Mangroves, which are coastal ecosystems located in transitional areas between the terrestrial and marine environments and subjected to tidal regimes, are highly productive environments associated with the provision of a wide range of ecosystem products and services (Barbier et al., 2011; Trégarot et al., 2021). Mangrove soils are characterized by pH values close to neutral, suboxic conditions on the surface and anoxic conditions in depth, and generally high contents of organic matter (OM), Fe oxyhydroxides on the surface, and Fe sulfides in depth (Otero and Macías, 2010). These properties and composition lead to a high reactivity, which promotes fixation of trace elements (Otero et al., 2010), turning these ecosystems into major sinks for pollutants (Marchand et al., 2016).

Todos os Santos Bay (BTS), the second largest bay in Brazil (Hatje and Andrade, 2009), hosts vast mangrove areas (Hadlich et al., 2008), many of them close to urban and industrial areas, oil refineries, ports, and sea terminals (Hatje et al., 2009). Previous studies have shown high concentrations of pollutants in this area, including hydrocarbons (Queiroz and Celino, 2008a, b; Silva et al., 2014) and trace elements, mostly copper (Hatje and Barros, 2012; Andrade et al., 2017). Despite pollution having been reported in the BTS (Milazzo et al., 2014; Rocha et al., 2016; Andrade et al., 2017), most of the studies carried out focused on total levels in soil and sediments, with works on bioavailability based mainly on the quantification of acid-volatile sulphides and metals extracted simultaneously (AVS-Me; Silva-Júnior et al., 2016; Brito et al., 2020). This technique has limitations with regard to the quantification of sulfides, with underestimation of concentrations in the system as well as of metallic bioavailability (Cooper and Morse, 1998; Luther, 2005), since it disregards other associations established between metals and other geochemical phases of soils/sediments (Huerta-Días and Morse, 1992; Otero and Macías, 2003). Unlike the AVS-Me technique, the sequential extraction of metals (Tessier et al., 1979; Rauret et al., 1998) allows analyzing the distribution of trace elements among the different geochemical fractions of the soil/sediment and favors a more comprehensive analysis of the mobility of each element and the processes that regulate its availability to the biota.

Copper can be found in association with different fractions, depending on geochemical conditions and the factors that control its speciation processes under conditions of higher or lower availability to the biota (Otero et al., 2000a; Chakraborty et al., 2015). Among the shellfish species extracted in the BTS, the crab Ucides cordatus is one of the main crustaceans exploited in this region (Soares et al., 2009; Mota et al., 2023) and one of the most consumed throughout the country (Pinheiro et al., 2016). It is considered a bioindicator of pollution in mangroves, as it bioaccumulates both organic pollutants and trace elements in its tissues (Nudi et al., 2007; Almeida et al., 2014; Silva et al., 2018; Ramos and Leite, 2022).

Considering the scarce information about Cu distribution among the geochemical fractions and its bioavailability, along with the importance of Ucides cordatus in the BTS, the objectives of this study were 1) to understand the geochemical behavior of this element in mangrove soils using a sequential extraction method and 2) to study the transfer of Cu to biota using the crab Ucides cordatus as a potential bioindicator species in this region.

The study was carried out in four mangrove sites (Figure 1). Two of them were located at the entrance of the bay (Cacha Prego, CP; and Ponta Grossa, PG), both in more undeveloped areas; another one located in the northeast region (Ilha de Maré, IM), close to industrial areas and maritime ports, and another one in the northern region (Pitinga, PT), in the Subaé River estuary, an area with a history of metal pollution associated with an abandoned metallurgical factory (Hatje et al., 2009; Rocha et al., 2012).

Mangroves in IM and CP are comprised of monospecific Rhizophora mangle (L.) forests, while PT and PG show mixed patches composed of R. mangle and Laguncularia racemosa (L.). These sites were selected to cover different sections of the BTS, thus analyzing potential spatial variations according to the degree of exposure to anthropogenic stressors, the composition of the different forests, and soil characteristics in each mangrove, both to establish baseline values and to better understand soil-to-biota transfer processes.

Twelve soil samples, each consisting of a pool of three individual samples, were collected at low tide during the dry season (DS: dry season 2020/2021) and during the rainy season (WS: wet season 2021) at depths of 0–5 cm (surface - S) and 15–30 cm (depth - D). Determinations of pH and Eh were performed in situ using a portable device (HI8424, Hanna Instruments) (Souza et al., 2007). The contents of sand and fine fraction (silt + clay) were determined by sifting through a 2 mm sieve to obtain the fine Earth fraction, then through a 0.05 mm sieve to separate the fine sand from the fine fraction (silt and clay particles) (Almeida et al., 2012. Souza et al. (2007), while total organic carbon (TOC) content was determined using an elemental analyzer (FlashEA1112, ThermoFinnigan). For TOC analysis, all soil samples had been previously subjected to acid attack using 10 mL HCl (1N) to remove carbonates (Schumacher, 2002; Nobrega et al., 2015). After adding the acid solution (HCl), samples were shaken for 1 h, then centrifuged to remove HCl, washed five times in deionized water, and dried in an oven at 60°C. All analyzes were carried out on the bulk sample, without separating the granulometric fractions.

