Lethal toxicity
Living animals are constantly faced with various environmental stresses that challenge their daily lives. Cu is an essential metal that participates in normal physiological process of crustaceans, but several studies have shown that crustaceans are adversely affected when exposed to high concentrations of Cu. LC50 value represents a common point at lethal physiological response to toxicity, which has been well-documented in many crustaceans. For example, the 96-h LC50 value for shrimps of Exopalaemon carinicauda, Echinogammars olivii, Sphaeroma serratum, and Palaemon elegans was 0.712 mg Cu/L, 0.25 mg Cu/L, 1.98 mg Cu/L, and 2.52 mg Cu/L, respectively20,22. In addition, for paddy field crab Paratelphusa hydrodromus and freshwater crab, Barytelphusa cunicularis, the 96-h LC50 values recorded were 15.70 mg Cu/L and 215 mg Cu/L, respectively23,24. Likewise, in freshwater crayfish, Procambarus clarkia, the 96-h LC50 value reached 162 mg Cu/L25. These large variations in sub-lethal effects to Cu toxicity in crustaceans appear to be species specific. In our present study, the 96-h LC50 value for Cu exposure in C. dauricus was 32.5 mg/L, which is much higher than those of the most crustaceans, but this species seems relatively less tolerant to Cu, compared to P. clarkia. This difference may also be attributed to the various biotic and abiotic factors like age, sex, weight, salinity, and temperature, besides the species. For example, Taylor26 compared the 96-h Cu tolerance of Cambarus robustus in Pike Creek and in Wavy Lake and concluded the environment differences could affect population sensitivity to Cu toxicity.
Oxygen consumption rate
The effects of heavy metal on the respiratory rate of marine and estuarine organisms have been well documented. Spicer and Weber27 showed that heavy metal could cause respiratory impairment in crustaceans. The results obtained in the present study confirmed this previous finding. Both acute and sub-chronic Cu exposure induced significant inhibition of OCR in C. dauricus, with the maximum decreases of 48.4% and 57.9%, respectively, compared to the control. Similarly, a declined OCR by heavy metal has been observed in shrimps, including Penaeus indicus19, L. vannamei28, F. paulensis29, and E. carinicauda20, and crabs, including Uca annulipes, U. triangularis30, and Cancer pagarus27, as well as crayfish, P. clarkia25. The levels of inhibition of the respiration rate were mainly dependent on the exposure time and exposure concentration. Those authors assumed that the ultrastructural impairments of gill epithelium were related to the decrease in respiration rate, thereby affecting the oxygen carrying capacity of gills. Besides the cytological damage of gill, heavy metals also inhibit mitochondrial energy production, thereby affecting the key metabolic pathways. By contrast, an increased respiration rate has been detected in freshwater shrimp, Paratya curvirostris21, and lobster Homarus americanus31. The authors argued that it was attributed to an elevated rate of glycolysis, a mechanism of expenditure of energy reserves characteristic of a stress compensation process. In all, the changes of oxygen consumption level were mainly dependent on the time and concentration of exposure to heavy metals.
Ammonia excretion rate
Amino acids are the main sources of ammonia production in vivo. Crustaceans have the ability to regulate the concentration of intracellular free amino acids in order to deal with environmental stress32. In the present study, AER in either acute or sub-chronic Cu exposure showed a declining trend with increasing exposure concentration and time to Cu. A maximum decrease in AER of 79.4% and 70.06%, respectively, were observed respectively after exposure to 16.48 mg/L for 96-h and 2.06 mg/L for 14 days, in comparison to the control (Fig. 1B). In a similar manner, Chinni19 also reported a significant decrease in AER in post larvae P. indicus when exposed to Pb for 30 days. It assumed that such a decrease may be due to reduction in the metabolic rate or an interaction of heavy metal with the pathways for the production of ammonia-N. By contrast, elevations of ammonia excretion in response to heavy metals exposure were reported in other crustaceans. For example, an increase in AER was found in juvenile E. carinicauda after exposure to Zn and Hg20 and in F. paulensis after exposure to Cd and Zn29. It was considered that the gill function was impaired by the metal exposure, resulting in the dysfunction of ammonium excretion control; therefore, outflow of ammonia excretion from the hemolymph to ambient water induced an increased ammonia concentration in the water. In addition, no change in ammonia excretion rate was obtained in Paratya curvirostris after 96-h acute and 10-day sub-chronic Cd stress21. Therefore, the questions of the relationship between heavy metal exposure and ammonia excretion needs to be properly investigated.
