METAL CONCENTRATIONS IN THE TISSUES OF BLUE CRABS IN THE ESTUARINE REGION OF THE SÃO FRANCISCO RIVER, NORTHEASTERN

Objective : This study aimed to evaluate concentrations of metals in the tissues of the midgut gland and muscles of males and females of Callinectes sapidus and C. bocourti from the estuarine region of the São Francisco River. Theoretical Framework : A critical aspect of the introduction of chemical substances into aquatic systems is the bioaccumulation within the food chain, particularly with respect to contamination by heavy metals. In estuaries, this situation may cause impacts on the biota and pose risks to public health. Method : Tissue samples were prepared following the procedures described by Sawidis (2011) and analyzed using flame atomic absorption spectrometry. Results and Discussion : The highest concentrations observed were of Copper, especially in the tissues of females, which may be related to the presence of hemocyanin in the blood, or the need for trace elements by the females to complete their life cycle. It was also found that the muscle tissue exhibited the highest concentrations, indicating potential bioaccumulation in the biota, explaining the concentrations above those established by legislation. Research Implications : This study has significant implications for public health, as it addresses aspects previously unknown about the fishing of this important resource, serving as a valuable contribution to the establishment of public health and environmental policies. The findings may assist in fishery management and public health measures. Originality/Value : This study represents the first analysis of metals in crustaceans from the lower São Francisco region and contributes to the existing body of knowledge, offering new insights into the presence of metals in invertebrates from one of the most important estuarine regions in the country.


INTRODUCTION
Decapod crustaceans display an impressive array of shapes, behaviors, and sizes, and they can be found at virtually all depths, spanning various environments (Melo, 1996).This taxon is extensively exploited worldwide (Scalco et al., 2014), and in Brazil, there is significant fishing potential (Lacerda et al., 2016).The various species of the Portunidae often coexist, sharing habitats in the western portion of the Atlantic Ocean, including shallow coastal waters and estuaries (Arnold, 1984;Williams, 1984;Melo, 1996).
Assessing the contamination of bioaccumulated metallic pollutants within decapod crustacean tissues holds profound significance, as it serves to delineate potential environmental hazards (Yilmaz et al., 2007;Simonetti et al., 2013).
Therefore, the current study seeks to assess the levels of metal concentrations in Callinectes crabs obtained from an estuarine area in northeastern of Brazil.

THEORETICAL FRAMEWORK
Metal contamination in fish stocks could potentially escalate into a global crisis, as rivers and seas are becoming increasingly susceptible to rising discharges of pollutants, acting as reservoirs for persistent chemicals like heavy metals (Ahmed et al., 2015;Abdelkarim, 2020).When these metals infiltrate aquatic systems, they tend to become trapped in the medium and accumulate in the sediments.This accumulation makes them directly accessible to benthic fauna or releases them into the water column, thereby posing a threat to the ecosystem (Pekey et al., 2006;Gribof et al., 2018).
Moreover, these contaminants can enter the food chain through the organisms' diet, whether they reside on the ocean floor or in the water column (such as zooplankton.phytoplankton.and benthos).Alternatively, contaminants can be absorbed through gills and skin (Ahmed et al., 2015;Ali & Khan, 2018).Thus, a critical aspect of introducing chemicals into aquatic systems is the potential for bioaccumulation within the food chain (Gribof et al., 2018;Ali & Khan, 2019;Shi et al., 2020).This circumstance has prompted numerous studies.focused on analyzing metal contaminants and demonstrating how persistent and widespread they can be in the environment.They interact with the natural components of biota, leading to harm in living organisms (Çogunm, 2017;Ahmed et al., 2019;Sabir et al., 2022).Metals, including Copper, Zinc, and Iron, which inherently and fundamentally compose the entirety of biological entities, are detectable in substantial concentrations within fish.This presence leads to detrimental impacts on both aquatic organisms and the organisms consuming them (Martins et al., 2006;Yang et al., 2007;Jesus et al., 2014).The accumulation of essential metals in water, sediments, and living tissue, like that of crustaceans, can pose risks to human health (Beltrame et al., 2011).This stress induces synaptic damage and leads to neurotransmitter malfunction in fish and other aquatic organisms (Weis & Weis, 1998;Lee et al., 2019;Zhao et al., 2020;Kim et al., 2021).
The Portunidae family consists of individuals with large carapaces and nine teeth on the anterolateral margin, along with a fifth pair of flattened, paddle-shaped pereopods.Because of their preference for habitats with sandy and muddy substrates along estuary and mangrove margins (Milne Edwards, 1879;Rathbun, 1896;Williams, 1984;Melo, 1996), these organisms can accumulate pollutants in their tissues, providing insights into the environmental quality of their habitats (Çoğun et al., 2017).
In addition to exerting substantial ecological and economic influence, they also play a role in maintaining the equilibrium of brackish water habitats and shorelines by regulating the abundance, diversity, and structure of diverse benthic communities.(Lima et al., 2013).

