JBCS

INTRODUCTION

Estuarine environments and coastal zones have increasingly impacted as primary receptors and sinks for chemical contaminants, particularly those carried by continental run-off.^1,2 Among these contaminants, metals are prevalent. They naturally occur in the earth's crust and aquatic environments, where their concentrations increase due to indiscriminate anthropogenic activities. Metals are widely recognized as potential pollutants due to their persistence, toxicity, abundance, non-biodegradability, and bioaccumulative behavior.^3-6

Once introduced into the aquatic environment, metals can be distributed across various compartments, depending on the environmental characteristics.⁶ Their availability, speciation, sorption (adsorption/desorption), precipitation/dissolution, complexation/decomplexation, and mobility are influenced by physical and chemical conditions such as pH, redox potential (Eh), suspended particulate matter (SPM), temperature, organic carbon and salinity.^7,8 Numerous studies^8-12 have particularly emphasized the influence of salinity on the mobility, bioavailability, and toxicity of metallic elements.

Metal bioaccumulation in aquatic animals occurs through different pathways; organisms can uptake contaminants from particulate and dissolved phases derived from the sediment or the water column, as well as from their food.¹ However, it is important to note that metal bioaccumulation strongly depends on the physiology of the animals.^10,13

Bivalves, as oysters, are known for their high resistance and ability to hyperaccumulate metals, playing an essential role in the fate of contaminants in the estuarine environment.^3,14,15 They have been adopted as target organisms for coastal environmental biomonitoring due to their wide geographical distribution, abundance, filtering nature, sedentary lifestyle, and tolerance to environmental stress.^16-19 The bioaccumulation of contaminant in bivalves tissues closely reflects the extent of contamination in each ecosystem.²⁰ Identifying suitable biomonitors for aquatic contamination is essential to assess contaminant bioavailability and toxicity. Since it is impossible to monitor all species in a given region, biomonitors are selected to represent exposure conditions and predict effects on other organisms.²¹

The mangrove oyster Crassostrea brasiliana (Lamarck, 1819) (sin. Crassostrea gasar Deshayes 1830, Adanson, 1757 = Crassostrea tulipa Lamark, 1819)^21-24 is known to tolerate a wide range of salinity, dissolved oxygen, and temperature fluctuations in the intertidal zone. This species is distributed in the Tropical Eastern Atlantic (from Senegal to northern Angola) and the Western Atlantic (from Venezuela to Brazil), where it is commonly consumed by coastal populations.²⁵

In this study, we aimed to evaluate C. brasiliana as a useful biomonitor for essential metals (Cr, Cu, Fe, Mn, and Zn) and non-essential metals (Al, Pb and Cd) in the Paciência River estuary (PRE) in northeastern Brazil, along the Amazonian Equatorial coast. The selected metals reflect the vulnerability of the estuary to pollution from urbanization and agricultural sources. Including these metals facilitates comparisons with other studies and enhances our understanding of contamination patterns.

Our objectives were to (i) measure metal accumulation in the whole-body soft tissues of C. brasiliana at five sites, (ii) compare metal bioaccumulation across space and seasons (rainy and dry), (iii) establish a reference baseline for future studies on oysters, and (iv) assess potential risks associated with oyster consumption from the PRE on the Amazonian Equatorial coast.

 

EXPERIMENTAL

Materials and methods

Study area

The Paciência River estuary (PRE) in the Amazon Region is situated on the northeast coast of São Luís Island (1,412.4 km²) in the state of Maranhão (02º23'05" - 02º36'42"S and 44º02'49" - 44º15'49"W), Brazil. It flows into the Arraial-São José Estuarine Complex and is under pressure from anthropogenic activities such as agriculture, e.g., fruit and vegetable cultivation, artisanal fishing, deforestation, and mineral exploration.

This region experiences a rainy tropical monsoon climate, characterized by two very distinct seasons marked by precipitation (Figure 1): a rainy season (January to June - summer) and a dry season (July to December - winter).²⁶ The hydrology is influenced by a vertically well-mixed semi-diurnal regime, with an average tide of 4.6 m (15 ft) and can reach heights of up to 7.2 m (23 ft).²⁷ As for coastal vegetation, extensive mangrove forests of Rhizophora mangle L., Laguncularia racemosa L. Gaerth f., Avicennia germinans L. Stearn, and Avicennia schaueriana Stapf are observed between tidal channels and streams.

