Effects of nitrate on aquaculture production, blood and histological markers and liver transcriptome of Oplegnathus punctatus

Nitrate, the end-product of nitrification, tends to accumulate in recirculating aquaculture systems (RASs). We investigated the 96-h acute and 28-d chronic toxicity of nitrate-N to spotted knifejaw, Oplegnathus punctatus. We exposed juveniles (1.3 +/- 0.6 g, 2.3 +/- 0.7 cm TL) to seven different nitrate-N concentrations (11.3 (control), 673.1, 873.9, 1052.9, 1471.1, 1887.1, or 2368.3 mg/L) in a triplicated experimental design for 96h; the estimated LC50 values for nitrate-N at 24, 48, 72, and 96 h were 774.4, 678.8, 586.3, and 484.5 mg/L nitrate-N - N, respectively. In an examination of chronic effects of elevated nitrate-N, juvenile O. punctatus (44.9 +/- 7.0 g; 7.3 +/- 0.6 cm) were exposed to 21.4 +/- 4.4 mg/L (treatment 1), 165.5 +/- 7.8 mg/L (treatment 2), or 299.4 +/- 5.8 mg/L (treatment 3) nitrate-N in triplicated RASs, and the impact of nitrate-N on performance, blood, histological, and gene expression markers was evaluated. After 28 days, final length, specific growth rate and feed conversion ratio did not differ (p > .05) among treatments. Final weight and mortality did not differ significantly between treatments 1 and 2, but was significantly (p < .05) different in treatment 3. Red blood cell count, white blood cell count, and plasma hemoglobin concentration were significantly (p < .05) lower for fish in treatment 3, and plasma nitrate-N concentration was higher. Plasma cortisol concentrations did not differ significantly among treatments. At the histological level, telangiectasia, hyperplasia, and lamellar shortening and fusion were observed in the gills of fish in treatments 2 and 3; blood congestion was observed in the sinusoids of liver and the hepatocytes exhibited vacuolation and atrophy. RNA-seq showed differential expression of genes in liver (p < .05) among treatments 1, 2, and 3. Gene ontology and assessment of relevant molecular pathways showed the involvement of the Wnt signaling pathway in fish response to nitrate-N exposure. Transcriptome analysis identified important stress response-related genes and signaling pathways that provide insights into mechanisms of response to nitrate-N toxicity. Our findings showed chronic health impacts upon juvenile Oplegnathus punctatus at 165 mg/L nitrate-N, and we recommend further toxicity trials at lower levels to inform prevention of chronic nitrate toxicity.