3.1 Biometric data and Cd, Hg, and Pb total levels
The maximum weight (gutted) detected was obtained for H. arguinensis (465.0 g), while the lowest was observed for H. forskali (45.9 g), both in autumn. Also, maximum and minimum lengths were showed by H. arguinensis (36.0 cm) and H. forskali (10.9 cm), respectively. H. mammata presented intermediate values (Table 1).
Significant differences were found in the weight (live and gutted) and the length among species (Kruskal-Wallis test, p < 0.05). These differences having been previously reported by studies carried out with the same species in other regions (González-Wangüemert et al., 2015; Olaya-Restrepo et al., 2017).
Mean values of Cd, Pb and Hg for H. arguinensis, H. forskali and H. mammata are below the values recommended by the EU (Commission Regulation, 2006 and its amendments) (Table 2).
Mean Cd values were not significantly different (p > 0.05) between H. arguinensis (0.009 mg kg− 1) which ranged from 0.003 to 0.027 (mg kg− 1) and H. forskali (0.010 mg kg− 1) < LoQ at 0.031 (mgkg− 1). H. mammata, on the other hand, had a slightly higher mean value (0.019 mg kg− 1) which ranged from 0.010 to 0.030 (mg kg− 1) being significantly different from the two previous species (p < 0.05). These low differences in Cd content can be associated with the environment and its variations, once all species were captured under similar environmental conditions. Jiang et al. (2014) showed that the higher Cd values found for Apostichopus japonicus sampled in different coastal areas of the northern of China compared to the results of Gao et al. (2014) who evaluated the Cd values in the water and sediment of these region, can be associated with the bottom with higher Cd contents in seawater and sediments along the Bohai and Yellow Sea. Also, the Cd solubilization is higher than the other elements, increasing its availability because there is greater solubilization of Cd compared to other elements (McAloon and Mason, 2003). Wang et al. (2016) evaluated the effect of Cd on the Apostichopus japonicus’ growth and its bioaccumulation, testing different concentrations of this metal in the diet. The results showed that a higher Cd content in the diet, was linked to its greater concentration in the sea cucumber tissues, showing the digestive tract the highest values, followed by the respiratory tree and body wall.
H. arguinensis, H. forskali and H. mammata had similar low mean Hg values (p > 0.05), between < LoQ and 0.02 (mg kg− 1). The maximum value was observed for H. mammata, reaching 0.05 mg kg− 1 (Table 2). None of these species showed an average concentration of Hg above the limit proposed by the EU (2006), 0.50 mg kg− 1. As far as we were able to determine the maximum values found in the literature, these are from the work carried by Fretes et al. (2020) who found average Hg levels to 2.1 mg kg− 1 of Hg in Stichopus herrmanni caught in Indonesia. Due to the nature of the element, Hg is persistent and toxic, causing serious concern in both the seafood market (Sheehan et al., 2014; Chen et al., 2008; Sanzo et al., 2001; Bjornberg et al., 2003) and the populations of animal species. Knowledge is little about the effects of this metal on different species of sea cucumbers, but a study carried by Telahigue et al., (2019) showed that high concentrations of Hg (> 80 µg L − 1) in H. forskali can induce to various physiological and morphological damages, such as substantial changes in fatty acid composition. Another harmful effect of high Hg concentrations was observed by Fretes et. al., (2020) in the intestinal villi of Sticopus herrmanni that had notorious damage.
H. arguinensis was the species that presented the highest concentration of Pb, with an average value of 0.30 (mg kg− 1) which varied between 0.03–1.20 (mg kg− 1), followed by H. mammata with 0.18 (mg kg− 1) and H. forskali with the lowest mean value (< LoQ) (Table 2). Although Pb was the element with the highest concentration and presented a significant difference among the analysed species (p < 0.05), these values do not exceed the maximum permitted limits (EU, 2006). Lead has a well-known affinity for calcareous skeletons and has been shown to be predominantly concentrated in the body walls of several echinoderms, considering the ossicles are formed inside cell vesicles and are composed mainly of calcium carbonate (Smiley, 1994) present in the body wall of these animals (Beeby, 1991) ossicles. González-Wanguemert et al. (2018a) also associated the high Pb levels detected in H. tubulosa (unless partially) with its feeding behaviour associated to sediment from seagrass meadows, which have a high potential for metal retention (Marín-Guirao et al., 2005; Warnau et al., 2006). Indeed, it has been suggested that due to its peculiar ecological characteristics, H. tubulosa could be used to complement the small set of bioindicators available so far for surveying metal pollution in the Posidonia oceanica ecosystem from the Mediterranean Sea (Warnau et al., 2006).
