What do you think of when you hear the terms, “global warming” or “climate change”? Melting polar caps? Rising oceans? More energy use for air conditioning? Withering farmlands? Lack of drinking water?

Only about 3% of the earth’s water is usable, but 2.5% of that is unavailable, locked in ice caps, glaciers, atmosphere, soil, is highly polluted, or too deep in the subsurface to obtain. That leaves about 0.5% to use and recycle (US Bureau of Reclamation).

In the western United States, “new” sources of fresh, usable water are quickly being tapped and are diminishing. The time has come for authorities to take action to optimize water use and “tap” into all possible sources for fresh, safe, potable water. California, Colorado, Arizona, Nevada, New Mexico, and Texas are actively applying or planning on regulations to govern direct potable reuse (DPR) of domestic wastewater.

To that end, the California State Water Board (SWB) sponsored an Expert Panel administered by the National Water Research Institute (NWRI). This expert Panel published a Final Report in August 2016, which identified knowledge gaps for developing criteria for DPR. Six research projects have been identified to address these gaps. The focus of the second of these research projects, DPR-2, is to assess the concentration of relevant pathogens in raw wastewater (RWW) via a 12-month monitoring campaign. The principal objectives of DPR-2 are:

• To collect empirical data on the concentration and variability of pathogens in raw wastewater for the purpose of verifying log removal values necessary to adequately protect public health in DPR projects.
• To develop recommendations for the collection and analysis of data on pathogens in raw wastewater. These recommendations may be used in future monitoring efforts.

The selected organisms are, Giardia, Cryptosporidium, enterovirus, adenovirus, norovirus, and male-specific coliphage. SARS-CoV-2 was added at the onset of the COVID-19 pandemic in California. RWW can be a challenging matrix and therefore, method modifications were explored during a pre-screening methods optimization study. Based on this information and subsequent multi-lab method validations, optimized SOPs were developed.

We know that RWW contains these infectious pathogenic microorganisms, so why go to the trouble and expense to develop methods to quantify them? The basis of the DPR regulations depends on knowing the concentration of microbial pathogens. Currently, the State of California is designing their regulations on the log-reduction value (LRV) of the combined treatment components at an advanced wastewater treatment (AWT) plant. Log-reduction values for microorganisms are based on a log base-10 scale; where 90% reduction = 1 LVR; 99% reduction = 2 LRVs, etc.

In California, the goal is to have the AWT process reduce pathogens by, 12-log reduction of virus, 10-log reduction of Cryptosporidium, and 10-log reduction of Giardia. One cannot calculate these LRVs without knowing the initial concentration in the RWW.

Again, we know these organisms are there, but the RWW matrix is extremely complex. Each organism type must be isolated from the matrix, treated to be free of method inhibiting components, isolated and assayed. The complexity of the assays ranges from direct observation of the target organism(s) to molecular biological techniques to cultivability. The culture method is the only one that can demonstrate not only presence but infectiousness, and this is only applied to the enteric and adeno-viruses.

Large projects involving multiple laboratories, independent organizations or departments and management teams, require the appropriate tools for maintaining schedules, ensuring samples are distributed and processed in a timely fashion, automatically centralizing data, preventing entry errors from data submitted from multiple sources.

Planning, scheduling, tracking, and centralizing data are critical to project success. Large projects such as this can benefit from the organizational and data handling power of a LIMS. The project presents additional duties for the operators at the utilities, so scheduling samples in advance is critical for sending and shipping supplies for samples that have a short hold time (<48 hours). Once at the lab, preparation of the samples is complex and subsamples are handled by many hands, all of which must be tracked. There may be specific requirements for analysis depending on the sample sites/locations, the LIMS tracks the required analytical methods and associated QC with those sites/locations and conveys this to the analyst. In some cases, the raw data from the assays must be adjusted to account for dilution factors, matrix spikes and recovery rates; all pre-programmed in the LIMS. The LIMS can automate the organization of the raw data and make the calculations that need to take place. Data in an Excel-type format from various laboratories can be automatically formatted and imported into the LIMS for centralized storage. Reports consist of data packages verified and uploaded to a shared project directory by laboratory and project quality managers. Finally, the LIMS, with its auditing tools, streamlines the quality assurance component of the project during independent auditing evaluations.

This monitoring program has been on-going since November 2019. Data are being accumulated and prepared for publication. Although most attention now is on the occurrence of the SARS-CoV-2 virus, pathogen monitoring projects like this will determine the future of the emerging science of wastewater-based epidemiology (WBE).