STUDY AREA AND SAMPLING
Sampling was carried out in July 2018 at estuarine region of the São Francisco River (10° 26' 46" S / 36° 25' 38" W) with the organisms being obtained from the artisanal fishing fleet (Figure 1).

Figure 1
The sampled area is situated within the estuarine region of the São Francisco River in

Northeastern Brazil
Source: Adapted from Google Earth, 2022.

LABORATORY PROCEDURES
The animals were transported while alive within polyethylene containers to the Estuary and Mangrove Research Laboratory.The crabs were subjected to cryoanesthesia prior to analysis.Subsequently, they were measured, sexually differentiated, weighed with analytical precision, and stored in plastic bags (with a precision of 0.01g).Finally, the crabs were dissected using non-metallic materials, including plastic and wood, to prevent potential metal contamination.
The samples were categorized as follows: Callinectes sapidus and C. bocourti; males and females; midgut gland tissue (MGT) and muscle tissue (MT).The collected tissue samples were encapsulated in polypropylene containers and chilled within an insulated container 6 supplied with ice, thereby facilitating their subsequent transportation to the Laboratory of Electrochemistry and Microsystems Analysis.

ANALYSIS OF IRON, COPPER, ZINC, AND CADMIUM
To mitigate contamination risks all glassware employed in this study underwent a prior immersion in 2% Nitric Acid (HNO3) for 24 hours.The sample preparation methodology was adapted from Sawidis et al. (2011).Tissue samples.partitioned by species sex and sexual maturation.were weighed at 1.0g using a COLEMAN BNV6 digital balance (0.01g precision), subsequently deposited within a 50mL beaker along with 5mL of concentrated HNO3.The beaker was hermetically sealed with PVC film and left at room temperature for a duration of 24 hours.Following this interval, the sample was translocated to a digester block (Digesdahl-Jundilab), equipped with a reflux condenser.The thermal process commenced at 50 °C, gradually escalating to 125 °C, and was sustained until complete desiccation.The residual liquid fraction was then subjected to filtration through a WHATTMAN filter paper (catalog No. 1001042), transferred to a 25mL volumetric flask, and supplemented with a 2% HNO3 solution to achieve the desired volume.
Following the dissolution process with the heated acid, the samples underwent metal content analysis utilizing a Varian AA 220FS flame atomic absorption spectrometer equipped with a monochromator.The spectrometer employed a scan rate of 33nms -1 , while hollow cathode lamps served as the radiation source, operational concurrently and selected through a motorized mirror.Flame stoichiometry was fine-tuned using programmable automatic gas flow control, as detailed in Amorim et al. (2008).Calibration of the spectrometer relied on standard samples fabricated from stock solutions supplied by Sigma-Aldrich, featuring concentrations on the order of 1000 (± 0.3%) mg kg -1 .
Individual crab samples were subjected to triplicate readings, with the average of these analyses serving as the resultant value for each specimen.