Low values of Cd, Pb and Hg found in H. arguinensis, H. mammata and H. forskali are similar to the low concentration of these elements in the sediment and seawater where they were captured. The three species were captured in a coastal zone between the mouth of the Sado Estuary and the city of Sesimbra. The possible source of contamination by Cd, Pb and Hg in this area would be due to the anthropic activity carried out in the Sado Estuary and on its banks, such as the Port of Setúbal, the copper mines of the Sado basin, intensive agriculture, mainly rice, fish farming and fish farms (Catarino et al., 1987; Freitas et al., 2007). Despite this intense activity around the Sado Estuary, there only one work that assesses contamination along its gradient, a study by Caeiro et al. (2005) at 153 sampling points along the estuarine gradient showed that only 3% of these stations were highly contaminated for cadmiun (6.1–8 mg/kg), lead (50.1–69 mg/kg) and mercury (0.61–0.7 mg/Kg). The coastal area where sea cucumber were caught is included in the section with partial protection from the Professor Luís Saldanha Natural Park, with little or no human activity and no significant connectivity with other potentially polluted areas along the closed Atlantic coast.
H. arguinensis, H. forskali and H. mammata showed Cd, Pb and Hg values below the values recommended (EU, 2006; 2021ab; 2022), being Pb the element with the highest concentration, and Cd and Hg having the lowest ones. However, the concentration of a given trace element does not seem to be constant in all sea cucumber species. For example, similar results to this present work were found in the study by Al-Najjar et al., (2018) for Actinophyga bannwarth and Holothuria impatiens (Jordan Coast), where the concentration of Pb (0.95 mg kg− 1; 1.05 mg kg− 1) was higher than Cd (0.09 mg kg− 1; 0.08 mg kg− 1) in both species, respectively. However, Marrugo-Negrete et al. (2020) found values of Pb (0.032 mg kg− 1) higher than Cd (0.021 mg kg− 1), but the element with the highest concentration was Hg (0.081 mg kg− 1) for H. floridana. A possible cause explaining the observed differences would be the dependence of the toxicity of these metals on environmental factors with the degree of metal concentration in the sediment (Griscom et al., 2000; Pesch et al., 1995; Ankley et al., 1991; Ankley, 1996) and organic matter content (Mason and Lawrence, 1999; Lawrence and Mason, 2001). For example, Lawrence (1998) found that as the percentage of organic matter in the sediment increases, the amount of Hg accumulated by the organism decreases by sea cucumber Sclerodactyla briareus. Another factor may be habitat preference, as it varies with the life stage of the sea cucumber, and environmental conditions when usually heavy metals are linked with fine fractions of sediments like silt and clay, and also with a high concentration of OM. (Mercier et al., 2000; Purcell., 2004a, b; Shiel, 2004; James, 2005; Yamana et al., 2006; Slater et al., 2010).
Sperman-hro correlation coefficients were calculated for specimens for each trace elements, length and weight (Table 3).
