyzed using inductively coupled mass spectrometry (ICP-MS Thermo-Fisher Scientific), while Hg was determined with a mercury analyzer (LECO AMA-254).

To ensure accuracy, the analytical procedures were validated by analyzing certified reference materials (MESS-3, PACS-2, and 1646a for sediments; TORT-1, TORT-2, DOLT-1, DOLT-2, and DOLT-3 for organisms). Procedural blanks were also analyzed to check for any possible contamination during the analytical procedure.

2.4. Data Analyses

To assess the contamination levels of the stations and estuaries, the ratio to reference or Contamination Factor degree (C_d) was used, which involves comparing the measured concentrations to background levels. The background concentrations were determined as the median values of surface sediments from coastal zones, as recommended by [25,26] and were obtained from [27]. This approach allowed for a reliable comparison of the contamination levels across the different study sites. C_d is calculated using the following formula:

$$C_d={\displaystyle \frac{C_{measured}}{C_{background}}}$$

where C_measured is of the contaminants measured at a specific station and C_background is the median background concentration of the same contaminant from uncontaminated reference areas, typically coastal areas. A C_d value greater than 1 indicates contamination, with higher values reflecting increasing levels of pollution relative to the natural background levels. By using this ratio, we can effectively normalize the data across different locations and time points, facilitating a consistent evaluation of contamination severity. This method also enables the identification of pollution hotspots and provides insight into the potential ecological risks associated with elevated metal concentrations.

On the other hand, the correlation between the concentrations of metals in the soft tissue of clams and the concentrations in sediments, commonly known as the bioaccumulation factor (BAF), was calculated based on [28]. BAF is typically expressed as the ratio of the concentration of a given metal in the organism (C_organism) to the concentration in the surrounding medium, which in this case is sediment (C_sediment). The formula used is:

$$BAF={\displaystyle \frac{C_{organism}}{C_{sediment}}}$$

where C_organism represents the concentration of the metal in the soft tissues of the clams (in mg/kg dry weight), and C_sediment is the corresponding metal concentration in the sediment (in mg/kg dry weight). This ratio provides insight into the extent of metal bioaccumulation from the environment into the organism.

3. Results

3.1. Elements in Sediment

Table 1 presents the average concentrations of As, Cd, Co, Cu, Hg, Ni, Pb, and Zn in the 14 southern estuaries shown in Figure 1. Huelva Estuary exhibited the highest levels for all studied elements, with Zn (1415) > Cu (1123) > Pb (260) > As (200) > Ni (23.91) > Co (18.87) > Hg (1.59) > Cd (1.25), while Co and Ni had greater concentrations in the Guadarranque Estuary, with 35.95 and 41.45 mg kg⁻¹, respectively. Palmones had the most pristine estuary with the lowest concentrations of Zn (31.54) > Cu (12.36) > Ni (10.31) > Pb (8.68) > Co (3.28) > As (1.43) > Hg (0.04) > Cd (0.02). The average metal concentrations in southern estuaries showed significant variations, particularly for Zn (364 ± 675 mg kg⁻¹), As (49.13 ±94.32 mg kg⁻¹), and Cu (269 ± 559 mg kg⁻¹) (Table S2).

Table S1 provides additional details on the concentrations of trace elements, organic carbon, and percentage of fine fraction analyzed in the 14 sediment samples collected. Station 10 in the Bay of Cádiz had the lowest metal concentrations and was used as a negative toxicity control due to its extensive use and characterization in previous toxicity studies [29,30]. Half of the studied stations had a percentage in fine grain size above 75%. The OC displayed a wide range of percentages, from 1.44 (st7) to 86.12% (st2), probably due to rivers’ input and proximity to harbors and dredging activities. Detritus precipitation in bays together with the fine fraction of sediment (ease to digest by detritivores, filter-feeder, annelids, and other benthic organisms living in the immediate layers of sediment) tend to be associated with contaminants under different chemical associations (adsorption, absorption, coagulation, etc.).

Table 1. Averaged metal/metalloid concentrations in sediments (mg kg⁻¹) of the different estuaries from Figure 1.

