TY - JOUR T1 - Addressing Confounding Factors in Algal Phosphorus Limitation Experiments JF - bioRxiv DO - 10.1101/298281 SP - 298281 AU - Whitney S. Beck AU - Ed K. Hall Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/04/09/298281.abstract N2 - Assessing algal nutrient limitation is critical for understanding the interaction of primary production and nutrient cycling in stream ecosystems. Nutrient diffusing substrates (NDS) have been used for over thirty years to quantify nutrient limitation. However, many NDS studies have shown a decrease in algal biomass on phosphorus (P) treatments compared to controls, which is inconsistent with the paradigm of freshwater P-limitation. While direct P toxicity may occur, P-inhibition of algal growth could also be the result of NDS preparation artifacts or stream environmental factors. We first used a quantitative review to determine how often NDS P treatments inhibit algal biomass. We then designed and deployed targeted experiments to determine whether algal growth inhibition with P additions could be explained by P toxicity, differences in phosphate cation (K vs. Na), differences in phosphate form (monobasic vs. dibasic), or production of H2O2 as an artifact of NDS preparation. In addition, we completed a quantitative review of published data to determine the potential for heterotrophic microbial competition or selective grazing to explain decreases in algal biomass with P additions. We found that 13.7% of NDS P experiments showed significant inhibition of algal growth, as compared to just 5.1% and 3.9% of N and NP experiments. Our field experiments did not show inhibition of autotrophic growth with P additions as is routinely reported. However, we did see increased heterotrophic biomass on treatments that used monobasic phosphate salts as compared to those that used dibasic phosphate salts. The quantitative review did not show significantly enhanced heterotrophy on NDS P treatments, nor did we find consistent evidence for grazer selection of P-rich algae. Past laboratory experiments and meta-analyses support the plausibility of direct P toxicity, cation toxicity, or phosphate form (monobasic vs. dibasic) leading to inhibition of algal growth. Given that multiple mechanisms may be acting simultaneously, we recommend practical, cost-effective steps for designing NDS experiments to avoid the potential for P-inhibition due to artifacts of the experimental design. We also outline future research that could lead to a better understanding of how resources interact with environmental conditions to regulate algal growth in stream ecosystems. ER -