Adaptation and optimization of the HPLC-based determination procedure of amino derivatives
Because the application of HPLC chromatographic conditions, which have been already described, did not allow effective separation of all seven predicted intermediates or final products of AAP degradation; several factors, such as the flow rate, mobile phase composition, and column temperature, were optimized. The introduction of two gradient domains to isocratic separation [the first one after 17 min, which coincides with the stable flow of mobile phase (1.3 mL min−1), and the second from 24 to 31 min, accompanied by a gradual decrease of the flow (from 1.3 to 1.0 mL min−1)] allowed the reduction of the entire time of HPLC separation to approximately 36 min. The temperature of the column was set at 35 °C and was stable during the entire process. As shown in Fig. 1, the optimized conditions assured the successful, simultaneous separation of all seven presumable products of degradation in one step.
Validation of the analytical procedure
Calibration curves were constructed based on analytical values obtained for aminopolyphosphonates dissolved in a fresh, sterile Bg11 medium, after 1 week of freezing of the samples, and in fresh and frozen 14-days post-cultured media. That approach allowed us to investigate both the impact of freezing and storage of samples at − 28 °C and the possible interference of analytes with microbial metabolites that are present in a post-culture media. Calibration curves for aminomethylphosphonic acid (AMPA), N-(methyl)aminomethanephosphonic acid (MAMPA), and N-phosphomethyl glycine (NPMG) were found to be linear in the concentration range from 0.8 to 100 μM. For 2–aminethylphosphonic acid (2-AEP) and glycine (Gly), the curves were linear in the range of 1.5 up to 100 μM, while for N-methylamine (NMA) and N-(methyl) glycine (SAR), the linearity was observed from 0.4 to 100 μM. The values of limits of detection (LOD), limits of quantification (LOQ), and correlation coefficients are given in Table 3.
The table includes the values of limit of detection and limit of quantification [expressed in (μM)] that are computed for stock solutions prepared in a fresh Bg11 medium (Bg11), after 7 days of freezing (Bg11 frozen), and stock solutions made in a 14-day-old post-cultured medium of Anabaena variabilis with cells being removed prior to the measurement (post-cultured Bg11).
The impact of freezing or the presence of cyanobacterial metabolites on the limit of detection was slight. The presence of cyanobacterial matrix had evoked the changes in the level of determination of MAMPA and SAR (compared with a Bg11 stock solution). Nevertheless, this difference amounted in only circa 10 nM. Freezing, in turn, influenced the LOD values mostly in the case of Gly (almost 50 nM). Nevertheless, the values of LOD and LOQ that are presented in Table 3 are of the same order of magnitude and are in a nanomolar concentration range. It should be noted that AMPA, NPMG, MAMPA, and SAR can be detected below the concentration of 50 nM. In regard to 2-AEP, Gly, and NMA, the detection levels were estimated in the range of 120–130 nM. Considering the simplicity of the procedure used and the complexity of the matrix, in which the compounds have been detected, the described analytical procedure seems to considerably simplify their tracking in natural waters.
Precision of the implemented HPLC method
The precision of the HPLC method was expressed as the percent relative standard deviation (%RSD) and determined for 0.025-mM stock solutions of the mixture of examined compounds (Table 4). For all batches, the values were below 5%, which proved that neither the freezing process nor the presence of cyanobacterial metabolites substantially affected the precision of the HPLC-based procedure used.
Stability of the tested aminopolyphosphonates in the examined liquids
The absence of direct methods of aminopolyphosphonate determination, particularly at the concentration level below 0.1 μM, compels us to express their stability as a formation of new compounds. Indisputably, the components of a Bg11 medium influenced the stability of tested aminophosphonates, which resulted in the formation of some of the postulated breakdown products (Table 5.). All examined phosphonates underwent chemical decomposition that is accompanied by the release of an aminomethylphosphonic acid (AMPA) molecule but with a varied intensity. The emergence of N-(methyl)aminomethanephosphonic acid (MAMPA) among other decomposition products of aminotris(methylenephosphonic) acid (ATMP) and N,N-bis(phosphonomethyl) glycine (GBMP) should be underlined, especially because this compound has not yet been deliberated as a product of the aminopolyphosphonate transformation processes. The vulnerability of aminopolyphosphonates to degradation in Bg11 medium was characterized by the number and concentration of the formed products. Based on these data, the following order of increasing stability of tested compounds, which correlates with a growing number of methylphosphonic groups in molecules, can be concluded as follows: GBMP < ATMP < HDTMP < DTPMP.