 

 

During the collection months, rainfall was 1.8 mm in the dry season of 2017 and 729.1 mm in the rainy season of 2018, both below their historical averages of 15.4 mm and 852.6 mm, respectively (Figure 1). It is worth noting that the pattern of rainfall and drought persisted throughout the sampling seasons. Rainfall data were obtained from INMET (National Institute of Meteorology, Brazil).²⁸

Collection sites and sampling strategy

Sampling was carried out across the PRE, during the dry season (campaign 1 and 2: October/November 2017) and rainy period (campaign 3 and 4: March/April 2018), during neap tide. Five distinct sampling sites were chosen (Figure 2) to account for physical and chemical gradients. Each sampling site was visually characterized based on the anthropogenic activities observed at the sites (Table 1).

 

 

 

 

Surface (20 cm below the air-water interface) and bottom water samples were collected in duplicate using polyethylene and Van Dorn bottles, respectively. Salinity, temperature and dissolved oxygen (DO) concentration were determined in situ using a previously calibrated refractometer (Instrutherm, model RTS­101 STC) and a portable multiparameter (Hanna, model HI 9146; temperature: ± 0.2 ºC and DO: ± 1.5% FS (full scale)), respectively. The water samples were immediately stored in an isothermal box with ice and transported to the Laboratory of Ecotoxicology (LabEcotox) at the Federal University of Maranhão (UFMA). In the laboratory, pH was measured using a calibrated benchtop pH meter (Hanna, model HI 2221; ± 0.01). Suspended particulate matter (SPM) was determined in filtered water samples using glass fiber filters (Millipore AP040, 47 mm diameter) according to gravimetric procedures.²⁹ All parameters were measured in duplicate.

According to Lopes et al.²⁴ only C. rhizophorae and C. brasiliana (= C. gasar) occur along the Maranhão coast. Following the criteria outlined by Amaral and Simone³⁰ and the observable shell features, juvenile oysters C. brasiliana (with a shell length of 4 to 6 cm) were carefully detached from mangrove roots (P1, P2, P3, and P5) and rock beds (P2, P3, and P4) using a stainless steel cleaver. After sampling, the organisms were placed on ice and immediately transported to the LabEcotox. Upon arrival, they were sorted (n = 10 at each site) to select juvenile organisms of similar sizes. Subsequently, the soft tissues were removed from the shells using plastic tongs and porcelain knives. They were then immersed in a saline solution (40) to facilitate the evacuation of feces, pseudofeces, and other particulate matter.³¹

Digestion and metals analysis

For metal analysis, the digestion procedures were based on the previous study by Ferreira et al.¹⁶ Briefly, the samples were dehydrated in an oven at 60 ºC until reaching constant weight, then powdered using mortar and pestle, and stored until further digestion. Approximately 0.1 g of dried sample was weighed into a Teflon tube and digested with 2 mL of HNO₃ (65%, Quimex) in a microwave oven (One Touch Technology, model Mars 6). After the samples cooled down to room temperature, the Teflon tubes were carefully opened, and the volume was adjusted to 30 mL with deionized water (Milli-Q) before metal analysis.

The determination of eight metallic elements (Al, Fe, Mn, Zn, Cu, Pb, Cd, and Cr) was conducted following the same precision and accuracy control outlined by Vilke et al.³² The metal content in the tissues of the oysters was subsequently analyzed using inductively coupled plasma optical emission spectrometry (ICP OES, ICPE Shimadzu 9800). Calibration of the results was performed based on standard curves constructed from ICP multi-element standard solutions (SpecSol), and the results were expressed in μg g^-1 of dry weight (dw). The recovery of the studied elements (Cd, Cu, Fe, Mn, Pb and Zn) performed with certified reference materials (CRMs) (TORT-3: Lobster Hepatopancreas and ERM^®-CE278k: Mussel) was 88, 87, 96, 88, 76 and 103%, respectively.

Health risk assessment

The estimated daily intakes (EDI), non-carcinogenic risk (target hazard quotient, THQ), and hazard index (HI) were calculated following standard assumptions for the calculations.^33-35 The EDI (μg person^-1 day^-1) of exposure doses of metals through the consumption of oysters was calculated using the Equation 1:

where, MC is the metal concentration (µg g^-1 wet weight), the consumption rate (g person^-1 day^-1) is based on the global average seafood consumption of 20.7 kg person^-1 year^-1 in 2022, according to Food and Agriculture Organization of the United Nations (FAO).³⁶ Since no specific data on oyster consumption are available for the study area or surrounding regions, seafood or fish consumption rates are commonly used in the literature to estimate human exposure to contaminants in edible aquatic organisms.^37-39 Following this approach, we considered two consumer scenarios: average level consumers (ALM, 56.7 g person^-1 day^-1) and high level consumers (HLM, 113.4 g person^-1 day^-1) for an individual with a body weight of 60 kg.

