1 Management of Non-Point Source Pollution CE 296B Department of Civil Engineering California State University, Sacramento Lecture #10, March 10, 1998 Sources of Pollutants - Part VI

2 Recall that we were looking at the six categories of pollutants: 1. Toxic inorganics - e.g. metals 2.Synthetic organics - e.g. solvents 3.Biostimulants - BOD, nutrients 4.Sediment - clay, silt, sand, gravel Left off here  5.Pathogenic organisms - viruses, bacteria, protozoa 6.Trash - use your imagination

3 And the framework for acquiring knowledge about each category: 1.What are the sub-categories in each category and what are representative members?  and here 2.What are the origins of pollutants? 3.How pollutants are introduced to the flow stream? 4.How pollutants behave in water?

4 C.Methodology for testing of indicator organisms in water samples. 1.Recall that it is difficult to identify specific pathogens in water samples. The current solution is to test for organisms that would likely accompany pathogens. These are called indicator organisms because they should indicate the presence of pathogens. 2.The central assumption with this approach is that the predominate source of pathogens is fecal contamination of water. VI.The fifth category of pollutant to examine is pathogens. (cont.)

5 Characteristics of the Perfect Indicator Organism - I 1.The organism must always be present in fecal material. Prevent False Negatives It would be preferred that one kind of organism came solely from humans and another kind from every animal but humans. Differentiate Types of Pollution 2.Once that organism was in a water body, it should no longer reproduce. Prevent False Positives 3.In a water body, that organism should remain viable for a reasonable amount of time. Too short and we would never find evidence of fecal contamination. False Negative Too long and all samples would appear contaminated. False Positive

6 Characteristics of the Perfect Indicator Organism - II 4.The organism should be easily and readily detectable by means that are inexpensive and easy to perform. Prevent False Negatives 5.The time from sampling to completion of test should be short. Enable Tracing to Source 6.Population density of organism in water should correspond to extent of fecal contamination. Enable Appropriate Response To Contamination

7 3.As bacteria are easy to culture and grow rapidly, they are the chosen type of microorganism. No viruses or protozoa. 4.Bacteria are classified by the dominate biochemical process(es) they perform. A classic method for identifying a bacteria type is to place a sample into an environment where the biochemical process in question is dominate. VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

8 5.Given that fecal contamination is assumed to be the source of pathogens, a biochemical process specific to intestinal tracts, the source of fecal material, would be the process of choice. 6.Around the turn of the century, a biochemical process specific to intestinal tracts was identified. VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

9 7.The only place that fermentation of lactose in the presence of bile salts takes place is the intestinal tract of warm-blooded animals. 8.At the turn of the century, it was difficult to obtain significant quantities of bile salts so lauryl sulfate was substituted as a chemical analog. VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

10 9.Several different types of procedures are available to test for indicator organisms. Total Coliform Organisms (Presumptive and Confirmation) Fecal Coliform Organisms Fecal Streptococcus (Presumptive and Confirmation) Enterococcus VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.) Increasingly Selective

11 Positive Test for Total Coliform Organisms - Presumptive Test Initially sterile Lactose broth containing Lauryl Sulfate {(C 12 H 25 O) 2 SO 2 } Inverted tube to capture CO 2 produced as part of fermentation Inoculated with water sample Incubate 48 hours at 37°C (body temp.) If gas appears in inverted tube, test is positive Thus, an organism was introduced that ferments lactose in the presence of lauryl sulfate at 37°C

12 Discussion Break In what ways does this test duplicate conditions in warm-blooded intestines? In what ways does this test not duplicate conditions in warm-blooded intestines? If an organism came straight from your lower intestinal tract, would it take the full 48 hours to yield a positive result? If an organism came from the American River and was several generations removed from fecal material, would it require the full 48 hours to yield a positive result?

13 Use of Confirmation and Presumptive Test for Total Coliform Each time a positive test is recorded, the population density of organisms reported increases significantly. To make sure a positive test is indeed positive, organisms from a positive test are transferred to a different medium. Although the medium is different, it relies on the same principle, fermentation of lactose in the presence of bile salts.

