Novel standard biodegradation test for synthetic phosphonates

Determination of biodegradation of synthetic phosphonates such as aminotris(methylenephosphonic acid) (ATMP), ethylenediamine tetra(methylenephosphonic acid) (EDTMP), or diethylenetriamine penta(methylenephosphonic acid) (DTPMP) is a great challenge. Commonly, ready biodegradability of organic substances is assessed by OECD 301 standard tests. However, due to the chemical imbalance of carbon to phosphorus synthetic phosphonates do not promote microbial growth and, thus, limiting its biodegradation. Therefore, standard OECD test methods are not always reliable to predict the real biodegradability of phosphonates. In the presented study, we report the development of a standardized batch system suitable to synthetic phosphonates such as ATMP, EDTMP, DTPMP and others. The novel standard batch test is applicable with pure strains, activated sludge from different wastewater treatment plants (i.e., municipal and industrial), and with tap water as inoculum. We optimized the required calcium and magnesium exposure levels as well as the amount of the start inoculum biomass. We demonstrated that our test also allows to determine several parameters including ortho-phosphate (o-PO₄^3-), total phosphorus (TP), ammonium (NH₄⁺) and total organic carbon (TOC). In addition, also LC/MS analyses of cell-free medium is applicable for determining the mother compounds and metabolites. We applied our optimized standardized batch with selected phosphonates and evidenced that the chemical structure has a major influence of the microbial growth rates. Thus, our novel batch test overcomes drawbacks of the OECD 301 test series for determination of easy biodegradability for stoichiometric imbalanced organic compounds such as phosphonates.