The THQ was used to assess the level of health risk (non-carcinogenic) in the local human population due to metal exposure. The oral reference dose (RfD_o) is the quantity that a consumer can be constantly exposed to without being affected. The THQ ≥ 1 indicates some noticeable potential risk to human health, requiring protective measures to be taken. However, THQ ≤ 1 means that the daily exposure at this level is unlikely to cause non-carcinogenic risk in humans. The THQ was described by Equation 2:

where, RfD_o values (µg person^-1 day^-1) are 1,000 for Al, 1 for Cd, 3 for Cr, 40 for Cu, 700 for Fe, 140 for Mn, 3.5 for Pb, and 300 for Zn.^40,41

The HI (Equation 3) was evaluated to assess the non-carcinogenic effects caused by the combined effect of metals on consumers. The HI was calculated as follows:

Data treatment and statistical analysis

The data were tested for normality using the Shapiro-Wilk test and for homoscedasticity using the Levene test. Analysis of variance (ANOVA, two-way, with Tukey's pairwise comparison) was employed to assess the significance of differences (p < 0.05) in concentrations among oysters from different sampling sites and seasons, considering the assumptions of normality and homogeneity of variances. In cases where the assumptions of normality were not met, the Kruskal-Wallis nonparametric test (p < 0.05) was utilized, with Dunn's post hoc test for pairwise comparisons between groups. The tests were performed using SigmaPlot, version 14.5 (Systat Software Inc.).⁴² Principal component analysis (PCA) and Pearson's linear correlation coefficient (r) were used to examine associations between metals and environmental parameters. The following classifications were applied for Pearson's coefficient: r = 0.1 to 0.3 (weakly correlated); r = 0.4 to 0.6 (moderately correlated); and r = 0.7 to 1 (strongly correlated).⁴³ Statistical analysis was conducted using the free software Jamovi, version 2.2.5 (Jamovi Project).⁴⁴

Metal values expressed in µg g^-1 wet weight (ww) were obtained by dividing µg g^-1 dry weight (dw) by the correction factor 6.8, which is commonly used for oysters of the genus Crassostrea.^45,46 This adjustment allows for comparison with the standards set by the Brazilian National Health Surveillance Agency (ANVISA)^47-49 and other international control agencies, such as Food and Agriculture Organization/World Health Organization (FAO/WHO),⁵⁰ Food Safety Authority of Ireland (FSAI),⁵¹ Canadian Food Inspection Agency,⁵² US Food and Drug Administration (FDA),⁵³ and Food Standards Australia New Zealand (FSANZ).⁵⁴

 

RESULTS AND DISCUSSIONS

Environmental parameters

Environmental parameters such as temperature, salinity, pH, DO and SPM were examined for two seasons (dry and rainy) along the estuary (Table 2). Water temperature showed minimal vertical stratification, with a difference of 0.8 ºC at site P5 during the dry season. Overall, temperature variations were 2.0 ºC during the dry season and 2.6 ºC across the dry and rainy seasons, respectively. Salinity and pH increased towards the lower part of the estuary (P1 to P5), while DO exhibited variations along this gradient, with lower values at P5 compared to P3 and P4 during the dry season. Salinity ranges from 0 to 22 (surface) and 11 to 25 (bottom) in the rainy season, while during the dry season, it varies from 9 to 36 (surface) and 17 to 36 (bottom). The pH values showed little variation from the surface to the bottom and across seasons, ranging from 7.30 to 7.80 in the rainy season, and 7.50 to 8.07 in the dry season. Dissolved oxygen (DO) concentrations range from 2.7 to 5.3 mg L^-1 in the rainy season while in the dry season they range from 4.4 to 7.6 mg L^-1. With no spatial marked pattern, the SPM was higher in the bottom with higher values at P4 (91.3 mg L^-1) and P5 (91.3 mg L^-1) in the dry season, while during the rainy season, the highest concentrations were observed at P2 (358.3 mg L^-1), and P3 (163.5 mg L^-1).

 

 

Water temperature followed a typical pattern for tropical estuaries in the equatorial zone, where small seasonal variations are expected in coastal waters.^55-57 Sampling revealed a salinity gradient between surface and bottom waters, likely resulting from increased fluvial inflow and limited saline intrusion, driven by seasonal variations. The pH measurements indicated that the waters of the PRE varied from the neutral to slightly alkaline range.

The highest DO values observed during the dry season may be attributed to increased photosynthetic activity, as reported by Cavalcanti et al.²⁷ in the PRE, who found diatom blooms and higher chlorophyll-α concentrations. In our study, field observations revealed widespread sewage discharge throughout the estuary, compounded by other contamination sources including shipyards, small vessel traffic, improper waste disposal, agricultural settlements, and urbanization.

