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Periphyton and water were sampled 1 km upstream of the Cookeville Wastewater Treatment Plant (PRupstream), 2 km downstream of the discharge (PRdownstream), and from a tributary (PO) 10 km south of the wastewater treatment plant. Percent composition of 113 algal taxa were documented. Assemblages of soft-bodied algae and diatoms at the PRdownstream site had more eutrophic taxa than assemblages at the other sites. Water at the PRdownstream site had >7-fold higher concentration of total phosphorus (193 μg∙L-1), yet <1.5-fold higher concentration of total nitrogen (1900 μg∙L-1) than water at the other sites. The total nitrogen to total phosphorous ratio (TN:TP ratio) at the PRdownstream site (9.8) was below the value hypothesized for N-limitation of algae growth (10), whereas the ratios at the PRupstream site (65.9) and PO site (62.4) were above. Phosphorus and nitrogen additions to in vitro growth assays using Raphidocelis subcapitata indicate in vitro carrying capacity for Raphidocelis subcapitata was P-limited in water from the PRupstream and PO sites. In vitro carrying capacity for Raphidocelis subcapitata was significantly greater and N-limited in water from the PRdownstream site. The results indicate that reduction of the TN:TP ratio of Pigeon Roost Creek changed the nutrient that limits in vitro carrying capacity for R. subcapitata from P to N, and imply that standard growth assays using Raphidocelis subcapitata to evaluate carrying capacity of P-enriched stream water may not indicate the extent of the impact of the P-enrichment to a watershed due to N-limitation of carrying capacity in vitro.