Comparative biochemistry and physiology, part C combined Zn and Mn affect osmoregulation of the freshwater teleost Prochilodus lineatus in distinct ways from isolated metals

Juveniles of the freshwater teleost Prochilodus lineatus were used to evaluate the osmoregulatory effects of the essential metals zinc (Zn), manganese (Mn) and iron (Fe), both individually and in different combinations. Fish were subjected to three independent 96-h exposure tests, each including its respective control group (CTR; fish exposed only to water): (i) Zn test, with exposures to 0.18, 1.0, and 5.0 mg L⁻¹ Zn; (ii) Mn test, with exposures to 0.1, 0.5, and 5.0 mg L⁻¹ Mn; and (iii) combined test with exposures to Fe (5.0 mg L⁻¹) individually, a mixture of Zn (1.0 mg L⁻¹) and Mn (0.5 mg L⁻¹), or the three metals combined (Zn + Mn + Fe). Gill enzyme activities were analyzed for carbonic anhydrase (CA), H⁺-ATPase (HATP), Ca²⁺-ATPase (CaATP), Na⁺/K⁺-ATPase (NKA), and Mg²⁺-ATPase (MgATP). Plasma concentrations of Na⁺, K⁺, Mg²⁺, Ca²⁺, Cl⁻, and plasma osmolality were also measured. Exposure to Zn resulted in decreased plasma concentrations of K⁺, Na⁺, and Ca²⁺ without affecting gill enzymes activities. Mn exposure altered only plasma Ca²⁺ concentration, decreasing at low Mn levels and increasing at high Mn levels, accompanied by elevated HATP and CA activities, suggesting the occurrence of acidosis. In fish exposed to Zn + Mn, a reduction in plasma Ca²⁺ concentration and an increase in CaATP activity were observed. In fish exposed to Zn + Mn + Fe, an increase in plasma Ca²⁺ concentration and a decrease in HATP activity were detected. The evaluated biomarkers proved effective for monitoring Zn, Mn, and Fe contamination, both individually and in combination, in Neotropical species such as P. lineatus. Zn and Mn disrupted osmoregulation through different mechanisms, while mixed exposures produced agonistic effects.