As confirmed by spiny-cheek crayfish research, Ca accumulated in the highest amounts in the exoskeleton, while Zn and Cu in the meat. The analyses of spiny cheek crayfish showed that the concentration of metals in abdominal meat and in exoskeleton was in this decreasing order Ca > Zn > Cu. The same results were observed by Protasowicki et al. (2013) in signal crayfish (Pacifastacus leniusculus), by Goretti et al. (2016) in red swamp crayfish (Procambarus clarkii) from Lake Trasimeno, and by Varol and Sünbül (2018) in mud crayfish. Higher level of Cu than Zn in exoskeleton was analysed by Nędzarek et al. (2020) in signal crayfish exoskeleton and by Mistri et al. (2020) in red swamp crayfish.
Calcium (Ca) is one of the macroelements responsible for building the skeletal system and maintaining a proper acid-base balance and as confirmed by the research of Lall (2002), muscle tissue is not the primary site of Ca accumulation, unlike the scales, bones and skin. It was confirmed by studies of a spiny-cheek crayfish, which showed calcium concentrations about 40 times higher in the exoskeleton compared to the muscle tissue. This is due to chitin, which builds the crustacean exoskeleton and has a high sorption capacity for metals, which is due to passive adsorption of metals from water (Mistri et al. 2020). So this tissue may be a rich source of Ca and can be used as a natural fertilizer for plants. Nędzarek et al. (2020) obtained from 2.4 to 3.8 times higher Ca level in the meat compared to the exoskeleton for signal crayfish. Our research did not confirm significant differences in the level of Ca in the exoskeleton between groups. Neither sex nor age were a significant factors affecting level of Ca in this tissue. As Wærvågen et al. (2016) confirmed, moult timing and frequency, as well as length increment per moult, have a major impact on exoskeleton calcification in Norwegian populations of the crayfish Astacus astacus (Linnaeus, 1758).
Zinc (Zn) is a microelement that plays an important role in the proper functioning of an organism. This metal is responsible for carbohydrates, proteins, nucleic acids metabolism, proper bone mineralization and the functioning of the immune system (Chavez-Sanchez et al. 2000). The degree of zinc accumulation in crayfish tissues depends on the concentration of calcium in the water and the correlation between these metals, moreover it is largely due to surface adsorption on the shell and gills (Bryan 1967). Our analyses of spiny-cheek crayfish demonstrated about 8.5 times higher concentration of Zn in meat compared to the exoskeleton. Nędzarek et al. (2020) found about 3 times higher Zn levels in signal crayfish meat compared to the exoskeleton. The same results were denoted by Mistri et al. (2020) in red swamp crayfish (the ratio was from 3.5 to 4.8 times). The studies of Jaafarzadeh et al. (2015) confirmed that the chitin extracted from the shrimp was an effective Zn adsorbent, but the contact time or individual predispositions strongly influenced the efficiency of biosorption.
Microelement which plays a significant role in the production of red blood cells, synthesis nucleic acids, hardening of collagen and keratinization of hair, haemocyanin biosynthesis and catabolism in aquatic arthropods and molluscs is copper (Cu) (Mistri et al. 2020). Our research showed almost 9 times higher concentration of this metal in meat compared to the exoskeleton. Significantly highest concentration of this metal in the exoskeleton was found in 3-year-old males of spiny-cheek crayfish. Nędzarek et al. (2020) found about 1.3 times higher Cu levels in signal crayfish meat compared to the exoskeleton. The same results were denoted by Mistri et al. (2020) in red swamp crayfish (the ratio was from 1.3 to 3.4 times). The studies of Balzani et al. (2021) on P. clarkii showed that the muscle tissue was characterized by the lowest degree of Cu absorption compared to other tissues, which confirms a greater degree of elimination than accumulation. No significant differences in Cu concentration in the abdominal meat between females and males of spiny-cheek crayfish within one age group were observed. The only significant differences were found in the Cu concentration in meat between 3- and 4-year old males. These differences are likely due to the ability of crayfish to clear metals rapidly and this is why these animals are useful for assessing Cu bioavailability in aquatic ecosystems in a short-term monitoring program (Kouba et al. 2010; Xiong et al. 2020). As demonstrated by Mistri et al. (2020), the large accumulation of Cu in the abdominal muscle may also be caused by a physiological response to external stress.
Statistically significant differences in the content of the determined metals between 3- and 4-year old crayfish were determined for Zn, Cu and Ca in the meat and for Cu and Zn in the exoskeleton. The analyses showed significant differences in the concentration of Zn in meat between 3- and 4-year old females and in the concentration of Cu between 3- and 4-year old males. As confirmed by Jafarzadeh et al. (2015) in studies performed on shrimp shells, the period of exposure to the environmental factor is responsible for the level of accumulation of metals such as Zn. This is consistent with our previous research, which confirmed the significant influence of the age of spiny-cheek crayfish on the Zn and Cu levels in meat (Stanek et al. 2017). Lower metal values in the meat of older individuals (for example Cu in 4-year old spiny-cheek crayfish) may be due to a dilution effect. When the growth rate is faster, as is often the case in younger organisms, then the growth dilution effect will be greater (Evans-Illidge 1997).