It is recognized that the physiological processes that influence the absorption, distribution and elimination of trace elements are influenced by the length and weight of the animal. This observation is particularly accepted for absorption of cadmium, lead, and mercury because levels of these elements generally increase in heavier/older aquatic animal species that are generally associated with higher concentrations as opposed to the low concentration found in smaller or younger aquatic animals (Raulinaitis et al., 2012; Nathaniel, 2002). However, there are studies that show a higher concentration of trace elements in small animals, such as a study carried by Al-Najjar et al. (2018) who analyzed the concentration of seven microelements in two species of sea cucumber with different sizes. These autor found significantly higher values in small animals, namely Cd in Actinophyga bannwarth (small < 25 cm 1.19 ± 0.26 µg/g; large > 30 cm 0.93 ± 0.21µg/g) and Holothuria impatiens (< 25 cm 0.86 ± 0.21µg/g; large > 30 cm 0.86 ± 0.21µg/g) and Pb in Actinophy-ga bannwarth (small < 25 cm 15.28 ± 1.81µg/g; large > 30 cm 14.69 ± 2.11µg/g) and Holothuria impatiens (< 25 cm 17.51 ± 2.36µg/g; large > 30 cm 11.2 ± 1.33µg/g). In our study, only H. mammata showed a positive correlation between the concentration of Pb and the weight of the animal (p < 0.05), whereas the length showed a negative correlation (-0.088) but not significant (p > 0.05) for Pb. H. arguinensis and H. mammata showed a positive correlation between weight and size of the animals, but this correlation was not significant (p > 0.05).
Pb and Cd were highly positively correlated (p < 0.01) in H. forskali. The correlation obtained values were similar than those reported by Tunca et al. (2016) for the H. tubulosa (0.560) and H. polli (0.542) caught off the Northern Mediterranean Sea, but different than those reported by Marrugo-Negrete (2020) for Holothuria floridana from Colombia (-0.006).
3.3 Metal concentration vs sex and tissue of sea cucumber
Regarding animal sex and Cd and Pb concentrations, significant differences were observed for H. arguinensis and H. mammata but not for H. forskali. On the contrary, no significant differences (p > 0.05) were found for Hg concentration between males and females of the three studied species. However, no clear pattern of variation with the concentration of the three metals was found (Fig. 3). Lead and Hg had the highest values in males in H. mammata and H. arguinensis, on the other hand, the highest concentrations of Cd were in females. As the Pb values for H. forskali were close to the detection limit (LoQ) it was not possible to verify the differences mentioned for this species. So far there does not seems to be any work on sea cucumbers that relates the metal content to both sexes, however, for other species the significant differences between males and females are not clear and seems to be more related to the metal and the tissue that properly matches the sex of the animal (Bat et al., 2012).
The variation in tissue metal concentration of three analysed sea cucumber species is also shown in Fig. 3. In general, the highest metal concentration in H. arguinensis, H. forskali and H. mammata is found in the muscle compared to the wall body. However, no significant differences were observed for Hg and Pb between males and females for H. forskali Previous studies reported that the highest levels of metals are in sea cucumber viscera (Liu et al. 2016; Warnau et al. 2006). A study carried out by Tunca et al. (2016) found significant differences between metal concentration and tissues of three Mediterranean species of sea cucumber (Holothuria polii, Holothuria tubulosa and Holothuria mammata), with a higher concentration in the viscera compared to the body wall. The sea cucumber gut is also known to play a role in solubilization and bioaccumulation (McAloon and Mason, 2003). Warnau et al. (2006) observed that H. mammata significantly accumulated some metals such as Cd in its body wall. In the work performed by Lin et al. (2018) in which they characterized trace elements in Acaudina leucoprocta, the body wall was the second tissue with the highest concentration of metals, behind only the intestine.
One reason for lower metal concentrations and different profiles on body walls may be that muscle tissue contains a limited number of metal-binding proteins (Guner 2007). Although the metal content was the lowest in the body wall of sea cucumber, this tissue comprises 58–81% of the total amount of most metals as it represents 85–90% of the entire body (Givianrad et al., 2014), these values may actually be higher than those found in the viscera (Liu et al., 2016). Unlike Japan which consumes the viscera in the form of konowata and konoki, in Europe normally the viscera are not used as food, so this tissue was not evaluated. So, a possible explanation for the high concentration of metals in the visceras can be because this is the first route of absorption of trace elements, that is, the first contact of the highest concentrations is carried out in the digestive tract (short period of time), and due to the high energy requirement for production of gametes, there is a directed flow of the metabolized to the gonads. However, the bioaccumulation of heavy metals is focused on the body wall (long period of time) with a lower concentration than in the viscera, but more constant over time, therefore the body wall is a better bioindicator than other tissues of trace elements pollution in the marine environment.