14 Positive Test for Total Coliform Organisms - Confirmation Test Initially sterile Brilliant green bile broth, lactose and bile salts. Inverted tube to capture CO 2 produced as part of fermentation Inoculated with loop from presumptive test. Incubate 24 hours at 37°C (body temp.) If gas appears in inverted tube, test is positive Thus, an organism was introduced that ferments lactose in the presence of bile salts at 37°C

15 10.Problems with total coliform procedure: Virtually every facultative anaerobic organism will test positive in the total coliform procedure. Impossible to assess fecal origin. Requires 72 hours to complete. Virtually no correlation with pathogen presence. VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

16 11.Positive attributes associated with total coliform procedure: Because it is so indiscriminate, serves as a good measure of disinfection completeness. Because it is so indiscriminate, it does a reasonable job of assessing overall bacterial population. As a matter of general policy, it is a good idea to be concerned when there is a lot of bacteria. VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

17 Positive Test for Fecal Coliform Organisms Initially sterile Lactose broth containing bile salts Inverted tube to capture CO 2 produced as part of fermentation Inoculated with water sample Incubate 48 hours at 44.5°C (112.1°F) If gas appears in inverted tube, test is positive Thus, an organism was introduced that ferments lactose in the presence of bile salts at 44.5°C

18 Discussion Break In what way is the fecal coliform procedure more selective than the total coliform procedure? If an organism came straight from your lower intestinal tract, would it take the full 48 hours to yield a positive result? If an organism came from the American River and was several generations removed from fecal material, would it require the full 48 hours to yield a positive result?

19 12.Problems with fecal coliform procedure: Many facultative anaerobic organisms will test positive in the fecal coliform procedure that reproduce in soil and water. Requires 48 hours to complete. Has minimal correlation with pathogen presence. VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

20 13.Positive attributes associated with fecal coliform procedure: Favored environment for positive testing organisms is intestinal tract of warm-blooded animals. Although most if not all will reproduce elsewhere, they will do so slowly. Will do the positive things the total coliform test does VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

21 Historical Note The initial total coliform test (presumptive form) was developed at the turn of the century to help confront outbreaks of serious diseases such as Cholera or Typhoid. The total and fecal coliform procedures as they currently exist were codified in They continued to be used as public health measures in confronting major outbreaks. Use in assessing completeness of disinfection was also used. Public health officials were aware of tests short comings and did not use them for recreational waters. Starting in the 50’s, total and fecal tests were used to assess quality of recreational swimming waters.

22 Positive Test for Fecal Streptococcus Organisms - Presumptive Test Initially sterile Glucose broth containing sodium azide (NaN 3 ). Sodium azide is highly toxic, but serves to select for streptococcus group. Inoculated with water sample Incubate 48 hours at 35°C (95°F) If contents of test tube become turbid, test is positive. Thus, an organism is present that is likely to be part of the streptococcus group associated with fecal material.

23 Positive Test for Fecal Streptococcus Organisms - Confirmation Test Initially sterile Incubate 24 hours at 35°C (95°F) Thus, it is confirmed that an organism is present that is likely to be part of the streptococcus group associated with fecal material. Streak with contents of positive presumptive test. Pfizer selective enterococcus (PSE) agar, complex organic media (bacterial candy), bile salts, and sodium azide. If brownish-black colonies with brown halos appear the presumptive test was indeed positive.

24 Positive Test for Enterococcus Organisms Initially sterile Brain-heart infusion broth containing 6.5% NaCl. Inoculated with brownish-black colony with brown halo from fecal streptococcus confirmation test. Incubate 24 hours at 45°C (113°F) If contents of test tube become turbid, test is positive. Thus, an organism is present that is likely to be part of the enterococcus group associated with fecal material.

