Splenda may solve our problem.

Your Coffee Sweetener May Help Make Recreation Water Safer.

R.H. Bennett Ph.D.

Applied Life Sciences LLC

Honaunau, Hawai‘i

The coffee and tea drinkers that like it sweet but don't use sugar are giving us a handle on a greased watermelon that has slipped through our hands for decades.   The artificial sweetener Splenda may turn to provide a marker of wastewater and sewage that “flows” into the environment.  Sweden, Arizona, Maine, and Florida scientists confirm that Splenda, Sucralose is a reliable wastewater indicator.  Our work, slated for this fall, will likely show the assay reliable in Hawaii too.

In this era of great scientific achievement, we can identify a single gene on one strand of human DNA.  Advanced analytical methods can detect toxicants as low a few parts per trillion in water or food. ( one part per trillion is one second in 31.7 thousand years)  Molecular proteomics can detect tumor proteins long before an MRI will ever visualize the tumor.

 Given these exceptional abilities, it would be reasonable to expect we can detect traces of sewage in the waters of Hawai‘i; paradoxically we cannot. It has been that way for almost four decades with no advances. Confirmation of a sewage spill or leak still requires a visual assessment.  Alternatively, we assume elevated levels of Indicator Bacteria reveal sewage contamination.  Unfortunately, all the bacterial indicators used over the last  100 years are replete with false positives and negatives for the assessment of pathogen presence in water (Noble 2003).

 The currently used official indicator test for the bacterial genus Enterococci is written in stone in both Federal and State law.  Refer to Colford (2012) for a detailed review of the shortcomings of the Enterococci test to predict illness risk.  In short, the test has one chance in two for detecting sewage pathogens in surface waters without known sewage outfalls like the one at Sand Island Oahu.  Would we feel safe driving our cars if the break failure indicator light only worked half the time?

Other Sewage Indicators

Water quality researchers have taken several approaches to find a valid sewage contamination indicator.  Fairly recently, certain pharmaceuticals, caffeine, and nicotine metabolites showed some promise. However, most do not persist across the vast array of sewage processing and disposal technologies.  Sewage treatment operates processes to break down the components of sewage and the indicators are typically not resistant to degradation.

A reliable indicator must persist through most treatment processes and endure over time.  They must resist photodegradation from sunlight, and they must resist microbial decomposition.  A valid indicator must not cross-react with other chemicals in the sewage milieu and create false positives.

Advanced microbial detection of human fecal pathogens using DNA/RNA technology shows much promise and precision. Both  PCR and Nextgen sequencing offers excellent precision and accuracy. However, the technique requires a commercial laboratory equipment expertise that is not currently available. The biggest problem is the long turnaround times needed to produce actionable data for the regulatory agencies.

Sucralose as an ideal Indicator: The Evidence

The artificial sweetener Sucralose (Splenda) is a very widely used sugar substitute. It is an ingredient in over 4000 consumer products worldwide.  It is a simple carbohydrate; however, it has three chlorine molecules attached.  As a chlorinated carbohydrate, it is not digestible and not metabolized by bacteria in the gut or the environment.

In the figure below, its chemical structure is similar to table sugar, sucrose, yet note the three chlorine molecules (green).  As such it is a member of a class of chemicals called chlorinated organics.  This class of compounds tends to be very stable and persistent in the environment.

In humans ingesting Sucralose, it is poorly absorbed, and the mean residence time is 19 hours.  Half-life is 13 hours.  Most is excreted in the feces, 78% and 15% in the urine. It is mainly excreted unchanged. Only 2.6% is excreted as a glucuronide conjugate (Roberts 2000).

Sucralose survives a wide variety of wastewater treatment processes with minimal and insignificant degradation from oxidation or UV irradiation.  It is not degraded by anaerobic or aerobic microbial wastewater treatment. Sucralose survives waste treatment essentially unchanged in wastewater effluent at concentrations ranging from 1800 to 3800 nanograms per L. (Torres 2011).

In waters receiving wastewater in any form, Sucralose exists in fresh and marine waters.  Extensive studies in Sweden Sucralose show 400 to 900 ng/L downstream of a wastewater treatment plant Upstream it was less than 4ng/L (the minimum detection limit or MCL) (Brorström-Lundén (2008).  Sucralose testing in the open ocean and the marine waterways of Florida demonstrated its wide distribution. In the ocean waters of the Florida Keys, Sucralose was routinely detected at concentrations ranging from 147 ng to 393 ng per liter (Mead 2009).