Conversely, the low DO values recorded during the rainy season suggest degradation of organic matter from adjacent areas, such as mangroves, and inputs from domestic and urban sewage. According to the limits established by Brazilian environmental legislation (Resolution CONAMA No. 357),⁵⁸ the DO in class 2 brackish waters should not be lower than 5 mg L^-1 in any sample. During the rainy season, DO values were generally below this threshold, except for P5, while in the dry season, DO levels at P1, P2, and P3 (bottom) were below 5 mg L^-1. Oliveira et al.⁵⁹ found a high number of pathogenic organisms near P1 and P2, where the lowest DO values were observed in our study. According to Neff et al.,⁶⁰ DO values as observed at P1 (min 2.9 mg L^-1) could be classified as a moderate hypoxia condition (2 to 5 mg L^-1).

The observed patterns of high SPM and low DO concentrations suggest a spatial shift in the turbidity maximum zone (TMZ) between the dry and rainy seasons, indicating sediment entrapment due to hydraulic dam due to the change in water masses density. In Amazonian estuaries, this phenomenon may arise from interactions between tidal variations and river discharge.⁵⁶ During the rainy season, in addition to tidal fluctuations, fluvial inputs prevail, serving as a significant source of particles for the estuary.

Consequently, these factors contributed to the entrapment and redistribution of the suspended particles near the narrower and more confined region of the estuary, according to Yu et al.⁶¹ in the macro-tidal estuary of the Yalu River. The bottom sediment resuspension is likely a significant contributor to metal release and, consequently, to the contamination of macro-tidal estuaries and coastal lagoons, including oysters habitats.¹

Metal bioaccumulation

The bioaccumulation of essential metals (Cr, Cu, Fe, Mn and Zn) in the soft tissue of C. brasiliana was found in higher concentrations than non-essential metals (Cd and Pb), except for Al, which exhibited higher concentration among all the metals analyzed.

Regarding the spatial pattern of metals bioaccumulation, Al, Fe, and Zn (Figures 3a­3c) increased towards the upper estuary (P5 to P1) in both seasons, while for Mn and Pb, this pattern was observed only in the dry season (Figures 3d and 3h), and for Cr only in the rainy season (Figure 3m). In contrast, the bioaccumulation of Cd (in both seasons), Cr (dry season) and Cu (dry season) increased significantly towards the lower estuary (P1 to P5, Figures 3g-3f). Manganese, Pb and Cu showed no spatial pattern in the rainy season, with the highest levels of Mn, Pb and Cu observed at P3 (Mn 16.9 and Cu 36.3 µg g^-1) and P5 (Pb 0.26 µg g^-1).

 

 

During the rainy season, Al, Fe, Zn and Cr (except at P5) and Cu (except at P4 and P5) showed higher bioaccumulation compared to the dry season. However, Mn bioaccumulated more during the dry season at P1, P2, and P4. Cd did not exhibit significant seasonal variation in space, with an exception for P4, which increased bioaccumulation during the dry season. For Pb, no differences were observed at P2, P3, and P4, with higher bioaccumulation noted at P1 during the dry season and at P5 during the rainy season.

Although limited (n = 5), the sampling sites provided valuable initial insights into the relationships between physicochemical parameters and metal bioaccumulation. The sample size was appropriate for the objectives of the study, allowing us to identify meaningful trends that can guide future research with a broader sampling approach. Spatial oyster bioaccumulation revealed patterns across seasons that help identify the sources and behavior of metallic elements in the estuarine environment. During the dry season, elements like Al, Fe, Zn, Mn and Pb exhibited a negative correlation with salinity, pH, DO and SPM gradients. In the rainy season, Al, Fe, Zn, and Cr exhibited an increase in bioaccumulation values along the estuary, showing a negative correlation with salinity, pH and DO, and a positive correlation with SPM. These results clearly showed the contribution of freshwater discharge on metal distribution in estuaries and a significant input of these elements during the rainy season.

They also demonstrate the protective effects of environmental parameters, such as salinity, pH, and DO, on metal bioavailability due to the influence of these parameters on metal partitioning and speciation.^62-64 The positive correlation of Al, Fe and Zn with SPM during the rainy season and negative correlation during the dry season suggests that SPM acts as both a source and a sink for metals, influencing their availability to suspension-feeding organisms like oysters.³³

In contrast, the bioaccumulation of Cd (in both seasons), Cr (dry season) and Cu (dry season) exhibited an unexpected spatial pattern where the bioavailability and bioaccumulation of elements increased with salinity, pH, DO and SPM gradient. Therefore, during the dry season, the penetration of the salt wedge is greater, and organic matter concentrations are higher, favoring the formation of organometallic and chlorine complexes.