		As	Cd	Co	Cu	Hg	Ni	Pb	Zn
Guadiana River Estuary	av	14.2	0.08	9.97	24.5	0.15	19.94	12.32	74.16
	sd	7.88	0.07	5.24	16.5	0.13	11.95	4.75	37.07
Huelva Estuary	av	200	1.25	18.8	1123	1.59	23.91	260	1415
	sd	117	0.96	8.80	795	1.20	11.77	177	913
Guadalquivir River Estuary	av	6.83	0.07	6.60	27.0	0.05	18.12	10.8	53.2
	sd	2.41	0.10	5.20	26.9	0.04	17.97	6.40	46.2
Bay of Cádiz	av	6.62	0.07	5.00	25.1	0.13	13.60	11.4	52.5
	sd	4.68	0.06	3.73	20.6	0.11	11.05	7.07	44.5
Barbate		13.7	0.14	9.16	160	0.09	28.86	23.1	191
Palmones Estuary		1.43	0.02	3.28	12.4	0.04	10.13	8.68	31.5
Guadarranque Estuary	av	4.10	0.13	35.9	22.9	0.12	41.46	11.3	110.8
	sd	3.04	0.13	37.5	19.8	0.13	28.28	7.98	115.9
Southern estuaries	av	49.13	0.33	13.5	269.1	0.42	22.18	65.4	364.4
	sd	94.32	0.63	15.6	559.7	0.79	15.17	126	675.1
Marine Sediment Quality Values *		7.40	680	n.a.	18.7	130	15.90	30.24	124

* US environmental protection values from [31,32]. n.a.: not available.

Table 1. Averaged metal/metalloid concentrations in sediments (mg kg⁻¹) of the different estuaries from Figure 1.

		As	Cd	Co	Cu	Hg	Ni	Pb	Zn
Guadiana River Estuary	av	14.2	0.08	9.97	24.5	0.15	19.94	12.32	74.16
	sd	7.88	0.07	5.24	16.5	0.13	11.95	4.75	37.07
Huelva Estuary	av	200	1.25	18.8	1123	1.59	23.91	260	1415
	sd	117	0.96	8.80	795	1.20	11.77	177	913
Guadalquivir River Estuary	av	6.83	0.07	6.60	27.0	0.05	18.12	10.8	53.2
	sd	2.41	0.10	5.20	26.9	0.04	17.97	6.40	46.2
Bay of Cádiz	av	6.62	0.07	5.00	25.1	0.13	13.60	11.4	52.5
	sd	4.68	0.06	3.73	20.6	0.11	11.05	7.07	44.5
Barbate		13.7	0.14	9.16	160	0.09	28.86	23.1	191
Palmones Estuary		1.43	0.02	3.28	12.4	0.04	10.13	8.68	31.5
Guadarranque Estuary	av	4.10	0.13	35.9	22.9	0.12	41.46	11.3	110.8
	sd	3.04	0.13	37.5	19.8	0.13	28.28	7.98	115.9
Southern estuaries	av	49.13	0.33	13.5	269.1	0.42	22.18	65.4	364.4
	sd	94.32	0.63	15.6	559.7	0.79	15.17	126	675.1
Marine Sediment Quality Values *		7.40	680	n.a.	18.7	130	15.90	30.24	124

* US environmental protection values from [31,32]. n.a.: not available.

Table S1 shows that the highest concentration of all the studied metals was found at stations 3 and 4 in the Ría of Huelva, which is a heavily industrialized area located at the combined mouth of the Odiel–Tinto River Estuary (Figure 1). This area has been receiving acidic fluvial water discharges with high concentrations of metals since the Roman Empire times, several centuries ago, resulting in one of the highest levels of metal pollution in Europe [33,34]. All the studied elements, except for As, were above the probable effect threshold (PEL) calculated by [35]. Based on several sediment quality guidelines [31,32,36], only the stations located in Huelva could be associated with biological effects.

The C_d index was used to determine the contamination degree (Figure 1). The most contaminated stations were located in Huelva (3, 4) for all the studied elements (As, Cd, Cu, Hg, Pb and Zn) with a very high degree of contamination and considerable contamination for Ni. However, contamination appeared to decrease towards the ocean (station 5) with considerable contamination for As but low for Cd and Ni. The least contaminated station was located in the inner bay of Cádiz (station 10), with a low contamination degree for the studied elements, followed by the outer stations of the estuaries from Guadiana (station 2), Guadalquivir (station 7), Palmones (station 12), and Guadarranque (station 13). Except for the Huelva stations, all the sampling points showed low contamination for As and Cd and from moderate to very high contamination of Cu.

