A major issue affecting land-based, closed containment Atlantic salmon Salmo salar growout production in water recirculation aquaculture systems (RAS) is precocious male maturation, which can negatively impact factors such as feed conversion, fillet yield, and product quality. Along with other water quality parameters, elevated nitrate nitrogen (NO₃-N) has been shown to influence the reproductive development and endogenous sex steroid production in a number of aquatic animal species, including Atlantic salmon. We sought to determine whether elevated NO₃-N in RAS can influence early maturation in post-smolt Atlantic salmon in an 8-month trial in replicated freshwater RAS. Post-smolt Atlantic salmon (102 ± 1 g) were stocked into six RAS, with three RAS randomly selected for dosing with high NO₃-N (99 ± 1 mg/L) and three RAS set for low NO₃-N (10 ± 0 mg/L). At 2-, 4-, 6-, and 8-months post-stocking, 5 fish were randomly sampled from each RAS, gonadosomatic index(GSI) data were collected, and plasma was sampled for 11-ketotestosterone(11-KT) quantification. At 4- and 8-months post-stocking, samples of culture tank and spring water (used as “makeup” or replacement water) were collected and tested for a suite of 42 hormonally active compounds using liquid chromatography/mass spectrometry, as well as for estrogenicity using the bioluminescent yeast estrogen screen (BLYES) reporter system. Finally, at 8-months post-stocking 8–9 salmon were sampled from each RAS for blood gas and chemistry analyses, and multiple organ tissues were sampled for histopathology evaluation. Overall, sexually mature males were highly prevalent in both NO₃-N treatment groups by study’s end, and there did not appear to be an effect of NO₃-N on male maturation prevalence based on grilse identification, GSI, and 11-KT results, indicating that other culture parameters likely instigated early maturation. No important differences were noted between treatment groups for whole blood gas and chemistry parameters, and no significant tissue changes were noted on histopathology. No hormones, hormone conjugates, or mycotoxins were detected in any water samples; phytoestrogens were generally detected at low levels but were unrelated to NO₃-N treatment. Finally, low-level estrogenicity was detected in RAS water, but a NO₃-N treatment effect could not be determined. The major findings of this study are i) the NO₃-N treatments did not appear to be related to the observed male maturation, and ii) the majority of hormonally active compounds were not detectable in RAS water.