1



### Appendix B.7: Analysis of NEEAR culture data: combining marine and
freshwaters

MEMO

– Analysis of NEEAR culture data: combining marine and freshwaters QA

- The calculations in this analysis have been conducted independently twice.

Bottom line: Marine and freshwater culture

- derived data do not reflect a pattern suggesting that they are different. Based on our observations, m arine and freshwater culture enterococci data
are combinable

. .

Rationale and Evidence :

o The main source of the fecal contamination (and thus, fecal indicators)

- POTW effluent

- is the same at all NEEAR study bea ches.

o The 1986 RWQC geometric mean (GM) indicator density, which is to be used as the reference point in the 2012 RWQC is (effectively) the same in freshwater and marine waters (GM 33 vs 35, respectively).

o The number of beaches evaluated in the NEEAR studi es was a relatively small (4 freshwater, 3 temperate marine, and 1 tropical marine) subset of all waterbodies that the RWQC will apply.

o The literature indicates that of the factors influencing enterococci fate in the environment,
there is more evidence tha t sunlight, temperature and predation are more important in controlling enterococci concentrations than the effects of salinity.

Analytical Approach for Comparing NEEAR Beaches:

To determine if beaches should necessarily be group ed by salinity (grouping beaches as freshwater vs marine) we conducted several analyses . First, t he observed range of culturable enterococci for each NEEAR beach was plotted in a box

- and

- whisker plot (Figure 1). As shown,

t here is substantial overlap in the observed densities across all beaches. There is also substantial
intra

- beach variability.

Next, we used a n ANOVA analysis of the NEEAR culturable enterococci to test for equality of
GMs between beaches. This test indicated that the means (Refer to Tabl e 1) between beaches were significantly different (p<0.0 01 ) . Since more than two beaches were compared, a statistically significant effect in the ANOVA only indicate s that the GMs are different between the beaches, but not which beaches are different and h ow.

Given the overlap of observed culturable enterococci among all beaches (Figure 1) we examine d the variability between individual beaches. A post

- hoc test (Tamhane’s test) was used to ascertain which pairs of GMs are significantly different. This test is suitable in this situation because the variances are not equal and the number of beaches in groups is not constrained to be equal. The major advantage of this test is that it accounts for both intra

- and inter

- variability in
GMs for beaches to inform t he groupings; another advantage is that it is a conservative test, accounting for multiple comparisons between beach GMs (the type I error α is constrained to be2

less than 0.05). As shown in Table 1, these statistically derived groups are not aligned stric tly by classification of whether the waterbody was a fresh or marine beach

.

Figure 1.

Table 1. Homogenous subgroups of NEEAR beaches based on culturable enterococci

results. (The log10 means of each beach are listed under the column of the homogenous group to which it belongs . Log10 standard deviations are shown in parentheses ).

Beach Water body
type N Group 1 * (cfu/100ml) Group 2

(cfu/100ml) Group 3

(cfu/100ml Surfside (SS) marine 530 0.48 (0.73) Goddard (GB) marine 426 0.56 (0.92) Boqueron (BB) marine 600 0.75 (0.81) West Beach (WB) fresh 336 0.83 (1.01) Edgewater (EB) marine 395 0.88 (0.95) Fairhope (FB) marine 431 1.32 (0.95) Washington Park(WP) fresh 421 1.39 (0.50) Huntington (HB) fresh 420 1.4 (1.01) Silver Beach (SB) fresh 423 1.49 (0.66)3

*Groups in the table are reflected by different colors and cross hatching in the figure
above.

Analysis Findings:

o Extensive data analysis of NEEAR enterococci culture densities indicates that there is not a compelling distinction between marine and freshwater.

o The observed enterococci culture densities across all beaches are within the same range.

o While the means between individual beaches were significantly different, their differences are not aligned strictly by classification of whether the water body was a fresh

or marine beach

.

Background information

The literature indicates that of the factors influencing enterococci fate in the environment, there
is more evidence that sunlight

, temperature

, and predation are more important in controlling enterococci concentrations than the effects of salinity . While numerous studies have documented these sunlight and predation, there are overall fewer studies documenting temperature and salinity effects on entero cocci persistence. While the results of all temperature studies illustrate that lower temperatures results in increased persistence, the results from the few salinity studies are conflicting

– sometimes showing no effect of salinity, sometimes increased pe rsistence at low salinities and sometimes increased persistence at high salinities. The most relevant study for the issue at hand on enterococci survival is Noble et al. (2004) 1 who tested the persistence of enterococci from a variety of sources in fresh and seawater exposed to sunlight and various temperatures. They found that inactivation rates in did not differ amongst various treatments executed in seawater and freshwater. The Noble et al. (2004) paper is perhaps the most direct
evidence of the lack of effect of salinity on the persistence of enterococci in the aqueous
environment.

