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Abstract Background Eutrophication of freshwater ecosystems resulting from nitrogen and phosphorus pollution is a major environmental stressor across the globe. In this systematic review, we compiled and synthesized literature on sestonic and benthic chlorophyll a (chl-a) responses to total nitrogen (TN) and total phosphorus (TP) concentrations in the water column in streams and rivers to provide a state-of-the-science summary of nutrient impacts on these endpoints. This review was motivated by the need for comprehensive information on stressor-response relationships for the most common nutrient and biotic response measures used by state-level environmental managers in the United States to assess eutrophication of lotic ecosystems and support environmental decision making. Methods Searches for peer-reviewed and non-peer-reviewed articles were conducted using bibliographic databases, specialist websites, and search engines. These returns were supplemented with citation mapping and requests for material from experts. Articles were screened for relevance using pre-determined eligibility criteria, and risk of bias was evaluated for each included article based on study type-specific criteria. Narrative summaries and meta-analysis were used to evaluate four primary stressor-response relationships: TN-benthic chl-a, TP-benthic chl-a, TN-sestonic chl-a, and TP-sestonic chl-a. Potential effects of modifying factors and study validity on review conclusions were assessed via sensitivity and sub-group analysis and meta-regression. Results Meta-analysis of 105 articles, representing 439 cause-effect pairs, showed that mean effect sizes of both benthic and sestonic chl-a responses to TN and TP were positive. Of the four stressor-response relationships examined, TP-sestonic chl-a had the most positive relationship, followed by TN-benthic chl-a, TN-sestonic chl-a, and TP-benthic chl-a. For individual U.S. states, mean effect sizes for the four stressor-response relationships were mostly positive, with a few exceptions. Chlorophyll measurement method had a moderately significant influence on mean effect size for TP-sestonic chl-a, with chl-a responding more strongly to TP if fluorometry versus spectrophotometry was used. Year of publication had a significant negative effect on mean effect size, as did mean nutrient concentration for both sestonic chl-a nutrient relationships. When the same study measured both TN and TP, chl-a tended to respond similarly to both nutrients. Sensitivity analysis indicated that conclusions are robust to studies with high risk of bias. Conclusions This systematic review confirms that nutrients consistently impact primary producer biomass in streams and rivers worldwide. It builds on previous literature syntheses evaluating chl-a responses to nutrient concentrations and confirms that benthic and sestonic chl-a respond positively to nutrients across a range of stream and river conditions, but also points to limits on these relationships (e.g., potential saturation at high nutrient concentrations). Lack of consistent reporting of contextual data limited our ability to examine how moderating factors influenced these stressor-response relationships. Overall, we provide nutrient managers responsible for protecting the quality of lotic ecosystems with a comprehensive evidence base for chl-a responses to TN and TP concentrations in the water column.