This highlights the contribution of the SPM phase as a source of these elements to oysters and suggests possible additional sources of metals along the estuary. Increases in water parameters such as salinity, lead to the association of metals with larger particles (< 0.7 μm), which are then concentrated in sediments acting as temporary reservoirs.⁶⁵ While SPM fluctuations showed an irregular pattern in the PRE, potentially due to sediment remobilization and increased water turbidity, the key to understanding metal bioaccumulation in oysters lies in their feeding behavior.

As filter feeders, oysters (C. gasar) ingest SPM from the water column, selectively retaining beneficial nutrients for growth.⁶⁶ Previous studies^1,67,68 indicate that the ingestion of sediments and microalgae is a more significant pathway for metal bioaccumulation compared to contributions from dissolved phases in the water. Analysis of stomach contents indicated that the diet of C. gasar mainly consisted of phytoplankton (72.7%, predominantly Diatomophycea at 33.5%), sediment particles (22.9%), detritus (1.4%) and protozoans (0.01%).⁶⁶ Previous studies^69,70 have shown that metal bioaccumulation in oysters tends to increase with their ingestion of phytoplankton.

Phytoplankton accounted for 30 to 75% of Cd and 30 to 80% of Zn assimilation in Crassostrea rivularis, with low elimination rates at a constant of 0.14 day^-1 (Cd) and 0.01 day^-1 (Zn),⁶⁹ which were the metals with highest concentrations in oysters from PRE. Similarly, with diatoms as food, the Cu assimilation efficiency was highest for oysters (85%) compared to clams (73%) and black mussels (67%).⁷⁰ Although there is no information on Cr assimilation via feeding on diatoms by oysters, previous findings⁷¹ indicate that Cr was the least assimilated metal, with assimilation efficiencies ranging from 10 to 16% in green mussels and 11 to 24% in clams. Neff⁷² reported that approximately 59% of the Cd bioaccumulated by Crassostrea virginica oysters comes from diatoms, influenced by environmental Cd concentrations.

Additionally, several authors have observed the highest filtration rates between salinities 20 and 35 for Crassostrea iredalei,⁷³ Crassostrea gigas,⁷⁴ Crassostrea corteziensis,⁷⁵ and Saccostrea glomerata.⁷⁶ Cavalcanti et al.²⁷ demonstrated that seasonal changes and environmental gradients affect the distribution and abundance of diatom blooms in the PRE, with the dry season providing better conditions for the growth, and higher growth near P4 and P5. This could explain the highest bioaccumulation at P3 to P5 of Cd (in both seasons), Cr, and Cu during the dry season, and the highest bioaccumulation of Pb during the rainy season.

Regarding Mn, Pb, and Cu in the rainy season, the observed bioaccumulation did not appear to be influenced by the physicochemical patterns (spatial) of the PRE, with the highest levels in P3 and P5, indicating possible point sources. Conversely, Cd and Pb (across all seasons) did not exhibit significant differences between seasons, suggesting the presence of perennial diffuse sources of these elements for the estuary. As a whole, sources of Cd, Cr, Cu, Mn, Pb, and Zn might be potentiated by anthropogenic activities in addition to natural inputs, such as erosion and weathering, into the aquatic environment. The discharge of industrial effluents, agricultural activities, urban runoff, and domestic sewage from the surrounding area are among the most common sources of these elements for the estuarine system,^77-79 and we observed similar sources in the PRE. Other anthropogenic sources for the environment include the incineration or combustion of fossil fuels, fertilizers, pesticides, fungicides, and leaching from landfills.⁸⁰

The Pearson correlation matrix and PCA analysis revealed distinct seasonal patterns in metal bioaccumulation in the estuary (Figure 4). During the dry season, Al, Fe, Mn, and Zn exhibited strong positive correlations with each other (r > 0.7) and strong negative correlations (r < -0.7) with Cd, salinity, and pH (Figure 4b). Additionally, Cd, Cr, Cu, salinity, and pH were significantly positively correlated. Chromium showed a strong positive correlation with SPM, while Pb correlated positively with Zn. In contrast, during the rainy season, Al, Cr, Fe, and Zn were positively correlated with each other and negatively associated with Cd, salinity, pH, and DO (Figure 4d), showing a pattern similar to the dry season, with slight differences.