In a recent Florida study, Sucralose levels above the MCL was detected in 78% of the samples, from slightly brackish to marine waters, ranging from 8 to 148 ng/L. A treated wastewater ocean outfall tested at 8414 ng/L in the Miami area (Batchu 2013).  These data confirm the assay method is sound for very diluted Sucralose in environmental waters.

Since estuaries in the United States are commonly used for both direct and indirect discharge of treated and untreated wastewater, questions about the suitability of using Sucralose as a wastewater proxy in estuaries may arise.  The question is answered by a recent study in the Narragansett Bay Estuary in Maine.  Researchers found that Sucralose was readily detected in estuarian waters. Sucralose concentrations had an r² = 0.88 correlation with salinity (Cantrell 2019).

Among the numerous chemical markers of sewage, including pharmaceuticals and other artificial sweeteners, Sucralose has the advantage of wider distribution and environmental persistence.  It is the preferred chemical indicator for sewage, wastewater, septage and cesspool leachate. In time, Sucralose may become the preferred indicator for the presence of fecal bacteria and virus. One study in a tropical urban environment demonstrated that Sucralose concentrations had the strongest correlation with the common sewage indicator bacteria counts over other chemical indicators.  The correlation values ranged from 0.40 to 0.47 (Ekklesia, 2015).  The low R values are attributed to the lack of precision in the bacteria test methods and not the chemical assay for Sucralose.

The Sucralose Assay

Sucralose is available in pure analytical grade making most any assay for it highly confirmable. The preferred analytical method is one that is precise and accurate while being rapid and lower in cost.  The technique developed by Batchu et al. (2015) includes online solid-phase extraction coupled with orbitrap high-resolution mass spectrometer.  This

method is rapid and lesser cost compared to HPLC mass spectrometry. 

The HRMS method is capable of discriminating between sucralose molecules that differ only by the deletion of one chlorine molecule.  Thus, its precision and accuracy allow detection of Sucralose down to 1.4 nanograms per liter (minimum detection limit).  A nanogram is one billionth of a gram. A billionth is three seconds in one hundred years.

This means even very little Sucralose diluted in household wastewater, then further diluted, in sewage and then even further diluted in nearshore ocean water is detectable. Mawhinney (2011) describes sucraloseʻs presence in drinking water systems, including the point of consumption.  These researchers used another method of detection and obtained excellent precision and accuracy with this second method.  It further attests to the validity of Sucralose as a wastewater indicator

But some may ask how precise the assay is?  As it turns out, Orbitrap High-Resolution Spectrometry is very precise (Hornshaw 2015).  As can be seen in the chemical structure diagram, Sucralose has three chlorine molecules.   What will this assay detect if we add some faux sucralose, one that has only two chlorine molecules?   The assay can distinguish the two chlorine faux sucralose form the three chlorine real thing.  This means the likelihood of a similar molecule becoming a false positive is highly unlikely.

The concepts of precision and accuracy may be a tad foreign.  The target analogy is a simple way to understand the terms.  The assay for Sucralose fits the pattern in the lower right.  The assay detects the amount correctly and does so consistently.  It is accurate an precise.  Reliable data derives from the type of assay technology and proper calibration with each use.

Given that two Sucralose assays demonstrate accurate and precise measurements and given that Sucralose is reliably detected in waters and climates from Sweden to Arizona, the assay is validated. 

What is needed now to enable Sucralose for the official EPA wastewater/sewage indicator in a human epidemiology study.  Such a prospective study could be conducted at a major urban beach to determine recreation water exposure.  Exposures such as wading vs. swimming vs. surfing associated with post recreation illnesses, including GI, skin, eye, and upper respiratory diseases.   The theory being Sucralose is present when wastewater is present.   Sucralose is stable, not inactivated by sunlight UV.  The concentration of Sucralose in recreation water may be directly proportional to the concentration of human virus and bacteria capable of causing disease.   This type of research is costly, yet an excellent investment in finding more effective means to monitor recreation water microbiological safety.  In Hawai‘i that is rather important, to say the least.

Sweet coffee anyone?

References

Batchu, Sudha Rani, Natalia Quinete, Venkata R. Panditi, and Piero R. Gardinali. "Online solid phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of sucralose in reclaimed and drinking waters and its photo degradation in natural waters from South Florida." Chemistry Central Journal7, no. 1 (2013): 141.

Brorström-Lundén, Eva, Anders Svenson, Tomas Viktor, Andreas Woldegiorgis, Mikael Remberger, Lennart Kaj, Christian Dye, Arve Bjerke, and Martin Schlabach. "Measurements of Sucralose in the Swedish Screening Program 2007: PART I; Sucralose in surface waters and STP samples." (2008).