In addition to GBMP, which is not stable in water, instability of aminopolyphosphonates may result from the presence of some transition metal ions, as has been already stated by Nowack. It has been proven that in the presence of Mn(II), some AAPs undergo oxidation, whereas the presence of ions of Fe(II) and Fe(III) leads to the formation of phosphonate complexes, which undergo photodegradation (Nowack 2003). The molecule of aminotri(methylenephosphonic) acid (ATMP) subjected to oxidation in the presence of manganese ions forms iminedimethylenephosphonic acid (IDMP) and N-formyl-iminodimethylenephosphonic acid (FIDMP) (Nowack 2002).
In contrast to multifaceted and time-consuming HPLC determination, in which 9-fluorenylmethyl chloroformate (FMOC) was applied as a derivatization agent (Nowack 2002), the method applied in this work did not allow us to determine the presence of iminodi(methylenephosphonic) acid (IDMP). Although, Mn(II) ions are present in a Bg11 medium at a concentration approximately 10 times lower than the tested phosphonates, and ATMP breakdown in the presence of manganese cations was observed for equimolar amounts of these substances (Nowack and Stone 2000), the formation of iminodiphosphonic derivatives cannot be disputed. In addition, Nowack and Stone (Nowack and Stone 2000) postulated that Zn(II) and Ca(II) ions considerably inhibit the reaction by competing for the phosphonate substrate. In our study, these ions are present in Bg11 medium at a final concentration of 0.765 μM and 0.24 mM, respectively, and the process of degradation still occurs. Although the pH of the Bg11 medium (~ 7.1) favors degradation via metal ion-catalyzed pathways, the same process can be hampered by other components of the medium such as EDTA and Cu(II) ions, which completely inhibit AAP decomposition via IDMP and FIDMP release. The appearance of an aminomethylphosphonic acid (AMPA) molecule in the solution of ATMP in a Bg11 medium (Table 5) can be related to the IDMP occurrence due to the chemical structure of the substrate. However, the composition of a Bg11 medium suppressed the appearance of AMPA with respect to GBMP. In this case, the final concentration of aminomethylenephosphonic acid was approximately nine-fold lower compared with its solution in water.
Tendency of aminopolyphosphonates for biodegradation
Aminopolyphosphonates that are placed in a sole Bg11 medium have shown the tendency for spontaneous degradation, probably as a consequence of the action of transition metal ions. However, to understand the environmental fate of AAPs, it is necessary to assess their interactions with organisms that are responsible for the cycle of elements in nature. Certainly, cyanobacteria rank highly in this respect (Cottingham et al. 2015). The usefulness of several phosphonates as sources of phosphorus for aquatic microorganisms has been already proven (Drzyzga et al. 2017, Forlani et al. 2011, Ravi and Balakumar 1998, Studnik et al. 2015). Concerning the impact of cyanobacteria on the stability of AAPs, it is worth noting that among the detected intermediates, there are compounds that have been identified after 2 weeks of incubation in a sole Bg11 medium. Nevertheless, the concentrations of these substances in cyanobacterial cultures are significantly higher, even up to four- or five-fold in some cases (Table 6).
Aminotri(methylenephosphonic) acid (ATMP) molecules underwent degradation with a release of aminomethylphosphonic acid (AMPA), regardless of the presence of cyanobacteria and a deficiency in inorganic phosphate. The main metabolite of ATMP was detected in a post-culture media at a concentration of approximately 100 μM. Only in the culture of Chroococcidiopsis 049 was this intermediate identified at a lower concentration (41.3 μM), similar to that measured in the substrate control (34.0 μM). Thus, in this case, the formation of AMPA should be considered catalyzed by metal ions and not as an action of cyanobacteria. However, when examined, polyphosphonate was the sole available source of phosphorus, and aminomethylphosphonic acid was present at a concentration of approximately 127 μM. A similar increase in AMPA release in Bg11-P, in comparison to a Bg11 medium, was also noticed for Nostoc 129. In both media, N-(methyl)aminomethanephosphonic acid (MAMPA) was detected in nearly identical amounts (~ 3 μM). It is consistent with the data presented in Table 5 and indicates the lack of a microbial contribution in ATMP decomposition with the N-(methyl)aminomethanephosphonic acid production.