 

 

The PCA biplot analysis further illustrated these relationships, with Al, Fe, Mn, Pb, and Zn associated with each other and inversely related to Cd, Cr, Cu, salinity, and pH during the dry season (Figure 4a). Conversely, in the rainy season, Al, Fe, Cr, Zn, and SPM were associated with each other and inversely related to Cd, Mn, Pb, DO, salinity, and pH (Figure 4c). Sampling sites P1 and P2 (upper estuary) were similar but differed from P4 and P5 (lower estuary), with P3 (middle estuary) fluctuating between these groups.

The positive associations illustrated by PCA and Pearson's correlation can indicate that some metals probably originate from the same sources, such as Al-Fe-Mn-Zn, Cd-Cr-Cu, and Pb-Zn during the dry season, and Al-Fe-Cr-Zn, Cu-Mn, and Pb-Cd during the rainy season. For instance, Gu et al.⁸¹ attributed the grouping of Cd, Cr, and Cu mainly to chemical fertilizers in the Pearl river estuary (China), where phosphate fertilizers contain high levels of Cd derived from phosphate rocks. Similarly, higher concentrations of Cd in oysters from Cat Ba Island (Vietnam) have been associated with the intensive use of fertilizers in surrounding regions and the discharge of untreated domestic and municipal sewage effluent.⁸²

In a sediment core study conducted in the Arraial-São José Estuarine Complex, where the Paciência river discharges, Azevedo⁸³ noted significantly higher levels of Cu, Zn, Cr, and Pb at the mouth of the PRE (near P5), compared to other rivers within the estuarine complex. In the PRE region, particularly at P5 and its surrounding area, numerous land settlements are utilized for family farming and vegetable production. Similarly, Ribeiro et al.⁸⁴ found evidence of contamination by domestic sewage through Escherichia coli analysis in oysters at Cumbique Igarapé, a major tributary of the PRE near P5. Furthermore, they detected levels of dissolved Fe, Cd, Cr, and Pb in seawater above the limits permitted by Brazilian legislation (CONAMA No. 357, for class 2 waters), suggesting the potential influence of landslides as a source of these metals. It is plausible that the use of fertilizers, pesticides, fungicides, and the proximity to open dumps is more pronounced at P5, contributing to the increased bioaccumulation of these elements in oysters.

Overall, there is a weak correlation between the bioaccumulation of metals and toxic effects in bivalves.^85,86 Oysters can survive in environments highly contaminated with metals, bioaccumulating these elements rapidly without displaying visible toxic effects.⁸⁷ Comparing the metal levels found in the oysters from the PRE (Cd, Cr, Cu, Pb, and Zn) with those reported in the literature,^86,88,89 only Cr (0.1­5.5 µg g^-1 dw) was bioaccumulated in concentrations similar to those found in the gonads of Crassostrea hongkongensis (3.3­14.5 µg g^-1 dw).⁹⁰ These authors noted that such bioaccumulation led to adverse effects on the reproduction, recruitment, gametogenesis, spawning, and gonadal condition index of oysters.

In light of these findings, there is a critical need for further studies to assess the physiological and population-level effects of metal bioaccumulation in oysters from the PRE. Additionally, understanding the tolerance thresholds of these contaminants is essential, as they not only pose risks to human health but also threaten the recruitment and sustainability of oyster populations, which are vital from an ecological and economic point of view.

Health risk assessment

Brazilian Health Regulatory Agency (ANVISA) establishes maximum metals concentration in bivalve mollusks (Cd = 2.0 and Pb = 1.5 µg g^-1 ww);⁴⁷ (Cr = 0.1, Cu = 30.0 and Zn = 50.0 µg g^-1 ww)^48,49 for human consumption. Oysters naturally accumulate certain metals, such as Cu and Zn, particularly in their gills, as part of their physiological processes.⁹¹ However, the levels of Cr and Zn exceed the allowable limits set by ANVISA in almost all sites (except for Cr at P1, P3 - Oct/2017; Zn at P5 - Nov/2017) and sampled seasons (Table S1, presented in Supplementary Material). Therefore, for Cr, 56% of the analyzed oysters exceeded the safety limit in October and 100% in November, March and April. For Zn, the percentages were 89% in October, 88% in November, 100% in March, and 84% in April (Table S2, Supplementary Material).