Cantwell, Mark G., David R. Katz, Julia Sullivan, and Anne Kuhn. "Evaluation of the artificial sweetener sucralose as a sanitary wastewater tracer in Narragansett Bay, Rhode Island, USA." Marine Pollution Bulletin 146 (2019): 711-717.

Colford Jr, John M., Kenneth C. Schiff, John F. Griffith, Vince Yau, Benjamin F. Arnold, Catherine C. Wright, Joshua S. Gruber et al. "Using rapid indicators for Enterococcus to assess the risk of illness after exposure to urban runoff contaminated marine water." Water research 46, no. 7 (2012): 2176-2186.

Ekklesia, E., Shanahan, P., Chua, L., & Eikaas, H. (2015). Associations of chemical tracers and faecal indicator bacteria in a tropical urban catchment. Water research, 75, 270-281.

Hornshaw, M. (2015) Why is the Orbitrap Mass Analyzer so Amazing? http://analyteguru.com/why-is-the-orbitrap-mass-analyzer-amazing/

Loos, Robert, Bernd Manfred Gawlik, Kristin Boettcher, Giovanni Locoro, Serafino Contini, and Giovanni Bidoglio. "Sucralose screening in European surface waters using a solid-phase extraction-liquid chromatography–triple quadrupole mass spectrometry method." Journal of Chromatography A 1216, no. 7 (2009): 1126-1131.

Mawhinney, Douglas B., Robert B. Young, Brett J. Vanderford, Thomas Borch, and Shane A. Snyder. "Artificial sweetener sucralose in US drinking water systems." Environmental science & technology 45, no. 20 (2011): 8716-8722.

Mead, Ralph N., Jeremy B. Morgan, G. Brooks Avery Jr, Robert J. Kieber, Aleksandra M. Kirk, Stephan A. Skrabal, and Joan D. Willey. "Occurrence of the artificial sweetener sucralose in coastal and marine waters of the United States." Marine Chemistry 116, no. 1-4 (2009): 13-17.

Noble, Rachel T., Douglas F. Moore, Molly K. Leecaster, Charles D. McGee, and Stephen B. Weisberg. "Comparison of total coliform, fecal coliform, and enterococcus bacterial indicator response for ocean recreational water quality testing." Water research 37, no. 7 (2003): 1637-1643.

Oppenheimer, Joan, Andrew Eaton, Mohammad Badruzzaman, Ali W. Haghani, and Joseph G. Jacangelo. "Occurrence and suitability of sucralose as an indicator compound of wastewater loading to surface waters in urbanized regions." Water research 45, no. 13 (2011): 4019-4027.

Roberts, A., A. G. Renwick, J. Sims, and D. J. Snodin. "Sucralose metabolism and pharmacokinetics in man." Food and chemical toxicology 38 (2000): 31-41.

Torres, César I., Smitha Ramakrishna, Chao-An Chiu, Katherine G. Nelson, Paul Westerhoff, and Rosa Krajmalnik-Brown. "Fate of sucralose during wastewater treatment." Environmental Engineering Science 28, no. 5 (2011): 325-331.

Water Quality Monitoring Needs Reinvention

Water Quality Monitoring Needs Reinvention

R. H. Bennett Ph.D.

Applied Life Sciences LLC

The recent editorial by the Director Bruce Anderson of the Hawaii Department of Health makes some assurances that the agency is monitoring recreation water bacteria, and all is well. Unfortunately, all is not well, and it is not that the agency is doing something wrong. The methods are grossly obsolete, and there is ample scientific evidence to that fact. The method is written into the US Beaches Act and to qualify for the financial support the state must use the method. Once again, politics usurp science.

The indicator bacteria measured is the genus Enterococci. In recreation waters where there is no known discharge point of treated human wastewater, the test is less than 50/50 for predicting risk from the presence of human disease agents. Those agents are typically virus that causes gastroenteritis. In the genus Enterococci, there are 30 plus species detected by this test. Only two species are common in feces of most animals, and humans. What makes this test very imprecise is the fact that these two species of Enterococci as well as many others grow readily in the environment and not just soil. Just about any place, there is moisture and organic debris these microbes can be found. Concrete storm drains, sewer pipes, coastal wetlands, taro fields, and stream sediments all can support the growth and persistence of the bacteria.

The Director asserts that this soil growth issue is unique to tropical Hawaii. Unfortunately, that is not accurate. This indicator species are common in the environment with human activities, in places like Alaska, and the lakes of Michigan, to mention places that get rather cold (5,6).

