Combined effects of waterborne copper exposure and salinity on enzymes related to osmoregulation and ammonia excretion by blue crab Callinectes sapidus

  • Eduardo Guerreiro Gomes
  • Lívia da Silva Freitas
  • Fábio Everton Maciel
  • Marianna Basso Jorge
  • Camila de Martinez Gaspar MartinsEmail author


Copper is essential, but can be toxic to aquatic organisms when present in high concentrations. In freshwater crustaceans, copper inhibits enzymes related to ionic and osmoregulation and to the ammonia efflux, that leads to Na+ imbalance and inhibition of ammonia excretion. In the animals inhabiting estuarine or seawater, mechanisms of copper toxicity is not clear, but had been described as disruption of ionregulation and metabolism. To clarify the mechanism of copper toxicity in crustaceans inhabiting variable salinity, this work investigated whether copper affects ammonia excretion and enzymes used for ammonia balance and osmoregulation in the blue crab Callintectes sapidus acclimated to salinity 2 and 30 ppt. To achieve this, juveniles of the blue crab were exposed to 63.5 µg/L of copper at both salinities for 96 h. This is an environmentally realistic copper concentration. Results of ammonia efflux, free amino acids and Na+ concentrations in hemolymph, Na+/K+-ATPase, H+-ATPase and, carbonic anhydrase (CA) activities in gills were consistent with the osmoregulatory pattern adopted by the blue crab, which hyperosmoregulates at salinity 2 ppt and osmoconforms at 30 ppt. At 30 ppt copper reduced free amino acid in hemolymph of crabs, suggesting an effect of the metal on osmotic performance. At 2 ppt, copper significantly increased the H+-ATPase activity involved in ammonia excretion. This may be a compensatory response of crabs to maintain low levels of ammonia in their hemolymph; which can be increased by copper exposure. Results presented here are useful for the improvement of the Biotic Ligand Model (BLM) to predict copper toxicity for saltwater environments.


Biotic Ligand Model Crustacean Gills Metal Toxicity 



Authors wish to thank Dr. Adalto Bianchini from Universidade Federal do Rio Grande – FURG, RS / Brazil and Dr. Chris Wood from University of British Columbia, BC / Canada for scientific assistance. This work was supported by the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul from Brazil (FAPERGS - Proc. 1700-12-2). E. G. G. is a graduate fellow from the Brazilian CAPES. M. B. J. was a research fellow of the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS – Proc. 1635-2551/13-1) during the development of this work. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.