To analyze total metal contents, 0.5 g of each sample were weighed and digested using 9 mL ultrapure nitric acid (HNO₃ 65%) and 3 mL ultrapure hydrochloric acid (HCl 37.5%) in a closed-vessel microwave system (Milestone; ETHOS EASY). Geochemical partitioning of Cu was performed using the BCR method (Rauret et al., 1998) and based on the sequence proposed by Tessier et al. (1979), as described below:

• Fraction 1: soluble, exchangeable fraction associated with carbonates (ExCa), extracted using 30 mL acetic acid (0.11 mol L⁻¹; pH = 4.5) and shaking for 16 h at 25°C.

The residual fraction (FR) value was calculated as the difference between the total concentration after microwave digestion and the sum of the bioavailable fractions (Σ F1 -> F4).

Twelve U. cordatus specimens were collected from each mangrove, washed in distilled water, and subsequently dissected to extract muscle, hepatopancreas, and gills. Tissues were freeze-dried for 72 h, ground, and digested on a digestion block with 7 mL ultrapure HNO₃ and 2 mL ultrapure H₂O₂. Mili-Q water was added to the digested material up to a total volume of 25 mL. Copper concentrations in each fraction, total content in soil, and contents in animal tissues were determined by inductively coupled plasma-mass spectrometry (Varian 820-MS ICP-MS). The analytical method used was validated using certified reference material for soil (SO-3, Canadian Certified Reference Material Project, Canada), leaves (NIST-1547), and animal samples (NIST-1577b, bovine liver), with recovery rates over 85%. The limit of detection for Cu was 0.005 ± 0.001 mg L⁻¹, determined as 2.5 times the standard error of the blank (Bruland et al., 1979).

The results were analyzed using descriptive statistics (Sigmaplot 12.0), and a Kruskal-Wallis non parametric test was applied at a 5% level of significance (XLSTAT 2014) to compare the results obtained from the different study sites. This non-parametric test was selected due to its higher robustness and lower statistical assumption requirements (Reimann et al., 2008). Likewise, discriminant analysis was applied to verify possible relationships between the studied variables.

The results of the discriminant analysis indicate hydrodynamic differences among mangroves, with higher sedimentation of organic matter, silt, clay and Fe oxides and hydroxides in lower energy environments such as IM (Lessa et al., 2000; Hatje and Andrade, 2009). Mangroves exhibited spatial variability, both in terms of soil parameters and of Cu contents in soils and biota. Except for PT, mean pH values were close to neutral, similarly to the results found in other study areas (Otero et al., 2017; Araújo et al., 2022), and redox conditions varied between anoxic (deep soil layers in IM, PG, and CP) and suboxic, within the commonly observed range in northeastern mangroves (Bomfim et al., 2015; Ferreira et al., 2022). A different temporal pattern was observed in PT, with an effect of seasonality on pH and redox potential, showing highly acidic (pH < 5) and neutral values during the dry and the rainy season, respectively, and with significantly higher redox conditions during the dry season. This seasonal behavior may be associated with its more sandy texture and higher relief with respect to the riverbed, factors that could promote faster drainage during drought periods and higher levels of soil aeration (Du Laing et al., 2009; Ferreira et al., 2022).

The seasonal pattern observed in this mangrove influenced geochemical partition of Fe and Cu, with higher contents in reduced fractions observed during the rainy season, consistently with the lower Eh values (Otero et al., 2000a; Otero et al., 2000b; Thanh-Nho et al., 2019). The more reduced conditions observed during this period may have promoted reduction of Fe and precipitation of sulfides, mainly pyrite (Berner et al., 1985; Huerta-Díaz and Morse, 1992; Otero et al., 2009), as well as gradual Cu pyritization (Huerta-Díaz and Morse, 1992; Morse and Luther, 1999). Cu contents in more mobile phases, such as Cu associated with CuExCa fractions or easily reducible Cu (CuCr), were only detected during the dry season, consistently with sulfide oxidation during this period, leading to soil acidification and to higher Cu solubility (Otero et al., 2000b; Machado et al., 2014; Araújo-Júnior et al., 2016). IM soils showed significantly higher total Cu contents than the remaining areas, and the values were above the background value for the BTS proposed by the Bahia Environmental Resources Center (Centro de Recursos Ambientais da Bahia, CRA) (23.0 ± 5.2 mg kg⁻¹, CRA, 2004), as well as above the legally established preventative threshold in Brazil (60 mg kg⁻¹, CONAMA, 2009), above the TEL (threshold effects level, or the minimum threshold for potential risk of toxic effects to biota: 18.7 mg kg⁻¹), and above the ERL (effects range low: 34.0 mg kg⁻¹) established by NOAA et al. (1999). These results are related to the location of the IM mangrove, close to the Port of Aratu, in Aratu Bay, through which a wide variety of products are transported, including fertilizers and Cu concentrates associated with the mineral exploitation of copper sulfides, thus leading to abnormally high Cu concentrations in this bay and its environment (Rocha et al., 2016; Andrade et al., 2017).