In parts of West Maui and West, Hawaii streams are not prevalent. However, elevated indicator bacteria are commonly detected. In coastal waters, the major source of Enterococci is the beach sand itself. Entero persists there very well. Any event, including Spring Tides that stirs the sand, can suspend the bacteria to the water column (2,3,4). If beach water is tested soon after such an event, high indicator numbers can be found. The health risk is negligible in most cases. The “Entero” test provides no distinction of the relative risk.

To be forthright, we cannot project the disease risk from any bacterial indicator measurement. There are far too many false positives and false negatives. If a medical blood test were only correct half the time, it would never get FDA approval. The EPA scientists we know would love to work on this problem, but there is no political will at the top.

Amidst all this uncertainty, some may feel uncomfortable recreating in our ocean. There is some reassurance. The sun is the greatest disinfector ever known. The UV light penetrates deeply in clear water and will reduce bacterial and viral counts in a matter of a few hours of midday sun (7). Critical is keeping the nearshore waters clear. We are not doing well in this regard. The human waste nutrients that flow into the sea, in contaminated brackish groundwater, streams and rivers, nourish marine phytoplankton, and they grow. The effect is a reduction in light penetration and visibility. Many of our beaches are Federally Listed as Impaired for turbidity. As this happens, the UV of the sun becomes less and less effective, and disease risk will slowly increase as turbidity goes up. This means we need to stop dumping human waste nutrients into the sea. Maui County is hoping the Supreme Court will permit them to continue dumping wastewater indirectly into the sea. Why Hawaii would allow this is not an oversight.

Many communities on the East Coast are actively addressing the problem because their waters turned foul and putrid. Their economy suffers accordingly. So, are we waiting for foul and putrid water too?

Modern innovative approaches to measuring disease risk are coming. The Director mentioned the Phylochip. It is an exact tool that can measure the entire fecal microbiome distinguishing its source. At that time when the test is incorporated into public law and commercialized, the costs will be very competitive.

Another indicator of human waste is a chemical marker called Sucralose. It is known as Splenda, the artificial sweetener. It is omnipresent in human wastewater, very stable and persistent. Tests in Florida waters show it can be detected precisely at very low levels in fresh and marine waters (1). The test method is straight forward, and most analytical labs could conduct the test with some minor investments.

Needed now is the funding from private and public partnerships to get this done and protect Hawai‘iʻs most precious resource, the sea.

References

1.      Batchu, Sudha Rani, Cesar E. Ramirez, and Piero R. Gardinali. "Rapid ultra-trace analysis of sucralose in multiple-origin aqueous samples by online solid-phase extraction coupled to high-resolution mass spectrometry." Analytical and bioanalytical chemistry 407, no. 13 (2015): 3717-3725.

2.      Boehm, Alexandria B., and Stephen B. Weisberg. "Tidal forcing of enterococci at marine recreational beaches at fortnightly and semidiurnal frequencies." Environmental science & technology 39, no. 15 (2005): 5575-5583.

3.      Bonilla, Tonya D., Kara Nowosielski, Marie Cuvelier, Aaron Hartz, Melissa Green, Nwadiuto Esiobu, Donald S. McCorquodale, Jay M. Fleisher, and Andrew Rogerson. "Prevalence and distribution of fecal indicator organisms in South Florida beach sand and preliminary assessment of health effects associated with beach sand exposure." Marine pollution bulletin 54, no. 9 (2007): 1472-1482.

4.      Goodwin, Kelly D., Melody McNay, Yiping Cao, Darcy Ebentier, Melissa Madison, and John F. Griffith. "A multi-beach study of Staphylococcus aureus, MRSA, and enterococci in seawater and beach sand." Water research 46, no. 13 (2012): 4195-4207.

5.      Mutter, Edda A., William E. Schnabel, and Khrystyne N. Duddleston. "Partitioning and Transport Behavior of Pathogen Indicator Organisms at Four Cold Region Solid Waste Sites." Journal of Cold Regions Engineering 31, no. 1 (2016): 04016005.

6.      Ran, Qinghong, Brian D. Badgley, Nicholas Dillon, Gary M. Dunny, and Michael J. Sadowsky. "Occurrence, genetic diversity, and persistence of enterococci in a Lake Superior watershed." Appl. Environ. Microbiol. 79, no. 9 (2013): 3067-3075.

7.      Sinton, Lester W., Rochelle K. Finlay, and Philippa A. Lynch. "Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater." Appl. Environ. Microbiol. 65, no. 8 (1999): 3605-3613.