Human health index values (Table 3), such as EDI, were estimated using general seafood consumption rates due to the lack of specific data on oyster intake in the study area. This ensures a comprehensive assessment, though differences in dietary habits could influence the final values. EDI exceeded the corresponding value of the RfD_o for Zn (300 µg person^-1 day^-1) in the HLM category at P1 (384), P2 (520), and P3 (360 µg person^-1 day^­1) in the rainy season. The THQ, exceeded permissible limits (THQ > 1) for Zn in the HLM category at P1 (1.3), P2 (1.7), and P3 (1.2 μg person^-1 day^­1), suggesting a potential risk to human health in the rainy season. A THQ < 1 does not imply the absence of risk from oyster ingestion, since the cumulative impact of the elements may lead to a HI ≥ 1. In this way, the highest HI values for ALM were observed during the rainy season at P1 (1.1), P2 (1.3), and P3 (1.0 μg person^-1 day^-1). For HLM, the highest HI values were observed at all sites during both the dry and rainy seasons (Table 3).

 

 

Considering that oysters from PRE are used as a source of food and income, the bioaccumulation of metals in these organisms can pose a risk to the local population, as evidenced during the sampling campaign. The concentrations of Cr (to all sites during March and April 2018) and Zn (at P2 in April 2018) exceeded the tolerance limit allowed by Brazilian legislation by more than 5.2 and 5.7-fold, respectively.

As previously reported by Yuan et al.⁹² for Zn, its excess can be toxic to humans due to the generation of hydroxyl radicals. Consequently, Zn can induce symptoms such as nausea, bloody diarrhea, epigastric pain, abdominal cramps, altered blood lipoproteins (increasing low-density lipoprotein LDL and decreasing high-density lipoprotein, HDL), copper deficiency, anemia, and altered immune response (decreasing lymphocyte stimulation, chemotaxis, and phagocytosis) in humans.⁹³ In organisms, Cr³⁺ is recognized as an essential element, however, excessive Cr³⁺ intake can disrupt essential mineral balance (e.g., copper, zinc, calcium, and iron) and damage cell organelles.⁹⁴ Unlike its trivalent form, Cr⁶⁺ is considered more toxic and is linked to serious health issues, including cancer and deoxyribonucleic acid (DNA) damage.^92,95 Regarding Cd, despite the element being below the limit established by the ANVISA for human consumption, it is classified as group 1 of the most carcinogenic metals⁹⁶ and can be toxic even in low concentrations.⁹⁷

Although the THQ did not indicate a non-carcinogenic health risk for most of the elements studied (except for Zn at P1, P2 and P3 in rainy season), the HI revealed a risk (> 1) due to the co-exposure of metals in oysters from all PRE stations (in both seasons) when the maximum daily level of consumption is considered. It is worth mentioning that the estimated daily intakes (EDI) of metals through the consumption of oysters were calculated using the average and high levels of global consumption per day (56.7 and 113.4 g, respectively, according to FAO).³⁶ However, the daily seafood consumption per capita in riverine populations may be higher depending on the global region. According to Isaac et al.,⁹⁸ riverine communities from the Amazon consumed an average of 463 g of seafood per capita per day.

One possible alternative to reduce risks to communities that depend on seafood for subsistence is the depuration process before consumption. Butler and Timperley⁹⁹ observed that C. gigas eliminates half of the ingested Cd within 23 to 60 days after ingestion as a measure of physiological control. Geffard et al.¹⁰⁰ observed a reduction of half of the Cd bioaccumulated in transplanted oysters from a polluted site to a relatively clean site, between 86 and 251 days. For Zn, Wang et al.⁸⁶ observed that oysters can eliminate 50% of the Zn content bioaccumulated in 16 days (Crassostrea angulata normal) and 30 days (C. angulata green-colored) under purification in clean seawater. Moreover, Tan et al.¹⁰¹ used a toxicokinetic model to predict the biological half-life in C. hongkongensis; they found Zn (58 days), Cd (94 days), Cr (105 days), Pb (114 days), and Cu (408 days), inferring a long residence time of months to even longer. However, it is essential to recognize that depuration does not mitigate the broader ecological threats. Without substantial efforts to reduce contaminant releases and protect oyster populations, the long-term availability of oysters and other marine resources may be jeopardized.

Comparative analysis with other estuarine regions

The comparative analysis of metal bioaccumulation in estuaries across different regions (Table 4), highlights both the similarities and differences in metal oyster bioaccumulation across the estuaries. In our research, while Zn and Cr exceeded Brazilian legislation, metals concentrations in both seasons were notably lower than those reported in other research in Brazil, except for Al. However, the higher Al levels in PRE were attributed to the natural richness of Al₂O₃ in the sedimentary basin.¹⁰² Similarly, Zn levels were comparable to those reported in Mailao Habour (Taiwan)⁸⁵ and Gironde estuary (France).¹¹¹

 

 

Similarly to the present study, Amado-Filho et al.¹⁰³ observed higher levels of Al, Fe, Cd, Cu, and Zn in C. rhizophorae from Todos os Santos Bay (TSB, Brazil) during the rainy season compared to the dry season. This increase was potentially attributed to freshwater discharge that carries anthropogenic inputs from surrounding drainage basins. Unlike PRE, TSB is situated in a metropolitan area with over 2.6 million inhabitants and significant industrial activities.