Sunlight references

M. N. Nevers and A. B. Boehm. 2010. Modeling Fate and Transport of Fecal Bacteria in Surface Water. A chapter in The Fecal Indicator Bacteria edited by Michael Sadowsky and Richard Whitman. ASM Press. 328 pages.

Liu, L.; Phanikumar, M. S.; Molloy, S. L.; Whitman, R. L.; Shively, D. A.; Nevers, M. B.; Schwab, D. J.; Rose, J. B., Modeling the transport and inactivation of E. coli and enterococci in
the near

- shore region of Lake Michigan. Environmental Science & Technology 2006, 40, (16),
5022

- 5028.

1 Noble, R. T., Lee , I.M. and Schif f, K. C. Inactivation of indicator micro

- organisms from
various sources of faecal contamination in seawater and freshwater

. Journal of Applied Microbiology 2004, 96, 464

– 472

.4

Walters, S. P.; Yamahara, K. M.; Boehm, A. B., Persistence of nucleic acid markers of health

relevant organisms in seawater microcosms: Implications for th eir use in assessing risk in recreational waters. Water Research 2009, 43, (19), 4929

- 4939.

Boehm, A. B., S. B. Grant, J. H. Kim, S. L. Mowbray, C. D. McGee, C. D. Clark, D. M. Foley, and D. E. Wellman. 2002. Decadal and shorter period variability of su rf zone water quality at Huntington Beach, California. Environmental Science and Technology 36:3885

- 3892.

Boehm, A. B., K. M. Yamahara, D. C. Love, B. M. Peterson, K. McNeill, and K. L. Nelson. 2009. Covariation and photoinactivation of traditional and novel indicator organisms and human viruses at a sewage

- impacted marine beach. Environmental Science & Technology 43:8046

- 8052.

Sinton, L. W., R. K. Finlay, and P. A. Lynch. 1999. Sunlight inactivation of faecal bacteriophages and bacteria in sewage

- polluted seawater. Applied and Environmental Microbiology 65:3605

- 3613.

Sinton, L. W., C. H. Hall, P. A. Lynch, and R. J. Davies

- Colley. 2002. Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabil ization pond effluent in fresh and saline waters. Applied and Environment al Microbiology 68:1122

- 1131.

Davies

- Colley, R. J.; Donnison, A. M.; Speed, D. J.; Ross, C. M.; Nagels, J. W., Inactivation of
faecal indicator micro

- organisms in waste stabilizati on ponds: interactions of environmental factors with sunlight. Water Research 1999, 33, (5), 1220

- 1230.

Noble, R. T., Lee , I.M. and Schif f, K. C. Inactivation of indicator micro

- organisms from various
sources of faecal contamination in seawater and freshwater

. Journal of Applied Microbiology 2004, 96, 464

– 472

.

Temperature R eferences

Noble, R. T., Lee , I.M. and Schif f, K. C. Inactivation of indicator micro

- organisms from various
sources of faecal contamination in seaw ater and freshwater

. Journal of Applied Microbiology 2004, 96, 464

– 472

.

Wait, D. A., and M. D. Sobsey, Water Sci. Technol ., 43, 139

– 142, 2001.

Predation References

Enzinger, R.M., Cooper, R.C. 1976 . Role of bacteria and protozoa in the removal of

Esc herichia coli from estuarine waters. Applied and Environmental Microbiology 31(5):758

- 763.

McCambridge, J. McMeekin, TA. Effect of solar radiation and predacious microorganisms on the survival of fecal and other bacteria. Appl. Environ. Microbio. 1981, 4 15, 1083

- 1087.5

Chamberlin, C. E. & Mitchell, R. 1978 . A decay model for enteric bacteria in natural waters. In Water Pollution Microbiology (R. Mitchell, ed.), Vol. 2, pp. 325

- 348. John Wiley & Sons, New
York.

Menon P, Billen G, Servais P . 2003. Mortali ty rates of autochthonous and fecal bacteria in natural aquatic ecosystems. WATER RESEARCH, 37, 4151

- 4158.

A. B. Boehm, D. P. Keymer, G. G. Shellenbarger. 2005. An analytical model of enterococci inactivation, grazing, and transport in the surf zone of a marine beach. Water Research , 39,
3565

- 3578.

Barcina, I., P. Lebaron, J. Vives

- Rego. 1997 . MiniReview. Survival of allochthonous bacteria in aquatic systems: a biological approach. FEMS Microbiology Ecology 23(1):1

- 9.

Salinity References

Hanes, N. B. and R. Fragala. 1967 . Effect of seawater concentration on survival of indicator bacteria.
Journal of Water Pollution Control Federation 39:97

- 104.

Noble, R. T., Lee , I.M. and Schif f, K. C. Inactivation of indicator micro

- organisms from var ious
sources of faecal contamination in seawater and freshwater

. Journal of Applied Microbiology 2004, 96, 464

– 472

.