25 14.Problems with fecal streptococcus - enterococcus procedure: Requires 96 hours to complete. Involves difficult lab procedures From the perspective of some, the fact that the test is rarely positive is a bad thing. Discussion Item: Why would that be the case? VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

26 15.Positive attributes associated with fecal streptococcus - enterococcus procedure: Positive organisms reproduce very slowly outside the intestinal tract of humans. Is a good indicator of fecal contamination!! Does not pick up a lot of organisms from non- human fecal matter. From the EPA science side, enterococcus is the best organism for assessing waters for recreational use with respect to pathogens. VI.The fifth category of pollutant to examine is pathogens. (cont.) C.Methodology for testing of indicator organisms in water samples. (cont.)

27 Discussion Break Most, if not all, basin plans use the fecal coliform test as the standard for water quality objectives. If the enterococcus group does a better job, why isn’t it specified? How about using a heterotrophic plate count in conjunction with the enterococcus test? Political pitfalls? There are some who feel that the problem of people getting sick from recreational water use has been exaggerated. What do you think? Why?

28 D.Methodology for counting of indicator organism population density in water samples. 1.Until now, we have been concerned only with what is in the water, not how much. 2.It is a fair assumption that the greater the population density of valid indicator organisms, the greater the possibility of pathogen transmission. 3.An widely accepted method for enumerating bacteria is the multiple- fermentation tube (MFP) method. VI.The fifth category of pollutant to examine is pathogens. (cont.)

29 4.What ever test, total coliform, fecal coliform, etc. the procedures in the MFP process are the same. VI.The fifth category of pollutant to examine is pathogens. (cont.) D.Methodology for counting of indicator organism population density in water samples. (cont.)

30 Principles of Enumerating Bacterial Population Densities Using the Multiple Fermentation Tube Technique - I Assume the following: –Each bacterium is a suspended entity, not attached to particulate material and not growing in clumps. –All bacteria are equally viable. –If introduced to a fermentation tube, a bacterium will register as positive. The idea is to dilute the sample in question to the point where the bacteria are just about extinct. This is done with a series of 1:10 dilutions. Thus the name serial dilutions.

31 Principles of Enumerating Bacterial Population Densities Using the Multiple Fermentation Tube Technique - II Within each dilution, an equal amount of sample is placed in 5 separate fermentation tubes. The process is repeated for three successive serial dilutions. 15 tubes total. SampleFermentation Tubes, #1- #5 10 mL Each Tube

32 Principles of Enumerating Bacterial Population Densities Using the Multiple Fermentation Tube Technique - III When the incubation period is over, the number of positive tubes in each dilution are counted. What we need is a mathematical tool that will: Describe the occurrence of unlikely events in a large number of independent repeated trials. The French mathematician, Siméon Denis Poisson, , developed just such a distribution. The distribution has been reduced to a series of tables. One counts the number of positive tubes in each dilution and looks up the results on the table.

33 Principles of Enumerating Bacterial Population Densities Using the Multiple Fermentation Tube Technique - IV As an example, consider the following possibility: For this combination, the Poisson Distribution yields the following results: –The most probable number (MPN) of bacteria is: 21 bacteria per 100 mL –There is a 95% probability that the correct number is within the range: bacteria per 100 mL Set #1, Sample Set #2, 1:10 Dilution Set #3, 1:100 Dilution 4 Positive 1 Positive 2 Positive

34 Principles of Enumerating Bacterial Population Densities Using the Multiple Fermentation Tube Technique - V If in the previous example, the initial set was a 1:10 dilution, the second set was a 1:100 dilution and the third set was a 1:1000 dilution, the results would be: –Most probable number: 210 bacteria per 100 mL –There is a 95% probability that the correct number is within the range: bacteria per 100 mL

35 Discussion Break The Central Valley Basin Plan uses a Fecal Coliform Standard of 200 MPN / 100 mL for water quality objectives most directly related to contact recreation. Recall the previous example: –210 MPN / 100 mL –95% confidence limits / 100 mL Thoughts on why dischargers get bent out of shape with respect to this water quality objective?