Considering the Brazilian legislation and the sum of Zn, Cu, Cr, Cd, and Pb accumulated in oysters from each region in Brazil, the most contaminated environments were Santa Cruz Cabrália, Santos Bay, and Todos os Santos Bay. Conversely, the least contaminated environments were the Laguna Estuarine System, Meirim estuary, Lagoon Complex, and PRE. Only the Laguna Estuarine System exhibited values below the consumption limits for all elements analyzed (Zn, Cu, Cd and Pb). Cd concentrations in all studies remained consistently below regulatory limits. While Pb and Cu exceeded permissible levels exclusively in Todos Santos Bay and Santa Cruz Cabrália, respectively. In contrast, Cr and Zn emerged as the primary environmental concerns, with their accumulation rates surpassing consumption thresholds in most of the investigated regions, except for Zn in the Laguna Estuarine System and Cr in the Potengi estuary.

Oysters are known to be the most efficient bioaccumulators of Zn among coastal invertebrates.¹⁰⁴ Bodin et al.¹⁰⁵ found that Zn is the predominant element in C. gasar, constituting about 97% of the total analyzed metals in Sine Saloum estuary (Senegal). On the other hand, Cr is not typically considered to be highly accumulated by oysters, which implies that its presence in high levels is due to environmental inputs by human activities.¹⁰⁶

By contextualizing PRE contamination levels within a broader regional framework, this comparative analysis provides valuable insights for future research and pollution management in estuarine systems. Understanding these regional differences is crucial for accurate risk assessment and the development of targeted mitigation strategies adjusted to specific environmental conditions.

 

CONCLUSIONS

In conclusion, oysters have proven to be effective biomonitors for assessing contamination in estuaries. The Paciência River estuary is a dynamic environment significantly impacted by metal contamination in both seasons. Our study reveals substantial variations in metal bioaccumulation within C. brasiliana oysters, with higher levels recorded during the rainy season. These findings suggest how environmental parameters (salinity, pH, DO, and SPM) may influence contamination patterns and underscore the potential impact of various contamination sources, ranging from freshwater and continental inputs upstream (at P1) to downstream sites near the estuary mouth (P5).

Chromium and Zn were the most environmental issues exceeding the maximum tolerance limits set by ANVISA for consumption; however, the values for human health risks due to oyster consumption may be overestimated, as the ingestion rates were based on the consumption of fish. Further investigation is needed to understand the factors and mechanisms that regulate the concentrations of Cr and Zn in oysters, beyond the contamination levels present at the sites. Regarding health risk assessment, the EDI and THQ values raise concerns specifically for Zn. Additionally, the HI values greater than 1 at some sites during both seasons suggest non-carcinogenic health risks for consumers. The elevated Cr levels also raise concerns about potential reproductive implications for oysters in the PRE, as observed in comparative analyses with global literature.

In summary, this study underscores the importance of environmental biomonitoring to mitigate contamination issues and provides a reference background for future investigations. Long-term biomonitoring efforts encompassing various aquatic compartments are crucial to accurately assess pollution levels in the PRE. Future research endeavors should prioritize the investigation of factors such as the depuration times of oysters intended for human consumption and the evaluation of ecological and physiological responses, including biomarker assessments, to metal contamination in the PRE.

 

SUPPLEMENTARY MATERIAL

Tables 1S and 2S related to the results obtained in this work are available at http://quimicanova.sbq.org.br, as PDF file, with free access.

 

ACKNOWLEDGMENTS

We would like to thank Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA), Universidade Federal do Maranhão (UFMA), specifically the Postgraduate Program in Oceanography (PPGOceano), Laboratory of Ecotoxicology (LabEcotox), Laboratory of Ficology (LabFic), Laboratory of Coastal, Estuarine Hydrodynamics and Inland Waters (Lhiceai), Pesticide Waste Analysis Center (NARP) and Chemistry Analytical Center (CCET/UFMA), for providing infrastructure and technical assistance throughout the study. Special thanks are due to the dedicated team at LabEcotox for their invaluable assistance during fieldwork and sample processing. This work was supported by FAPEMA (universal process - 01315/17; MSc scholarship - BM 02181/17) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, agreement 23038.051628/2009-98), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 440850/2020-7).

 

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Associate Editor handled this article: Cassiana C. Montagner