Recovery of rainbow trout (Oncorhynchus mykiss) after subchronic nitrite exposure

The aim of the study was to assess the effect of elevated nitrite concentration on the haematological and biochemical profile of blood of rainbow trout and to evaluate the ability of fish to recover under varying conditions. Fish (n = 72) were exposed for 14 days to elevated nitrite concentration (1 mg·l-1). Thereafter the exposed fish were divided into four groups and monitored for 7 days in the following conditions: water containing 1 mg·l-1 NO2 (group E), water containing 1 mg·l-1 NO2 + 100 mg·l-1 Cl(group R1), nitrite-free water with 100 mg·l-1 Cl(group R2), or nitriteand chloride-free water (group R3). Nitrite exposure was associated with a significant increase (P 68%, pH 7.2–7.9, acid neutralisation capacity (ANC4.5) 1.2 mmol·l -1, chemical oxygen demand (CODMn) 1.5 mg·l -1, total ammonia 0.04 mg·l-1, sum of Ca2++Mg2+ 14 mg·l-1, Cl11 mg·l-1, and PO4 30.01 mg·l-1. Toxicity test protocol A prolonged toxicity test was carried out using a modified test design of OECD Direction No. 204 Fish, Prolonged Toxicity Test and ISO 10229 (2007) – Water Quality. Fish were exposed to 1 mg·l-1 NO2 -, a concentration that commonly occurs in intensive aquaculture systems. The desired concentration was obtained by addition of sodium nitrite. A chloride concentration of 100 mg·l-1 was obtained by addition of sodium chloride. Nitrite and chloride concentrations were checked daily during the test, and measured values did not differ from those stated for test purposes by more than 10%. The test baths had constant aeration. Oxygen saturation, pH, and temperature were measured daily. Experimental protocol Forty-eight fish were divided into two groups (control and experimental). Control and experimental group consisted of 12 and 36 fish, respectively. The supply water and diet used were the same as those during the initial acclimation period. The bath was renewed daily. During the 14 days exposure period, experimental fish (n = 36) were exposed to 1 mg·l-1 NO2 and controls (n = 12) were maintained in nitrite-free tap water. At the end of the exposure period, four experimental and four control fish were sampled. Group E continued to be exposed to 1 mg·l-1 NO2 and the remaining fish from the experimental group were divided into 3 groups (R1–R3) to evaluate recovery from nitrite poisoning. Water treatments following initial exposure to nitrite were: water containing 1 mg·l-1 NO2 + 100 mg·l-1 Cl(group R1); nitrite-free water with 100 mg·l-1 Cl(group R2); nitritefree tapwater (group R3). This post-exposure period continued for 7 days, with sampling of 4 fish per group on day 2 and at the conclusion of the test. Entire experiment was done in duplicate. The experimental system is shown in Fig. 1. Fish sampling Blood samples were taken from the caudal vessels into heparinised syringes, and the blood was stabilised with 40 IU of sodium heparin (Heparin inj., Leciva, Czech Republic) per 1 ml blood. Immediately after sampling, the erythrocyte count (RBC), haematocrit (PCV), haemoglobin (Hb), methaemoglobin (MetHb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and leukocyte count (WBC) were determined according Svobodova et al. (1991). Plasma was separated from cells by centrifugation (10 min at 12 000 × g) at 4 °C, and plasma ammonia (NH3), urea nitrogen (UREA), uric acid (URIC), glucose (Glu), total protein (TP), albumin (ALB), total globulin (Glob), calcium (Ca2+), magnesium (Mg2+), and inorganic phosphorus concentrations (PHOS) were determined using the VETTEST 8008 Analyser (IDEXX Laboratories, Westbrook, ME, USA). Plasma Na+, K+, and Clconcentrations were measured with an automated clinical chemistry analyser, Radiometer ABL90 FLEX. After blood sampling, the fish were quickly stunned with a blow to the head, and dorsal muscle samples of approximately 1 g were excised. The muscle and plasma samples for nitrite determination were kept at 74 Groups sampled in day: