Partners for Clean Water; Business and Organizsation Contributors


Wednesday, August 5, 2009

The Targeted Bacteria


The BWTF program is designed to detect the presence of enterococcus bacteria, in particular e. faecalis and e. faecium. These are commensal bacteria in the mammalian intestinal tract, meaning that although humans are not born with them they do quickly colonize the human gut during the neonatal months and in healthy individuals soon achieve their part of balanced intestinal ecology. In return for us providing a warm, wet, and reasonably safe place for them to reproduce they assist in the further breakdown of proteins passed on by the stomach so that we can absorb more nutrition from food than otherwise. These bacteria are so important in this secondary digestive phase that they are sometime included as an ingredient in probiotic supplements given to those patients whose intestinal ecology has been severely unbalanced or destroyed by extreme antibiotic treatments or chemotherapy. In a healthy individual about half of the dry weight of human feces is actually bacteria, largely enterococcus.
This of course causes problems if fecal matter is not disposed of properly. Returned to the ground these bacteria quickly are ingested by other bacteriophagic organisms resident in the soil, and rendered harmless. Released into water they can rapidly become a significant problem due to several characteristics of this genus of bacteria: they are facultively anaerobic, meaning they can survive and reproduce in environments that either contain oxygen or not; they are very tolerant of high levels of dissolved salt, and can survive and reproduce in water far more salty than typical sea water; they remain viable during prolonged dessication, and under laboratory conditions can survive and reproduce after 60 days of being completely dry. All this means that once released into fresh or saltwater environments they easily reproduce as long as food is available, and the only other natural controls to their growth are predatory bacteriophages or UV radiation in sunlight. More worrisome is a characteristic shared by all bacteria, which although they reproduce by ‘cloning’ or dividing into two with complete and identical sets of DNA each, they are also able to ‘horizontally transfer’ chromosome sets within the species. This lateral DNA transference is how bacteria evolve to survive new environmental challenges. The flu ‘bug’ continues to survive the best medical treatments because sometime, somewhere, one of the bacterium mutate sufficiently to tolerate whatever new drug is being administered, and passes the DNA of that survival trait on to others of that genus. A certain drug resistance was recently traced thru hospitals and tracked- it took 6 months for the trait to get passed on completely around the planet.
With regards to enterococcus this ability for lateral transference is particularly alarming since it is a common target of antibiotic treatments and has developed a tolerance for a wide spectrum of treatment drugs. Certain particularly virulent strains have multiple resistance, and their infections are extremely difficult and expensive to treat. There is no good reason to assume these strains of bacteria are not being released into our coastal waters by faulty sewage treatment practices, nor that this resistance characteristic is not going to be passed on to other strains of enterococci. That is why the legally ‘safe’ limits for these bacteria are very low- 35 viable bacteria per 100 ml of water in Puerto Rico, only 7 per 100 ml in Hawaii. The federal limit is 104 per 100 ml, although this will likely change as new legislation is enacted.
What are the medical problems associated with water borne fecal bacterial contamination? This depends of course on how healthy you are, and where the infection starts. A healthy person’s immune system is generally capable of dealing with minor quantities of ingested enterococcus, but if these defenses are overwhelmed then a wide variety of gastro-intestinal symptoms are likely. The real problem starts if the bacteria get into your mucous membranes via eyes or ears, or subcutaneous tissue and/or bloodstream via cuts, scratches or open wounds. Membrane infections can range from mild pink-eye to urinary tract infections to fatal meningitis. Tissue infections can range from surface impetigo to necrotic decay of deeper layers. In the bloodstream a wide range of bactericemic symptoms can develop, most notably of heart tissue. The ‘at risk’ group for these more severe illnesses are the very young, the elderly, those using immunosuppressant drugs, and those already suffering some type of systemic infection or otherwise with a compromised immune system. These people are far more susceptible to health problems at much lower levels of exposure to bacteria.
Given all that, how to you know the water is safe to swim in? Basically, you don’t. You cannot see or smell the presence of enterococcus at unsafe levels. The only way to know what the bacteria count is, is to test the water
To repeat: Here in Rincón our coastal waters are usually very clean, about 60% of the time no matter what season. About 10% of the time they are not, dangerously so, and this is not strictly dependent on rainfall. Whether this risk factor is enough to mobilize the community in supporting a regular water testing program remains to be seen.

For more information, or to volunteer to assist with this program, please contact Steve at

How Clean Are The Rincón Beaches?


This is certainly one of the basic questions the general public has regarding the program, and despite its apparent simplicity is not actually that easy to answer yet. Certainly the survey has shown that no specific beach is consistently and constantly contaminated, but beyond that fact is there any way to determine if any beaches are ‘safer’ than others in the Rincón area?
One simple way is to compare how many times specific beaches registered bacteria counts greater than the 104 MPN level.

But this is not a totally satisfactory yardstick, for a couple of reasons. Some beaches were sampled less frequently than others- for instance the Rio Grande site, due to its difficulty of access, was tested less often and could very well be far more contaminated than the survey shows. Some beaches were sampled more intensely- the Corcega site was examined much more than the others, and so results from there could be an ‘artifact’ of the cumulative results of various research efforts. And it does not deal at all with the fact that bacteria levels below the 104 level but above the 35 MPN level could be unsafe for those swimmers in the ‘at risk’ category.
A better, if less simple, method is to compare the statistics generated by the 17 month survey. Actually, the cumulative statistics here are the true fruit of the survey (aside from the weekly email postings of the test results) and will be used to shape the second phase of the testing program, once the survey has been completed. These numbers are what ‘justifies’ the program in terms and time and money spent, so they are worth examining closely.

For a larger view of this image, click here
The average bacteria count per beach is a simple number, the total of the test counts divided by the number of test dates for that beach. Unfortunately they are not an adequate yardstick for the question of ‘safety’, since one high reading can really skew a long series of 0 counts, and we have definitely had some high readings. So this number doesn’t accurately reflect what the water quality may be at any given time. However, it will come in useful.
The next yardstick is the standard deviation of bacteria counts per beach. This number becomes the workhorse of future statistical evaluation since it gives you an idea of the variability of the test results for that beach. The higher the SD means the wider the swings of bacterial levels detected, generally higher rather than lower. For bathers, this number indicates how likely it would be that, if the coast has been contaminated, each beach would compare to an average. A higher SD suggests that beach would more contaminated than average, for a number of reasons not clearly understood yet. After a heavy rainfall you might want to avoid swimming at these beaches for a longer period as well. Standard deviation will also be used in our continuing search for ‘bellwether’ beaches, sites we can test that would indicate the general water quality for much larger sections of the coast. Again, beaches with a high SD are totally unsuited for this purpose. Standard deviation also answers the problem of, since bacterial levels do fluctuate so much, how do we judge what an ‘unusual’ reading is? For any single bacteria count from a sample taken from a specific beach, 68% of the time it should be within one SD plus or minus from that beach’s average (in our case, always added-our beaches are fairly clean I have no doubt there are places on this island where such averages and SDs are high enough that subtracting would be possible, and would not recommend even wading there) and 95% of the time the result should fall within two standard deviations from the average. Anything beyond that automatically becomes unusual, interesting or, as is said in the trade, statistically significant. As I’ve said, standard deviations are going to power a lot of the shaping of second phase of the testing program. To be completely honest, since the survey hasn’t been completed yet, what we should be using here is a number called the sample standard deviation, which involves calculating a margin of error for these results. That is why you see those little + or - percentages on the bottom of the opinion polls that are so popular these days, as it is very difficult to poll enough people for a statistically valid sampling. The same thing happens in water sampling, but I’m keeping these numbers as simple as possible for the time being.
The final statistical yardstick is the geometric mean per beach. This is a bit more complicated, but more accurate, way of comparing water quality when using something like bacteria counts which do vary so much from week to week, and may have seasonal or yearly cyclical fluctuations as well. Calculating the geometric mean is a logarithmic function, but the easiest way to explain the number is that it is a mathematical method of minimizing the impact of high and low readings and focusing on the midrange test results. As we see, the GMs are extremely low for the surveyed beaches. Federal standards define a geometric mean of more than 35 MPN of enterococcus as dangerous, requiring public notice/flagging/beach closing, and a GM can only be calculated after a minimum of five sequential tests. Have we ever had times when test results showed over this level? Unfortunately yes, during the very first month of testing If you go back to the main chart you’ll see that the beaches we did test 5 times that month were far over, or darn close to, this. The relevant part of the spreadsheet follows:

or a larger view of this image, click here
Basically that month the whole coastline should’ve been flagged and closed. This was the event that spurred us to really upgrade the program’s data collection parameters beyond the typical BWTF requirements. It is also, sad to say, what we must be prepared to respond to at any time. When and if another contamination event of this magnitude happens we should be ready to do increased testing, carry out some offshore current measurements (beyond the surf break) on our own, and try to get our water samples to more complete lab facilities for confirmation of our results, complete speciation of the bacteria in the water, and any chemical analysis we can afford while we plaster the beaches and town with posters. This would be our version of a 5-alarm fire It has happened once, and can happen again at any time, so it seems. And the only way we’ll ever know is to have a continuing testing program and constant monitoring of a few beaches at least.

Using all these various yardsticks we can compare the surveyed beaches to create an informed, if only very tentative, opinion as to their relative freedom from bacterial contamination. To repeat, this are tentative conclusions based on an incomplete survey and insufficient data But better than nothing of course.
With this in mind, it would seem that (from north to south) Rio Grande, Pools and Marias would be the least safe beaches at any given time. Domes, Steps and Parcelas would seem to be the most safe.
I should note that I don’t include the South Barrero site in these calculations as there weren’t enough samples taken to generate meaningful numbers. I also note that Rio Grande has the second highest average, the third highest geometric mean, while having a standard deviation in the mid range. Thus statistically it would be the closest we’ve found to a consistently and contaminated site, which is certainly suggestive and bears further research. But, to repeat again, at this time it is only a suggestive and interesting observation.

For the complete spreadsheet page showing cumulative statistics by season for the survey period click here
For more information, or to volunteer to assist with this program, please contact Steve at

A Rough Outline of the Survey Program


The actual monitoring program itself is fairly easy to set up and run, and consists mostly of determining a convenient time and day to collect samples and get them processed within six hours, and finding enough dedicated volunteers reliable enough to do this. To accomplish the first aspect we quickly chose Tuesday afternoon to do the testing, so that volunteers could get their samples on lunch break, or after classes or work, and the lab work could be finished before nightfall. The second aspect, for the Rincón program at least, was less of a problem- there almost always were sufficient volunteers, and most participated for months. Once the routine was established volunteer time per person or team per week for sampling was an half hour; sample processing and lab work was two hours; and acquiring test results, record keeping and results posting on internet and the national database was another hour and a half. Once the survey began lab and test work time increased due extra samples and data processing required as well as processing the samples delivered by Rescate Playas Isabela.
Once the survey started we brought the program as close as possible to comply with federal requirements for volunteer water sample collecting (yes, there are standards for this) by getting duplicate samples from one or two beaches per week. We were not able to get 10% of our samples duplicated and tested by an outside agency, despite efforts in this direction. A negative lab control (distilled water and reagent) was always processed each testing date to verify the lab equipment and techniques were not affecting test readings. This is not called for in the EPA evaluation of the IDEXX Enterolert test protocols, but was done to avoid any doubt that those protocols worked under less than ideal conditions.
The duplicate sampling is not required by the national BWTF protocols, but it was the only way to verify the volunteer collectors were adequately following the protocols while acquiring samples. It was also useful for another basic reason- the bacteria we were monitoring for are not distributed evenly in the water column Although they tend to be found in the upper water layers, fluid turbulence and current flow tend to arrange them in ‘steamers and pools’ formations and chances are very good the bacteria count will differ from a sample taken a meter away or a minute later. So duplicate sampling is the quickest way to verify a general contamination event rather than a random fluctuation. For this reason building up a ‘profile’ for each beach is a long term project. When we did the parallel test with Dr. Otero we were all taking our sample from the same 12 liter container and each of the results differed But they were all within two standard deviations from the mean, acceptable variation for the test- and to develop accurate numbers for the entire coastline requires a long time- line of monitoring.
When it comes to attempting to determine where any contamination may be coming from, Rincón faces particular challenges. First, our water is just very clean usually Even after 17 months we hadn’t acquired enough non-zero test results for any serious statistical work (the typical requirement is about 30 numbers, or 30 non-zero testing dates) beyond the basics. Second, high bacteria counts dissipate quickly along our coasts, usually within the 24 hr incubation period needed for the test. So we can’t just go back the next day and begin back tracking. Third, our coastline is particularly complicated, with two separate marine systems meeting, and is not very well studied. Tracing bacterial contamination in water is very difficult anyway, and is only done with any success in smaller enclosed systems such as lakes or rivers. The best our program as it is designed can do in this regard is eliminate possible sources when we can, and collect enough data to suggest further research directions. Fourth, the majority of published studies using the IDEXX test method, or similar to our program, concern testing in temperate climates and may not be all that relevant to the tropics. Fifth, we have very little opportunity to verify our results through some other method. Due to the ‘information vacuum’ that exists for our area we do not have previous studies to use for comparison, nor are public health statistics available for water borne illness, nor accurate local weather data. These restrictions also require us to amass a fairly large database before even the simplest patterns of contamination apparent and then confirmed statistically.
The only possible way to speed up this process was designing the survey to collect as much data as practical. As an analogy, the typical BWTF program would provide what you could consider as a scattering of pixels on a computer screen, and it would take a long time to add enough detail pixel by pixel to discern a pattern or picture being formed. By ‘framing’ those same pixels with more information, like providing them with their background colors, the picture becomes visible much more quickly.
However, the strength of the program really lies in using this very accurate, industry standard test method and the commitment of volunteers to acquire valid samples, and justification of our confidence in this has been amply demonstrated.

For more information, or to volunteer to assist with this program, please contact Steve at

Water Quality Survey, Acknowlegements&Dedication


I would very much like to thank all the many volunteers who cheerfully gave their time and efforts towards this program, and without whom none of this would have been possible. It was a pure pleasure working with you all, and I am very grateful you could take time out of your busy lives to give something back to the community. This survey is really your work, and is dedicated to you all.
Water sampling stars of the survey are (in order of appearance): George & Marylou Maldonado, Milton Medina, Leon Richter, Jack Miller, John Foldan, Roger Wagner, Sally Fleming, Miguel Conty, Gina Mikan, Robert Watts, Jennette Rice & Rich Taylor.
An extra special mention and gold halo are given to: Colleen Donovan, Corey Senise, and Judith Anne Tamar for their excellent work, particularly for their heroic efforts to collect samples and get them to the lab during the deluge caused by tropical storm Olga on Dec. 11 2007 Such dedication to a long term project is all too rare, and I cannot thank them enough for providing real-time sampling during an extreme rain event. Volunteer collector extra ordinaire Susan Wruble not only gets a halo but a shiny gold star for her commitment and copiously detailed notes during her participation
I would also sincerely like to thank Dr. Luis A. Rios Hernandez, Assistant professor of Biology at the University of Puerto Rico, Mayagüez, for his support and interest in the program. I am particularly grateful for two in-depth discussions and lab tours of the laboratory side of water quality testing, his inspection of our facilities, and the donation of lab supplies so we could do some basic research in the Rincón area.
Equally sincere thanks to Dr. Ernesto Otero, Research Associate at the Department of Marine Sciences, UPR Mayagüez, for a very interesting morning while doing parallel testing and being shown standard field collection technique- a short course in microbial ecology at the beach He also inspected our facilities and sample processing routine, and made valuable suggestions regarding the practical aspects of our program. Both these gentlemen have been very kindly bringing to my attention whatever studies and papers they come across pertinent to our program, but more noted still is their willingness to actually go out into the community with their work and experience, both in Rincón and elsewhere on the island. We all cannot thank them enough for that.
I am very grateful to Dr. Jorge Capella, the Modeling Coordinator for CaRA (Caribbean Regional Association for Integrated Coastal Ocean Observing), for a very clear and concise explanation of the mechanics of current mapping and coastal modeling, and a virtual tour of his databases, showing what little is known so far about our area. His willingness to collaborate in future projects is also appreciated

I would also like to gratefully thank the following, all from UPR-Mayagüez, for their input into our program:
Dr. Amos Winters, Commonwealth Climatologist, for a fascinating morning discussing climate modeling, statistical analysis, and the affects of fecal contamination on acopora palmata, as well as lunch.
Dr. Mark Jury for a very informative discussion of hydrological modeling and the tools available on the internet, as well as recounting tales of life and teaching in Ethiopia.
Dr. Dallas Alston for the tour of lab facilities available in Magueyes, and several enlightening discussions on the affects of contamination in marine ecosystems carried on while kayaking, amongst many other things.
Dr. Robert Watts, recently of the Mayagüez campus, for his interest, support, and participation in the program, in addition to providing the geological explanation of how our coastline was formed.
I would give my special thanks to Mara Dias, the Water Quality Coordinator of Surfrider Foundation, for answering far too many questions when the survey began, and faithfully forwarding articles of interest to me.

For more information, or to volunteer to assist with this program, please contact Steve at