Aquatic Toxicology 170 (2016) 129–141 Contents lists available at ScienceDirect Aquatic Toxicology j ourna l ho me pa ge: www.elsevier.com/locate/aquatox Differential modulation of ammonia excretion, Rhesus glycoproteins and ion-regulation in common carp (Cyprinus carpio) following individual and combined exposure to waterborne copper and ammonia Amit Kumar Sinha a,∗ , Mumba Kapotwe a , Shambel Boki Dabi a , Caroline da Silva Montes a,b , Jyotsna Shrivastava a , Ronny Blust a , Gudrun De Boeck a a Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium b Laboratory of Immunohistochemical and Cellular Ultrastructure, Federal University of Para, Campus Guamá, Rua Augusto Corrêa 1, 66075-900, Belém, Pará, Brazil a r t i c l e i n f o Article history: Received 17 October 2015 Received in revised form 18 November 2015 Accepted 19 November 2015 Available online 24 November 2015 Keywords: Ammonia excretion rate Rhesus (Rh) glycoprotein Ion-regulation Na + /NH4 + exchange metabolon Plasma ions a b s t r a c t The main objective of this study was to understand the mode of interaction between waterborne copper (Cu) and high environmental ammonia (HEA) exposure on freshwater fish, and how they influence the toxicity of each other when present together. For this purpose, individual and combined effects of Cu and HEA were examined on selected physiological and ion-regulatory processes and changes at transcript level in the common carp (Cyprinus carpio). Juvenile carp were exposed to 2.6 M Cu (25% of the 96 h LC 50 value) and to 0.65 mM ammonia (25% of the 96 h LC 50 value) singly and as a mixture for 12 h, 24 h, 48 h, 84 h and 180 h. Responses such as ammonia (J amm ) and urea (J urea ) excretion rate, plasma ammonia and urea, plasma ions (Na + , Cl - and K + ), muscle water content (MWC) as well as branchial Na + /K + -ATPase (NKA) and H + -ATPase activity, and branchial mRNA expression of NKA, H + -ATPase, Na + /H + exchanger (NHE-3) and Rhesus (Rh) glycoproteins were investigated under experimental conditions. Results show that J amm was inhibited during Cu exposure, while HEA exposed fish were able to increase excretion efficiently. In the combined exposure, J amm remained at the control levels indicating that Cu and HEA abolished each other’s effect. Expression of Rhcg (Rhcg-a and Rhcg-b) mRNA was upregulated during HEA, thereby facilitated ammonia efflux out of gills. On the contrary, Rhcg-a transcript level declined following Cu exposure which might account for Cu induced J amm inhibition. Likewise, Rhcg-a was also down-regulated in Cu-HEA co-exposed fish whilst a temporary increment was noted for Rhch-b. Fish exposed to HEA displayed pronounced up-regulation in NKA expression and activity and stable plasma ion levels. In both the Cu exposure alone and combined Cu-HEA exposure, ion-osmo homeostasis was adversely affected, exemplified by the significant reduction in plasma [Na + ] and [Cl - ], and elevated plasma [K + ], along with an elevation in MWC. These changes were accompanied by a decline in NKA activity. Gill H + -ATPase mRNA levels and activities were not affected by either Cu or HEA or both. Likewise, NHE- 3 expression remained unaltered but tended to be numerically higher during HEA exposure. Overall, these data suggest that at equitoxic concentrations (25% of 96 h LC 50 ), the individual effect of Cu is more harmful while HEA induces quicker adaptive responses. Our findings also denote a competitive mode of interaction, exemplified by the inhibition of HEA -mediated adaptive responses in the presence of Cu. © 2015 Elsevier B.V. All rights reserved. ∗ Corresponding author. Fax: +32 32 653 497. E-mail address: sinha cife@rediffmail.com (A.K. Sinha). 1. Introduction Pollution of natural and culture water systems by metal and nitrogen waste (typically ammonia) are of global concern. Toxic concentration of copper (Cu) in natural water systems occur mainly through mining, smelting, industrial and agricultural activities while sewage effluents, industrial wastes and agricultural run-off http://dx.doi.org/10.1016/j.aquatox.2015.11.020 0166-445X/© 2015 Elsevier B.V. All rights reserved.