Statistically significant differences in the content of the determined metals between males and females were determined for Ca in the meat and for Zn in the exoskeleton. Hagen and Sneddon (2009) and Nędzarek et al. (2020) showed no significant difference between males and females crayfish in Zn and Cu content in the meat. Naghshbandi et al. (2007) confirmed differences in Zn and Cu content in the muscle between sexes for mud crayfish. Chen et al. (2005) confirmed the lack of significant differences in Cu concentration between individuals of different sexes in any tissue. One of hypothesis is that Cu is an essential element for the blood pigments of crustaceans, and its requirement should not differ between the sexes. Differences in the concentration of this metal may appear in small crustaceans or juveniles with little or no haemocyanin, which may result in an increase in the concentration of Cu in the body as the size of the animal increases. Kaya et al. (2015) confirmed by scanning electron microscopy (SEM) that the male chitin surface structure contained 25–90 nm wide nanofibers and 90–250 nm nanopores, while no pores or nanofibers were observed in the chitin surface structure of the majority of females (nanofibers were observed only in M. desertus females). Moreover, they confirmed the differences in dry matter of chitin between species (these values ranged from 4.71–11.84%) and they observed that the quantity of chitin was greater in males than in females. The research of Jussila et al. (1995) on noble crayfish confirmed differences in the metal content of the exoskeleton between males and females may be due to the difference in the frequency of moulting during the year. This was confirmed by the research of Nędzarek et al. (2020) on the level of heavy metals in the exoskeleton of signal crayfish from the Wieprza River. Differences in the metal content of the meat between males and females may be due to differing growth rates between sexes (Evans-Illidge 1997). Moreover, research concerning Nephrops norvegicus show that males have higher feeding rates than females and concentration of metals may be higher (Canli and Furness 1993).
According to Tao et al. (2012), a BCF value > 1 – means that the organism has the potential to accumulate a chemical substance and BCF value > 100 means high accumulation capacity. Other literature sources indicate that a BCF value of < 1000 means that the chemical is not significantly bioaccumulative; with BCF in the range of 1000–5000, the chemical substance has a potential to bioaccumulate, and with BCF > 5000, the chemical substance shows high bioaccumulation (Costanza et al. 2012; Varol and Sünbül (2018). The calculated BCF values for Zn were consistent with the data obtained, by Nędzarek et al. (2020) for signal crayfish, and BCF values for Cu were the same as obtained by Varol and Sünbül (2018) for mud crayfish. Our analyses confirmed that Zn and Cu accumulate mainly in the muscle, and Ca in the exoskeleton. This is because Ca is the main constituent of the exoskeleton as calcium carbonate (30–40%), in addition to chitin (20–30%) and protein (20–30%) (Chen et al. 2020). The analyses of spiny-cheek crayfish showed a much higher bioaccumulation capacity of Zn and Cu in the meat from water (BCF > 1000), and a greater bioaccumulation capacity of Ca in the exoskeleton from sediments (BCF > 3). Since the shellfish exoskeleton is in direct contact with the environment, metals accumulate in this tissue through passive adsorption from water or sediment rather than bioaccumulation (Mistri et al. 2020). The differences in the bioaccumulation of metals in a tissue by detoxification compared to other tissues can be expressed as the ratio of the levels found in these tissues (for example ratio Exoskeleton/Meat). BCF values for Zn and Cu were similar and were below 1, while in the case of Ca, BCF value was in the range from 20.98 to 74.24. Two factors influence the BCF values in different tissues: different biochemical functions of elements and different physiological functions of individual organs (Nędzarek et al. 2020). In order to confirm this tendency of metal accumulation in the analysed crayfish specie, studies on individuals obtained from reservoirs with various degrees of eutrophication should be carried out.
The lack of significant relationships between the length of the body and the concentration of metals (negative correlations) in the meat may result from the dilution of the concentration of metals as the animal's body grows, moreover, it may indicate faster metabolism in younger animals compared to older ones (Balzani et al. 2021), and it may also results from the growth-hindering effect of metals. Conversely, positive correlations indicate a trend of metal accumulation as the animal grows (Ergen et al. 2015). A significant negative correlations between the concentration of metals and TL in the exoskeleton may result from cyclic moulting, which results in the removal of metals. Our analyses on spiny-cheek crayfish confirmed this. The degree of absorption of Zn depends on the amount of Ca in the environment. Our analyses confirmed a positive and significant relationship between Zn and Ca concentration in the meat of 3-year-old males (high degree of correlation r = 0,822835).