  1. Beaumont MW, Butler PJ, Taylor EW (2003) Exposure of brown trout Salmo trutta to a sublethal concentration of copper in soft acidc water: effects upon gas exchange and ammonia accumulation. J Exp Biol 206:153–162CrossRefGoogle Scholar
  2. Bianchini A, Martins SEG, Barcarolli IF (2004) Mechanism of acute copper toxicity in euryhaline crustaceans: implications for the Biotic Ligand Model. Int Congr Ser 1275:189–194CrossRefGoogle Scholar
  3. Blanchard J, Grosell M (2006) Copper toxicity across salinities from freshwater to seawater in the euryhaline fish Fundulus heteroclitus: is copper a ionorregulatory toxicant in high salinities? Aquat Toxicol 80:131–139CrossRefGoogle Scholar
  4. Charmantier G, Charmantier M, Voss-Foucart MF, Jeuniaux C (1976) Les acides amines libres de l’hemolymphe des isopodes rnarins Sphaeroma hookeri, Sphaeroma serrtum (Flabellifera) et Idotea balthica (Valvifera). Arch Int Biochem 84:989–996Google Scholar
  5. Chen J-C, Chen C-T, Cheng S-Y (1994) Nitrogen excretion and changes of hemocyanin protein and free amino acid levels in the hemolymph of Penaeus monodon exposed to different concentrations of ambient ammonia-N at different salinity levels. Mar Ecol Prog Ser 110:85–94CrossRefGoogle Scholar
  6. Chen J-C, Cheng S-Y (1993a) Haemolymph osmolality, acid-base balance and shift of ammonotelic to ureotelic excretory pattern of Penaeus japonicus exposed to ambient ammonia. Comp Biochem Physiol C 106:733–737Google Scholar
  7. Chen J-C, Cheng S-Y (1993b) Urea excretion by Penaeus japonicus Bate exposed to different concentrations of ambient ammonia. J Exp Mar Biol Ecol 173:1–9CrossRefGoogle Scholar
  8. Clark ME (1964) Biochemical studies on the coelomic fluid of nephtys hombergii (polychaeta: nephytdae), with observations on changes during different physiological states. Biol Bull 127:63–84CrossRefGoogle Scholar
  9. Drach P, Tchernigovtzeff C (1967) Sur la méthode de détermination des stades d’intermue et son application générale aux crustacés. Vie Millieu 18:597–607Google Scholar
  10. Freire CA, Onken H, McNamara JC (2008) A structure-function analysis of ion transport in crustacean gill and excretory organs. Comp Biochem Physiol A. 151:272–304CrossRefGoogle Scholar
  11. Grosell M, Blanchard J, Brix KV, Gerdes R (2007) Physiology is pivotal for interactions between salinity and acute copper toxicity to fish and invertebrates. Aquat Toxicol 84:162–172CrossRefGoogle Scholar
  12. Grosell M, Nielsen C, Bianchini A (2002) Sodium turnover rate determines sensitivity to acute copper and silver exposure in freshwater animals. Comp Biochem Physiol C 133:287–303Google Scholar
  13. Grosell M, Wood CM (2002) Copper uptake across rainbow trout gills: mechanisms of apical entry. J Exp Biol 205:1179–1188Google Scholar
  14. Henry RP (1991) Techniques for measuring carbonic anhydrase activity in vitro: the electrometric delta pH and pH stat assay. In: Dodgson SJ, Tashian RE, Gros G, Carter ND (eds) The carbonic anhydrases: cellular physiology and molecular genetics. Plenum, New York, NY, USA, p 119–126CrossRefGoogle Scholar
  15. Henry RP, Cameron JN (1982) Acid–base balance in Callinectes sapidus during acclimation from high to low salinity. J Exp Biol 101:255–264Google Scholar
  16. Henry RP, Lucu C, Onken H, Weihrauch D (2012) Multiple functions of the crustacean gills: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals. Front Physiol 3(431):1–33Google Scholar
  17. Jorge MB, Lauer MM, Martins CMG, Bianchini A (2016) Impaired regulation of divalent cations with acute copper exposure in the marine clam Masodesma mactroides. Comp Biochem Physiol C 179:79–86Google Scholar
  18. Kormanik GA, Cameron JN (1981) Ammonia excretion in the seawater blue crab (Callinectes sapidus) occurs by diffusion, and not Na+/NH4 + exchange. J Comp Physiol B 141:457–462CrossRefGoogle Scholar
  19. Laurén DJ, McDonald DG (1985) Effects of copper on branchial ionorregulation in the rainbow trout, Salmo gardineri Richardson. J Comp Physiol B 155:635–644CrossRefGoogle Scholar
  20. Lim M, Zimmer AM, Wood CM (2015) Acute exposure to waterborne copper inhibits both the excretion and uptake of ammonia in freshwater rainbow trout (Oncorhynchus mykiss). Comp Biochem Physiol C 168:48–54Google Scholar
  21. Mangum CP, Slverthorn SU, Harris JL, Towle DW, Krall AR (1976) The relationship between blood pH, ammonia excretion, and adaptation to low salinity in the blue crab Callinectes sapidus. J Exp Zool 195:129–136CrossRefGoogle Scholar
  22. Mantel LH, Farmer LL (1983) Osmotic and ionic regulation. In: Vernberg EJ, Vernberg WB (eds) The biology of crustacean, vol. 5. Academic, New York, NY, USA, pp 53–161Google Scholar
  23. Martins CMG, Barcarolli IF, Menezes EJ, Giacomin MM, Wood CM, Bianchini A (2011a) Acute toxicity, accumulation and tissue distribuition of copper in the blue crab Callinectes sapidus acclimated to different salinities: in vivo and in vitro studies. Aquat Toxicol 101:88–99CrossRefGoogle Scholar
  24. Martins CMG, Volcan DA, Marins LFF, Bianchini A (2011b) mRNA expression and activity of ion-transporting proteins in gills of the blue crab Callinectes sapidus effects of waterborne copper. Envinron Toxicol Chem 30:2006–2011Google Scholar