RESULTS AND DISCUSSIONS
This study represents the first attempt to employ a comparative approach within the context of Portunidae traded in the estuarine region of the São Francisco River.It is crucial to emphasize that the toxicity of each element, along with factors such as bioaccumulation efficiency, bioavailability, solubility, and biomagnification, will significantly influence its  Metal concentrations were individually assessed for each species and subjected to comparative analysis across species and their corresponding tissues.During the laboratory phase, spectrophotometric verifications proved inconclusive for two samples of midgut gland tissue, leading to their exclusion from the study.
Copper emerged as the metal with the highest concentrations in both C. bocourti and C. sapidus, registering values of 1618.5 mg/kg and 3050.2 mg/kg, respectively, within the midgut gland tissue (MGT).Conversely, Cadmium exhibited the lowest concentrations, measuring 13.6 mg/kg and 11.5 mg/kg (Tables 2 and 3).An exception to this trend was observed in the MGT of C. sapidus females, where the Cadmium concentration reached 83 mg/kg, signifying a 500% elevation compared to other tissues.
Regarding Iron, the greatest concentration values materialized within the muscle tissue of C. bocourti females, reaching 1500.4 mg/kg, while the lowest levels were evident in the muscle tissue of C. sapidus males, with a measurement of 10.3 mg/kg.For Zinc, the apex concentrations were observed in the muscle tissue of C. bocourti females, tallying at 812 mg/kg, while the nadir values were identified in midgut gland tissue of C. sapidus females, at 4 mg/kg (Tables 2 and 3).Among the metals investigated in this study, Copper had the highest concentrations in female tissues, covering both the glandular and muscular compartments.It is well established that the presence of hemocyanin, a vital respiratory pigment for numerous invertebrates, may be intrinsically linked to copper accumulation in benthic organisms (Capparelli et al., 2017).In addition, it has been proven that decapod crustaceans have adequate regulatory mechanisms that govern the copper content in their tissues, thus justifying their notable prevalence at high levels, a phenomenon attributed to their exceptional catalytic role in the assimilation of iron, an essential process for most living systems.Rainbow (2007) and Almeida (2009) in research involving Portunidae, report predominance of high concentrations of copper in female specimens, a phenomenon conceivably rooted in their greater capacity to accumulate metals, mainly in supporting functions related to sexual maturity.Furthermore, according to the findings reported by Woll (2006), it is worth noting that female Callinectes show a tendency towards migratory behaviors in search of food during their reproductive phases.This behavior arises from the critical need for trace elements, such as copper, which are essential for the successful completion of its life cycle.Regarding iron, intriguing correlations were observed between this metal and the muscle tissues of male and female specimens in both species.The highest concentrations of iron were identified in the muscle tissue of C. bocourti females, while the lowest concentrations were observed in the muscle tissue of C. sapidus males.These patterns align with similar observations made by Bordon et al. (2012) and Merfa (2010) in their respective studies, emphasizing the robust association between the enzymatic and respiratory functions of iron in crustaceans, particularly in the context of metal assimilation and detoxification.Iron also plays key roles in cellular homeostasis, oxygen transport.DNA synthesis and energy metabolism in these organisms, as explained by Delgadinho (2014).In addition, the phenomenon of higher iron concentrations in women has been documented in several other studies (Virga, 2008;Silva, 2009Silva, , 2016;;Çoğun et al., 2017).Discrepancies in metal levels between different species can be attributed to possible variations in bioaccumulation strategies related to food preferences and the unique space-time distribution of each species, as discussed in previous studies by Macfarlane et al. (2000) and Engel (2001).For Zinc, the presence of high concentrations in muscle tissue differs from other work for the group.For example, Çogun et al. (2017), observed that the highest concentrations of Zinc were in the glandular tissue of the midgut.On the other hand, low Zinc concentrations in the glandular tissue may be associated with different vital functions of individuals (Krężel & Maret, 2016).
Since the concentrations found in Callinectes from the São Francisco River were higher than those found in the literature for similar organisms (Duarte et al., 2016-178 mg/kg;Çoğun et al., 2017-104 mg/kg), can reflect on its bioaccumulation in the sediment and water column of this region.Rossi (2009), studying C. bocourti observed that Zinc concentrations varied seasonally in the different tissues so that February presented the highest concentrations due to the lower incidence of rainfall in the region that resulted in a lower volume of water leading, therefore, to a lower dilution of Zn from domestic and industrial effluents increasing its concentration in the organisms (Çogun et al., 2017).Such conditions may have contributed to the values found in the present study.
Although cadmium does not have metabolic functions in decapod crustaceans, this study revealed the occurrence of high concentrations of this metal in the tissues of Callinectes.
Although cadmium occurs in the marine environment at residual levels, most organisms, particularly mollusks and crustaceans, can rapidly accumulate this element (Liu et al., 2019).
Our investigation showed that this metal has remarkable concentrations, particularly in C. sapidus, where its presence in glandular tissue reached levels up to 500% higher than in any 10 other tissue.This observation can be attributed to a multitude of biotic and abiotic factors, as well as the intricate metabolic processes that govern cadmium bioaccumulation in these organisms (Engel et al., 2001).In addition, Brouwer & Lee (2007) postulate that cadmium concentrations in crabs are closely linked to the presence of metallothionein protein in the hepatopancreas and gills, serves as a key factor in the fixation, control and regulation of other metals within organisms, while neutralizing toxic levels arising mainly from the absorption of cadmium and mercury in stressful environmental conditions (Virga, 2008).Consequently, cells equipped with higher metallothionein concentrations are protected against heavy metal toxicity and oxidative stress (Wang et al., 2013;Adams, 2014).
In the comparative analysis of the concentrations identified in the present investigation with the limit concentrations stipulated in the existing regulatory frameworks for aquatic organisms, it becomes evident that, without exception, the recorded values exceed the established legal limits (Table 4).