Additionally, the location of this mangrove in the middle section of the BTS implies that hydraulic energy conditions are low (Lessa et al., 2000), thus promoting the deposit of fine particles and, consequently, the accumulation of metals (Kumar and Ramanathan, 2015; Bastakoti et al., 2018). The results from partitioning demonstrated that the majority of Cu is found in the oxidizable fraction, consistently with the high tendency of Cu to form stable complexes with organic matter, even forming strong complexes in reducing conditions (Zhou et al., 2010; Chakraborty et al., 2015), or/and to precipitate as sulfides in sulfate-reducing environments (Huerta-Díaz and Morse, 1992; Otero et al., 2000a; Otero et al., 2000b; Thanh-Nho et al., 2019). IM soils showed a higher concentration of Fe and Cu associated with the oxidizable fraction than the other mangroves. However, seasonal changes in redox status may interfere with the stability of this fraction and its role as a metal sink, since pyrite can undergo oxidative dissolution, thus releasing a major portion of the adsorbed elements into more labile phases (Otero and Macías, 2002; Araújo Júnior et al., 2016). However, our results, despite showing a lower percentage of Fe and Cu associated with the reducible fraction, did not show any significant seasonal changes (Figure 2). More specifically, factors such as local flooding patterns and activity of the biota may promote sulfide oxidization and Cu release in a readily available form (Machado et al., 2014; Araújo-Júnior et al., 2016). Likewise, oxygen flow through the aerenchyma of mangrove plants into the rhizosphere substantially affects redox conditions, promoting soil aeration and, consequently, the system’s evolution towards oxic environments (Pi et al., 2009; Sarker et al., 2021).

Consistently with Cu values in mangrove soils, high concentrations were detected in U. cordatus tissues in IM, mainly in the gills and hepatopancreas and during the dry season, which suggests higher Cu bioavailability in this area and during this season. The higher Eh values recorded for this period are consistent with higher soil aeration levels leading to sulfide oxidation and system acidification, with increased Cu bioavailability (Otero et al., 2000a). Moreover, the greater activity levels of U. cordatus during the dry season, related to its breeding period (Ferreira et al., 2007b; Pinheiro et al., 2016), could have promoted higher levels of burrowing activity (bioturbation) and, consequently, greater soil aeration, with an impact on biogeochemical conditions and on the distribution of metals among fractions (Ferreira et al., 2007a; Araújo-Júnior et al., 2016), which could also have contributed to higher Cu bioavailability and its consequent assimilation. High Cu concentrations were also found in crustaceans collected in PT, again in the gills and hepatopancreas and mainly during the dry season, although total contents in soils were low. Moreover, values in the more mobile fractions were highest during the dry season, related to higher Eh values, which suggests a higher bioavailability during this period (Otero et al., 2000a; Araújo-Júnior et al., 2016). The results observed in this mangrove suggest that the risks associated with bioavailability are mainly likned to biogeochemical processes in soils controlling metal partition instead of being only related to total contents in soils, with contents in the more labile fractions acting as better environmental indicators than total metal contents with respect to the risk of bioaccumulation.

The high values observed both in gills and in hepatopancreas are consistent with other studies (Vedolin et al., 2020; Ramos and Leite, 2022), that have also demonstrated a tendency towards a high accumulation of Cu in these tissues in environments with high exposure to metals. Firat et al. (2008), in Iskenderun Bay, an area with an intense agricultural and industrial activity, found high Cu concentrations in gills (827 ± 92.6 µg g⁻¹) and in hepatopancreas (935 ± 50.5 μg g⁻¹) in the crab Charybdis longicolis, with a distribution pattern and concentrations similar to those observed in IM and PT. Cu is present in the pigment hemocyanin and is required in small concentrations for the physiological processes associated with breathing (Taylor and Anstiss, 1999), and for this reason, it is found in greater amounts in branchial tissue. Excess Cu, due to its toxic potential, is usually detoxified by these organisms in the hepatopancreas in order to avoid its presence in high concentrations in a metabolically active condition in the body (Rainbow, 2002).

In summary, the presence of total Cu at concentrations above background levels and the high concentrations found in Ucides cordatus tissues in IM suggest a clear enrichment of this metal, which increases its bioavailability. However, the abnormally high Cu concentrations in crab tissues in PT indicate that total content is not a suitable proxy to assess Cu bioavailability in these intertidal systems (Otero et al., 2000a) and that bioturbation and seasonal changes substantially affect geochemical conditions in soils (Araújo-Júnior et al., 2016; Ferreira et al., 2021), thus promoting environmental acidification and increased concentrations of Cu associated with the most labile and bioavailable fractions during the dry season.

Although a large proportion of Cu is associated with the oxidizable fraction, seasonal factors associated with biota and physicochemical parameters of soils could compromise the stability of geochemical fractions, making this metal available to resident fauna. On the other hand, contents detected in crabs from IM and PT suggest potential ecotoxicological risks in these mangroves; this warrants the need to assess the consumption of Ucides cordatus and its associated impacts on human and environmental health in these areas.