Saenz et al. [37] previously reported the presence of Cr, Cu, Fe, and Zn in sediments from the Bay of Cádiz and Barbate in a residual fraction, while Cu, Zn, and Cd were found in saltmarshes in Huelva, which posed a potential threat to local organisms due to historical mining and industrial activities. Blasco et al. [38] found no seasonal variation in metal concentration in sediments from the Ria of Huelva, Ria Formosa, Guadiana River, and Bay of Cádiz. They identified Cu, Cd, and Zn in the mobile phase and Pb and Ni in the residual fraction in Huelva, while Ni and Hg showed maximum concentrations in the Bay of Cádiz. Bonnail et al. [39] also reported higher concentrations of As, Cd, Co, Cu, Pb, and Zn (except for Ni) upstream of the Guadalquivir River, which may be due to the nearby accidental mining spill of Aznalcóllar.

In the Algeciras Bay, Díaz-de Alba et al. [40] found average metal concentrations of 11 ± 5 mg kg⁻¹ As, 0.3 ± 0.1 mg kg⁻¹ Cd, 11 ± 4 mg kg⁻¹ Co, 17 ± 7 mg kg⁻¹ Cu, 65 ± 36 mg kg⁻¹ Ni, 24 ± 7 mg kg⁻¹ Pb, and 73 ± 26 mg kg⁻¹ Zn, which were higher than those in the current study (except for Zn, which was similar). They also evaluated metal bioavailability using the sequential extraction procedure and identified Cd, Zn, Pb, and As as the most available fractions. Additionally, they noted the influence of seasonality and anthropogenic activities, such as sewage from the city of Algeciras and harboring activities.

3.2. Bioaccumulation

The metal concentrations in the soft tissue of clams exposed to sediments (day 7, day 14, and day 28) and background concentrations before exposure, as well as the sediment concentrations, are presented in Figure 2. The bioaccumulation pattern of elements by the clam R. phillipinarum, under the conditions of the conducted bioassay, showed a decreasing gradient in the order of Zn > Cu > As > Pb > Ni > Co > Cd > Cr > Hg.

The clams from st4 (Huelva harbor) exhibited the highest concentration of As, Cu, and Zn in their soft tissues, whereas st3, also located in Huelva, had the maximum concentration of Cd.

The data presented in Figure 3 illustrate BAF values. Significant relationships were observed (p < 0.0001) between the concentration in the environment and the concentration stored in clams for Cd, Cu, Pb, and Zn, while As and Hg also showed a significant relationship (p < 0.05). However, the correlation was not statistically significant for Co and Ni (Table S2). Based on Pearson correlation analysis, the ranking of the correlation strength was as follows: Pb (0.9244) > Cd (0.9034) > Zn (0.8983) > Cu (0.88) > As (0.6040) > Hg (0.5861).

Figure 4 illustrates the bioaccumulation factor (BAF) calculated for each estuary studied, representing the statistical relationship between sediment and biota concentrations displayed in Figure 3. The average BAF values are represented by the dotted line. The BAF values for each element were as follows: Pb (0.03 ± 0.03) < Cu (0.13 ± 0.08) < Zn (0.75 ± 0.53) < Hg (0.83 ± 0.62) < As (2.72 ± 3.2) < Cd (3.81 ± 4.43). There is a clear pattern of bioaccumulation about the sediment’s elemental concentration. The BAF values for the Huelva and Barbate estuaries were below the average for all elements studied, indicating that the concentration in the environment was much higher than that in the tissue. Conversely, the Palmones estuary registered the highest BAF values for all elements. This trend was consistent for all elements, except for Hg in Cádiz due to higher contamination levels (Figure 1, Table 1).

Similar BAF values were reported by R. philippinarum in the Ria de Aveiro (Portugal) for Pb (0.06–0.16), Cu (0.8–2.2), and Cd (2.1–3.4), but higher for Hg and As, which were also greater than 1 [41]. Additionally, in another study from the same area, [42] reported similar BAF values for As, Cu, Ni, and Pb.

