In watersheds with high concentrations of confined animal production operations, agricultural soils can become phosphorus-enriched when repeated applications of animal manure were made over the years. The near-surface zone accumulates and shows elevated levels of inorganic and organic phosphorus. The buildup presents risks of potential offsite discharges of bioactive phosphorus and of impairment of the quality of nearby bodies of water. An enzyme hydrolysis and soil phosphorus desorption study was conducted to determine whether the rate of buildup of these forms of phosphorus differs between soils and cropping systems. Water-extractable labile forms and complexed forms that are potentially bioactive were measured in Unicorn and Christiana soils (Typic Hapludults) that have received long term additions of cattle (Bos taurus) manure. The in situ Phytase-Hydrolyzable Phosphorus (PHP) assay showed that repeated land applications resulted in soil storage of unextractable complexed organic PHP and a buildup of inorganic ligand-Exchangeable Phosphorus (EEP). The accumulation of EEP increases risks of potential mobilization of bioactive inorganic phosphorus whereas biological hydrolysis of organic PHP also potentially contributes more soluble phosphorus to the near-surface zone of these soils. Therefore, nutrient management practices to reduce phosphorus source solubility have been developed for managing phosphorus surpluses in phosphorus-enriched soils and animal manure. Manure additives and soil amendments include phosphorus-immobilizing agents such as lime and municipal by-products containing metal sesquioxides. These practices are gaining acceptance although the speciation and environmental behavior of the immobilized phosphorus is yet largely unknown. Water-extractable dissolved and complexed phosphorus forms and Mehlich-3 phosphorus were determined to measure the stability of soil phosphorus that was affected by iron hydroxide amendments during a 4-month incubation of two benchmark soils. The iron hydroxide additive reduced water-extractable phosphorus and Mehlich-3 phosphorus by over 90% when applied at a rate of 10 g kg^-1. However, potentially bioactive phosphorus indicated a reversal of the phosphorus immobilization process and that the use of Mehlich-3 phosphorus was not appropriately reflecting these internal changes in soil phosphorus pools and the susceptibility of immobilized phosphorus to re-solubilization. The phytase-hydrolyzable phosphorus fractionation method revealed that the additives' effect was transitory; increasing previously insoluble inorganic EEP was extractable and more organic PHP was exchangeable and susceptible to enzymatic dephosphorylation over time to revert back to initial soil levels about four weeks after the iron addition. Calcium carbonate amended at a liming rate to raise soil pH to near neutrality negated the environmental benefit of applying iron-rich P-immobilizing additives to both soils. The temporary suppression might resolve a short-term elevated soluble phosphorus condition, however, was not effective in mitigating the long-term risks of bioactive phosphorus losses from phosphorus-enriched soils. Therefore, we must depend upon a biological tool to detect internal biochemical changes in the susceptibility of soil phosphorus inorganic and organic pools over time as the traditional soil test method was insensitive to these internal pool transfer and changing stability of the water-insoluble phosphorus species. We postulated that mild biological and biochemical mechanisms more adequately reflect the availability of manure phosphorus pools to microorganisms and plants over time. Moreover, these mechanisms can reveal the underlying potential for the timed release of bioactive phosphorus and protracted impairment of aquatic ecosystems by agricultural phosphorus.