Table 4
Legal limits allowed for contamination of the organisms (FAO/ WHO (1997WHO ( , 2000)); BRASIL (2006,2013)) in comparison with the maximum and minimum values found in this study.The presence of heavy metals in Callinectes samples that exceed the legally permitted levels was unequivocally verified in this study.National and international laws and regulations prescribe concentration limits for various aquatic organisms, including fish, mollusks and crustaceans.Through a comparative evaluation between established regulatory values and the results of this investigation, we effectively illustrated that the concentrations of essential and non-essential metals in the examined species exceed the recommended limits, these findings serve as a crucial wake-up call for communities that depend on the consumption of these aquatic organisms.In the context of Brazilian legislation, Decree No. 55.871. of March 26, 1965, andOrdinance No. 685, of 1998.establish maximum tolerance values for inorganic contaminants in food.The government's commitment to addressing prevailing circumstances is exemplified by the 2013 decision by the Ministry of Health to reduce the maximum permitted limit of lead in fish in Brazil to 0.50 mg/kg (Brasil, 2013).This reduction was implemented due to the bioaccumulative tendencies of this metal and its adverse effects on both the population and marine organisms.
In this study, we elucidate the presence of heightened concentrations of essential trace metals, namely Iron (Fe), Copper (Cu), and Zinc (Zn), in the glandular and muscular tissues of C. bocourti and C. sapidus.These findings may be closely associated with the crucial roles that these elements play in various metabolic processes, including enzymatic, catalytic, and reproductive functions, as previously expounded upon (Çoğun et al., 2017).Conversely, the presence of Cadmium (Cd), which lacks any known physiological role in these organisms, at the concentrations observed in our study, raises concerns regarding its potential toxicity to this specific biota (Cheng et al., 2018).It is noteworthy that these elements exhibit similar environmental dynamics, undergoing speciation through the formation of organic and inorganic complexes (Rodrigues et al., 2022).
Regrettably, the absence of oversight by governmental agencies has enabled heavy metals to become accessible to biota, potentially leading to their bioaccumulation in tissues and an increase in the food chain for ultimate consumers.This contributes to the ingestion of large concentrations by these organisms, which can generate, in the long term, damage to the health of the population.Given this reality, further studies on metal contaminants in fish tissues need to be carried out to monitor the elevation of these values in biota and develop ways to prevent other organisms from being contaminated.

CONCLUSION
Through this study, it was observed that commercially traded blue crabs, Callinectes sapidus and C. bocourti, from the estuarine region of the São Francisco River, exhibited elevated concentrations of metals in their tissues.
The findings highlighted higher concentrations in females compared to males, as well as in muscle tissue.It is likely that the increase in agricultural and industrial activities, coupled with significant alterations to the riverbed in recent years, has contributed to the presence of these metals in the region, particularly non-essential metals such as cadmium.
These results underscore the importance of further investigations into metal contaminants in fish and other aquatic species tissues, including expanded and seasonal sampling in the area to explore the long-term effects of such concentrations on these organisms.
Additionally, there is a pressing need for more comprehensive assessments of water and sediment quality in the region.
Carapace widths encompassed a range of 8.03 to 13.43 cm.The overall mass of the organisms, inclusive of the carapace, exhibited variation spanning 34.5 to 178.7 g.Within both male and female categories, C. sapidus demonstrated the highest mean values (Table1 Metal Concentrations in the Tissues of Blue Crabs in the Estuarine Region of the São Francisco River, Northeastern Brazil ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.1 | p.1-16 | e04655 | 2024.7 elemental speciation and subsequent presence in both the environment and biotic organisms.These complex considerations are supported by the research of Taylor and Calabrese (2018), Rodrigues et al. (2021), and further corroborated by Rodrigues et al. (2022), collectively highlighting the intricate interplay of these elements in this ecological context.A total of 48 specimens were subjected to analysis, comprising 18 Callinectes sapidus and 30 Callinectes bocourti.
).Biotic parameters (mean ± standard deviation) of Callinectes sapidus and C. bocourti during the study period in the estuarine region of São Francisco River

Table 2
Concentration (mg/kg) of Fe, Cu, Cd, and Zn in dry weight tissue of Callinectes bocourti in estuarine region of the São Francisco River, Northeastern Brazil.

Table 3
Concentration (mg/kg) of Fe, Cu, Cd, and Zn in dry weight tissue of Callinectes sapidus in estuarine region of the São Francisco River, Northeastern Brazil.