4. Discussion

The study by [43] highlights the clam R. philippinarum as a useful tool for monitoring chronic exposure to high levels of Cu contamination, despite its susceptibility to adverse effects from short-term exposure. However, not all species are capable of monitoring all types of metal(loid) contamination and some may not be able to withstand high levels of contamination [44].

In the Guadiana River, [45] utilized Corbicula fluminea, an invasive freshwater clam, to investigate biomarker responses and Pb pollution from freshwater input. Machado et al. [46] detected elevated levels of Cu (7.0 µg g^–1) in M. galloprovincialis mussels from the same estuary, while [47] found high concentrations of Cu, Ni, and Pb in the polychaete Nereis diversicolor in the Guadiana River affected by the Iberian Pyrite Belt, particularly in comparison with the Bay of Cádiz.

In the highly contaminated Odiel Estuary, [35] deliberately employed the Asian clam C. fluminea to monitor Cr, Cu, and Pb in the sediment. Adult coleoptera from the Odiel-Tinto Estuary also demonstrated a high ability to accumulate As, Cu, and Zn [48].

The clam Ruditapes phillipinarum monitored metal contamination toxicity and bioaccumulation in the Guadalquivir River Estuary, with greater bioaccumulation of Zn and Cd observed under low salinity conditions and activation of metallothioneins in the presence of Cd, Cu, and Zn [49]. More recently, [50] analyzed data a decade after the Aznalcóllar mine spill and found bioaccumulation of Zn. Kalman et al. [51] evaluated the bioavailability of metals in the Bay of Cádiz by using Arenicola marina and metallothionein-like proteins. They found a low toxicity for the increased rate of Cu and Zn, and higher toxicity for Ni and Co. Casado-Martínez et al. [52] demonstrated the cost-effectiveness of the biodynamic model with A. marina for Ag, As, Cd, Cu, Pb, and Zn. The biomarker response of the polychaete Nereis diversicolor in the Guadiana River and the Cádiz was determined in other studies, which found Cd as a source of oxidative stress (lipid peroxidation) in the Bay of Cádiz, and Ni (metallothionein protein) in the Guadiana River and Ria Formosa Lagoon [47]. Buratti et al. [53] also concluded contamination in Algeciras Bay through a neutral red retention time (NRRT) test with Ruditapes philippinarum and the crab Carcinus maenas after 28 days of exposure.

Díaz de Alba et al. [54] revealed that As, Ni, Co, and Cr concentrations exceeded the quality guideline values, and the study calculated BCF for four species of fishes, showing greater correlations of Co and As with environmental concentrations in the Algeciras Bay. A high Ni bioavailability (oxidizable fraction) resulting from the installation of a steel manufacturing plant close to Palmones was found in barnacle samples [55].

5. Conclusions

The results of this study indicate that estuaries in southern Spain are subjected to varying degrees of metal contamination from a range of industrial activities. The highest concentrations of As, Cd, Co, Cu, Hg, Ni, Pb, and Zn were found in the heavily industrialized Huelva estuary, which receives acid fluvial water discharges with high concentrations of metals/metalloids. The Guadiana estuary was also found to be influenced by mining activities, with high levels of Cu and Pb detected. Similarly, the Guadalquivir estuary was found to be highly contaminated in the inner river. Other estuaries, such as Guadarranque, Barbate, and the Bay of Cádiz, were influenced by different industrial activities.

To assess the potential adverse effects of these contaminants, the clam R. philippinarum was exposed to sediments collected from the different estuaries with varying contamination levels. The results showed that the clam exhibited a strong bioaccumulation pattern for Cd, Cu, Pb, and Zn, with a positive correlation (r > 0.88, p < 0.01) between sediment and clam concentrations. The bioaccumulation factor (BAF) signature for all the elements followed a similar pattern along the estuaries, except for Hg.

Overall, these findings suggest that R. philippinarum is a valuable tool for assessing the bioaccumulation of contaminants in estuaries and their potential transfer through the food chain. The concentrations of metals were not found to be significantly toxic, indicating that the clam individuals can be used to determine the bioaccumulation of contaminants and their potential mobility through the food chain. Further studies could be conducted to evaluate the potential long-term effects of metal contamination in southern Spain’s estuaries and the effectiveness of different remediation strategies.