Development of a Rapid Microbial-Based Toxicity Assay Employing Ferricyanide as an Artificial Respiratory Electron Acceptor

The use of ferricyanide-mediated microbial reactions was investigated with a view to developing a rapid microbial-based toxicity assay to overcome the short falls of existing techniques currently employed in the water and wastewater industry. This assay, known as the ferricyanide-mediated toxicity (FMTOX) assay, employs ferricyanide in place of oxygen as an artificial electron acceptor during microbial respiration. The principle behind this assay is similar to conventional microbial-based toxicity assays that quantify the inhibition of the electron transport system of microorganisms. In this case, however, rather than monitoring changes in bioluminescence (Microtox), oxygen (OEeD 209 Activated Sludge Respiration Inhibition Assay) or the production of oxidised nitrogen (ISO 9509 Activated Sludge Nitrification Inhibition Assay), the FMTOX assay quantifies microbially produced ferrocyanide derived from ferricyanide cellular respiration using an electrochemical detection system. In order to ascertain the relevance and practicality of the newly developed FMTOX assay, a tool box was developed that included a list features that an ideal microbialbased toxicity assay would have when applied to the water and wastewater industry. An assessment of other available microbial-based toxicity assays against the ideal toxicity assay was also made. Throughout this study, the use of this tool box approach served as a means to direct systematic research on FMTOX development and optimisation and also to evaluate if this new assay improves on the currently available microbial toxicity assays. FMTOX method validation, proof of concept investigations and optimisation of key experimental parameters was facilitated employing, Escherichia coli and 3,5-Dichlorophenol (3,5-DCP) as a model microorganism and test substance respectively. Following a concentration-response experimental design using a range of organic and inorganic test substances, the versatility of the generic FMTOX assay developed using E. coli was successfully applied without further modification (excluding exposure temperature) to three other microbial test species (Pseudomonas jluorescens, Bacillus subtilis and Acetobacter calcoaceticus). Furthermore, when a test substance exerted a measurable inhibitory effect, the trends obtained were found to display classic sigmoid shaped concentration-response curves reported for conventional toxicity assays. The degree of similarity between the ranked IC50 values calculated for the FMTOX microorganisms and literature values reported for standard microbial-based toxicity assays were compared using the Bray Curtis similarity matrix and visually displayed using hierarchal cluster analysis. While the trends obtained revealed that each of the microorganisms within the assays have their own sensitivity profiles, some similarities were evident. For example, of the microorganisms investigated in the FMTOX assay, B. subtilis was generally found to be the most sensitive test microorganism and the toxicity profiles obtained were found to be 88% similar to the ranked IC50 values reported for the Microtox assay. Consequently, it was proposed that the FMTOX B. subtilis assay would be relevant for applications where the Microtox assay has been typically employed, such as those requiring high sensitivity. On the other hand, owing to generally higher IC50 values, the FMTOX assay using A. calcoaceticus was found to be more comparable the activated sludge based assays as evidenced by relatively high percent similarities (93%) for both the OECD 209 assay and the ISO 9509 assay. It was therefore suggested that FMTOX A. calcoaceticus would be more suitable for applications where the activated sludge based toxicity assays are employed, such as WWTP influent monitoring, WWTP process control, and compliance monitoring of industries discharging into sewers. The successful application of the single microorganisms in the FMTOX assay prompted the investigation and development of a FMTOX assay employing activated sludge obtained from a WWTP. The results of this investigation are significant for a number of reasons. Firstly, this is the first report that has demonstrated that a mixed microbial consortium of activated sludge is able to reduce the artificial electron acceptor, ferricyanide, to ferrocyanide during cellular respiration. Secondly, using the test substances employed for the FMTOX single microorganisms, 97% (45 and 75 minutes total exposure times) and 100% similarity (135 minute total exposure time) in the ranked IC50 values were found between the OECD 209 assay and the FMTOX activated sludge. Comparison with the ISO 9509 assay also yielded excellent agreement as percent similarities ranging from 87% to 90% were found for each of the three FMTOX activated sludge assay exposure times. Importantly, the FMTOX activated sludge assay was able to achieve these comparable results in a much faster time frame (45 min.-135 min.) compared to the OECD 209 assay (3 hours) and the ISO 9509 assay (4 hours). Finally, the FMTOX activated sludge assay was also found to be much simpler to perform and required significantly reduced preparation and analysis time. Based on these very promising results it was concluded that the FMTOX assay employing activated sludge assay would be more appropriate for assessing the impact of wastewaters to the specific activated sludge community inhabiting any WWTP. The flexibility of being able to modify the biocatalyst(s) in the FMTOX assay demonstrates the versatility of the assay as it means that biocatalysts can be selected based on the indigenous species present in the environment being monitored and/or on the sensitivity and selectivity profiles required for specific applications. Importantly, this flexibility together with other significant advantages means that the FMTOX assay shows the necessary attributes of an ideal microbial-based toxicity assay that would be relevant to a wide variety of applications in the water and wastewater industry. Nevertheless, it is acknowledged that additional developmental work is still required, such as, analysis of the day to day variability of the FMTOX assay, further investigations of more test microorganisms, including tailor made mixed microbial consortiums and test batteries; further investigations of more test substances including mixtures and real samples; and further investigations of possible interferences to the FMTOX detection system.