Energy metabolism
O:N is a useful value for evaluating the characteristics of nutrients utilized by animals and can provide information on changes in energy substrate utilization under various environmental stresses33,34. Theoretically, pure protein catabolism will produce an O:N ratio of 835, and equal proportions of proteins and lipid results in an O:N of 2436. An O:N ratio higher than 24 indicates an elevation in lipid and carbohydrate metabolisms. In this study, in comparison with the controls, high values of O:N were obtained in individuals of C. dauricus exposed to Cu for 96 h and 14 days (Table 1, Fig. 1C). In generally, protein catabolism for energy is less efficient than lipid/carbohydrate catabolism. A species that relies on lipid and carbohydrate metabolism will likely be able to better meet energy demands of toxicant exposure than a species that principally metabolizes protein. The mean O:N ratio higher than 24 in acute Cu exposure and lower than 24 in sub-chronic exposure (Table 3 and Fig. 1C) indicated the differences in energy utilization strategy in response to two patterns of Cu stress. This could be a mechanism explaining the differences in energetic responses to Cu exposure in C. dauricus, relative to other crustacean species.
Tissues accumulation
Cu is an essential trace element for biological processes, particularly as a component of the respiratory pigment, hemocyanin. The body Cu concentration in decapod crustaceans can be regulated and does not accumulate until certain environmental threshold levels are achieved37. In addition, as an economic species of crustaceans and in relation to food quality and safety assessment, organ-specific accumulation data, especially for the muscle, are markedly required. In this study, tissue-specific bioaccumulation of Cu observed, and the Cu accumulation in hepatopancreas and muscles were highly dependent on water Cu concentration and exposure time (Fig. 2; Fig. 3A, 3B). Hepatopancreas is the organ most associated with the detoxification and biotransformation process and in direct contact with toxicants in water. The hepatopancreas, containing metal-binding protein, is the main target organ for regulating Cu level38. The maximum Cu accumulation was observed in hepatopancreas, which increased 12.7 folds and 31.6 folds after 4-day acute exposure to 16.48 mg Cu/L and chronic 14-day exposure to 4.12 mg Cu/L, respectively, this indicated that C. dauricus had a great potential for rapid accumulation of Cu in fresh waters. The greatest Cu accumulation occurring in hepatopancreas had been reported for the crayfish species, Astacus leptodactylus39 and Procambarus sp.40 as well as for the freshwater prawn, M. rosenbergii38. Although the hepatopancreas could regulate the Cu level in the animal’s body to avoid toxicity and deficiencies, the high level of external water Cu breaks down the regulation of Cu and causes continuous Cu accumulation, which might lead to the loss of muscular control and eventually, death, for crustaceans.
In this study, there was no significant time-dependent trend in the accumulation of Cu in the muscle between 7 and 14 days of Cu stress in the lower concentration of 2.06 mg/L (Fig. 3A), this suggests that C. dauricus was able to regulate Cu in the muscle to a fairly constant level under low Cu exposure concentrations. However, C. dauricus exposed to concentration of 4.12 mg Cu/L showed increased accumulation of Cu in the muscle and the equilibrium of Cu accumulation was not reached at 14 days, which might show that the high level of Cu in the external water breaks down the regulation of Cu and caused a continuous Cu accumulation, leading to its toxicity at high concentration. Similar result had been reported in Procambarus sp.40. The author found that Cu uptake reached a kinetic equilibrium within 10 days of exposure to 0.31 mg Cu/L in five organs (gills, ovaries, exoskeleton, hepatopancreas, and muscles), but Cu was rapidly accumulated in the organs of most Procambarus sp., especially in the hepatopancreas, when exposed to higher concentration of 0.38 mg Cu/L after the 14-d exposure test. However, muscle tissue, as the main edible portion, accumulates Cu at a relatively lower rate (Fig. 2; Fig. 3A) and this is important from the angle of human food quality and safety.
Conclusion
In this study, we observed that the acute and sub-chronic toxicity of Cu had a dramatic impact on the survival, oxygen consumption rate, ammonia excretion rate and bioaccumulation of C. dauricus. C. dauricus mainly took the strategies of inhibiting respiratory metabolism and shifting energy utilization to adapt to copper stress. The C. dauricus had higher concentration-dependent accumulation ability of copper. Our future work will focus on the metabolic characteristics of copper and other heavy metal from the angle of human food safety. Therefore, our studies provided basic information for further understanding of the toxicological responses of this species to trace metals.
Source: Ecology - nature.com