The appearance of MAMPA as the main metabolite in the biodegradation process of N,N-bis(phosphonomethyl) glycine (GBMP) has been proven for the first time. In a Bg11 medium, after 2 weeks, its level was approximately 44 μM. However, the cultivation of cyanobacteria in the presence of 100 μM of GBMP led to the enhancement of the MAMPA concentration from 83 μM for Chroococcus 055, even up to 156 μM for Chroococcidiopsis. It can be stated that the weaker biodegradation rate of GBMP was noticed when it was the sole source of phosphorus. However, we should remember that starved microorganisms induce a number of genes that activate enzymatic pathways that are involved in P acquisition and assimilation (Quinn et al. 2007). Therefore, the detected lower concentration of MAMPA unambiguously does not mean a weaker decomposition of GBMP. The slightly higher AMPA and N-phosphomethyl glycine (NPMG) concentrations were measured after microbial treatments compared with a non-inoculated Bg11 medium. However, this is a consequence of chemical breakdown.
It is worth emphasizing that in the case of aminotri(methylenephosphonic) acid (ATMP) and N,N-bis(phosphonomethyl) glycine (GBMP) subjected to both microbial and chemical degradation in a Bg11 media, cyanobacteria enhanced the formation of characteristic products of degradation several times, including AMPA for ATMP and MAMPA in the case of GBMP. Therefore, it can be assumed that the final effectiveness of AAP decomposition is related to the parallel action of metal ions and microbial activity. The biodegradation and chemical oxidation products are indicative of C–N and C–P bond cleavage.
Scale inhibitor, diethylenetriamine penta(methylene phosphonic acid) (DTPMP), during the 2 weeks of incubation in a non-inoculated Bg11 medium, released approximately 8 μM of aminomethylphosphonic acid (AMPA). In the cultures of examined microorganisms, the amount of AMPA was lower, with an exception of Fischerella 067 in a full growth medium. Additionally, for the four out of five examined strains, small amounts of N-(methyl)aminomethanephosphonic (MAMPA) were detected. It is worth emphasizing that the DTPMP molecule has been already proven to be a substrate for cyanobacterial enzymes that originate from freshwater Anabaena variabilis cells (Drzyzga et al. 2017), which conceivably indicates that the entire degradation occurred intracellularly.
Interestingly, hexamethylenediamine-N,N,N′,N′ - tetrakis(methyl phosphonic acid) (HDTMP) seems unlikely to be stable in cyanobacterial cultures. It has been revealed that the presence of the additional peak of retention time is very close to that of NPMG. Due to the lack of unambiguous identification of its structure, the appearance of this substance is reflected based on its amounts, which are presented in area units (mAU) (Table 7). Bearing in mind the retention time of this substance in relation to other examined compounds, it should be expected that its molecule contains a secondary amine group and a phosphonic acid motif. Therefore, the formation of (3-hydroxypropyl)aminomethylphosphonic acid, as the main product of (bio)degradation of HDTMP, may be postulated. The lower concentration of this compound, which is observed in a Bg11-P medium, should be considered the effect of Pi starvation of cells, rather than the result of a weaker decomposition rate. We postulate the appearance of a similar phenomenon for the GBMP molecule.
The stability of aminopolyphosphonates in water can be monitored by tracking the occurrence of their degradation products. The successful use of adopted and modified chromatographic procedure, which is fast, relatively inexpensive, reproducible, and requires neither expensive detector (MS, MS/MS, TOF/MS, ICP/MS, etc.), systems (e.g., UHPLC) nor time-consuming sample preparation process, has undoubtedly proven that it is possible.