  25. Masui DC, Furriel RP, McNamara JC, Mantelatto FL, Leone FA (2002) Modulation by ammonium ions of gill microsomal (Na+,K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion. Comp Biochem Physiol C 132:471–482Google Scholar
  26. Paquin PR, Gorsuch JW, Apte S, Batley GE, Bowles KC, Campbell PGC, Delos CG, Di Toro DM, Dwyer RL, Galvez F, Gensemer RW, Goss GG, Hogstrand C, Janssen CR, McGeer JC, Naddy RB, Playle RC, Santore RC, Schneider U, Stubblefield WA, Wood CM, Wu KB (2002) The biotic ligand model: a historical overview. Comp Biochem Physiol C 133(1-2):3–35Google Scholar
  27. Péqueux A (1995) Osmotic regulation in crustaceans. J Crustac Biol 15:1–60CrossRefGoogle Scholar
  28. Pequéux A, Vallota AC, Gilles R (1979) Blood proteins as related to osmorregulation in Crustacea. Comp Biochem Pshysiol A 64:433–435CrossRefGoogle Scholar
  29. Piller SC, Henry RP, Doeller JE, Kraus DW (1995) A comparison of the gill of two euryhaline crab species, Callinectes sapidus and Callinects similis: energy production, transport-related enzymes and osmoregulation as a function of acclimation salinity. J Exp Biol 198:349–358Google Scholar
  30. Pressley TA, Graves JS, Krall AR (1981) Amiloride-sensitive ammonium and sodium ion transport in the blue crab. Am J Physiol 241:370–378Google Scholar
  31. Regnault M (1987) Nitrogen excretion in marine and fresh-water crustacea. Biol Rev 62:1–24CrossRefGoogle Scholar
  32. Ribani M (2004) Validacão em métodos cromatigráficos e eletroforéticos. Quim Nova 7(5):771–780CrossRefGoogle Scholar
  33. SANCO (2013) Guidance document on analytical quality control and validation procedures for pesticides residues analysis in food and feed. SANCO/12571/2013. European Commission Health & Consumer Protection Directorate General, 48.
  34. Skaggs HS, Henry RP (2002) Inhibition of carbonic anhydrase in the gills of two euryhaline crabs, Callinectes sapidus and Carcinus maenas by heavy metals. Comp Biochem Physiol 133C:605–612Google Scholar
  35. Stewart PA (1978) Independent and dependent variables of acid-base control. Resp Phytiol 33:0–26Google Scholar
  36. Towle DW, Holleland T (1987) Ammonium ion substitutes for K+ in ATP dependent Na+ transport by basolateral membrane vesicles. Am J Physiol 252:479–489Google Scholar
  37. Towle DW, Rushton ME, Heidysch D, Magnani JJ, Rose MJ, Amstutz A, Jordan MK, Shearer DW, Wu WS (1997) Sodium/proton antiporter in the euryhaline crab Carcinus Maenas: molecular cloning, expression and tissue distribuition. J Exp Biol 200:1003–1014Google Scholar
  38. Tsai JR, Lin HC (2007) V-type H+ATPase in the gills of euryhaline crabs during salinity acclimatation. J Exp Biol 210:620–627CrossRefGoogle Scholar
  39. USEPA (2007) Aquatic life ambient freshwater quality criteria – copper, EPA-822-R07-001, 2007 revision. US Environmental Protection Agency, Washington, USA, p 204Google Scholar
  40. Verdouw H, van Echteld CJA, Dekkers EMJ (1978) Ammonia determination based on indophenol formation with sodium salicylate. Water Res 12:399–402CrossRefGoogle Scholar
  41. Vitale AM, Monserrat JM, Castilho P, Rodrigues EM (1999) Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae). Comp Biochem Physiol C 122:121–129Google Scholar
  42. Weihrauch D, Becker D, Postel U, Luck-Kopp S, Siebers D (1999) Potential of active excretion of ammonia in three different haline species of crabs. J Comp Physiol B 169:25–37CrossRefGoogle Scholar
  43. Weihrauch D, Towle DW (2000) Na+/H+-exchanger and Na+/K+/2Cl cotransporter are expressed in the gills of the euryhaline Chinese crab Eriocheir sinensis. Comp Biochem Physiol B 126:S158CrossRefGoogle Scholar
  44. Weihrauch D, Wilkie MP, Walsh PJ (2009) Ammonia and urea transporters in gills of fish and aquatic crustaceans. J Exp Biol 212:1716–1730CrossRefGoogle Scholar
  45. Weihrauch F, Morris S, Towle DW (2004) Ammonia excretion in aquatic and terrestrial crabs. J Exp Biol 207:4491–4504CrossRefGoogle Scholar
  46. Wilson RW, Taylor EW (1993) Differential responses to copper in rainbow trout (Oncorynchus mykiss) acclimated to sea water and brackish water. J Comp Physiol B 163:239–246Google Scholar
  47. Wood CM, Camaron JN (1985) Temperature and the physiology of intracellular and extracellular acid–base regulation in the blue crab Callinectes sapidus. J Exp Biol 114:151–179Google Scholar
  48. Zimmer AM, Barcarolli IF, Wood CM, Bianchini A (2012) Waterborne copper exposure inhibits ammonia excretion and branchial carbonic anhydrase activity in euryhaline guppie acclimated to both fresh water and sea water. Aquat Toxicol 122 123:172–180CrossRefGoogle Scholar
  49. Zimmer AM, Jorge AM, Wood CM, Martins CMG, Bianchini A (2017) The effects of acute copper and ammonia challenges on ammonia and urea excretion by the blue crab Callinectes sapidus. Arch Environ Contam Toxicol 73(3):461–470CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Programa de Pós Graduação em Ciências Fisiológicas, Instituto de Ciências BiológicasUniversidade Federal do Rio Grande – FURGRio GrandeBrazil
  2. 2.Instituto de Ciências BiológicasUniversidade Federal do Rio Grande – FURGRio GrandeBrazil
  3. 3.Departamento de OceanografiaUniversidade Federal do Maranhão – UFMASão LuísBrazil

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