1

Appendix C: Analysis of NEEAR culture data: combining marine and 

freshwaters
2

Analysis of NEEAR culture data: combining marine and freshwaters

Bottom line: Marine and freshwater culture-derived data do not reflect a pattern suggesting that 

they are different. Based on our observations, marine and freshwater culture enterococci data 

are combinable.

Rationale and Evidence:

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

- is the same at all NEEAR study beaches.

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 versus 35, respectively).

o The number of beaches evaluated in the NEEAR studies was a relatively small (4 

freshwater, 3 temperate marine, and 1 tropical marine) subset of all waterbodies that the 

RWQC will apply to.

o 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.

Analytical Approach for Comparing NEEAR Beaches:

To determine if beaches should necessarily be grouped by salinity (grouping beaches as 

freshwater versus marine) we conducted several analyses. First, the observed range of culturable 

enterococci for each NEEAR beach was plotted in a box-and-whisker plot (Figure 1). As shown, 

there is substantial overlap in the observed densities across all beaches. There is also substantial 

intra-beach variability. 

Next, we used an ANOVA analysis of the NEEAR culturable enterococci to test for equality of 

GMs between beaches. This test indicated that the means (Refer to Table 1) between beaches 

were significantly different (p<0.001). Since more than two beaches were compared, a 

statistically significant effect in the ANOVA only indicates that the GMs are different between 

the beaches, but not which beaches are different and how.

Given the overlap of observed culturable enterococci among all beaches (Figure 1) we examined

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 the 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 be 

less than 0.05). As shown in Table 1, these statistically derived groups are not aligned strictly by 

classification of whether the waterbody was a fresh or marine beach. 
3

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 per 

100ml)

Group 2

(cfu per 

100ml)

Group 3 (cfu

per100ml

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)

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

above.

NEEAR Fresh and Marine Water Culturable Water Quality Results

(white, grey, hatched boxes represent statistically different groups)

HB SB WB WP EB FB GB BB SS

log10 enterococci (cfu/100mL)

-2

-1

0

1

2

3

4

5

6

7

Freshwater POTW-impacted sites Marine water

POTW-impacted sites

Marine water

non-POTW

impacted site
4

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 (Davies-Colley et al., 1999; Sinton et al., 1999; Boehm et al., 2002; Sinton et al., 

2002; Noble et al., 2004; Liu et al., 2006; Boehm et al., 2009; Walters et al., 2009; Nevers and 

Boehm, 2010) and predation (Enzinger and Cooper, 1976; Chamberlin and Mitchell, 1978; 

McCambridge and McMeekin, 1981; Barcina et al., 2997; Menon et al., 2003; Noble et al., 2004; 

Boehm et al., 2005), there are overall fewer studies documenting temperature (Wait and Sobsey, 

2001; Noble et al., 2004) and salinity (Hanes and Fragala, 1967; Noble et al., 2004) effects on 

enterococci 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 persistence 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) 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. 

References

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.

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 surf zone water quality at 

Huntington Beach, California. Environmental Science and Technology 36:3885-3892. 

Boehm, A.B., 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.

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 
5

viruses at a sewage-impacted marine beach. Environmental Science & Technology 43:8046-

8052. 

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.

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

faecal indicator micro-organisms in waste stabilization ponds: interactions of environmental 

factors with sunlight. Water Research 1999, 33, (5), 1220-1230. 

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

Escherichia coli from estuarine waters. Applied and Environmental Microbiology 31(5):758-

763.

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.

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. 

McCambridge, J. McMeekin, TA. Effect of solar radiation and predacious microorganisms on 

the survival of fecal and other bacteria. Appl. Environ. Microbio. 1981, 415, 1083-1087. 

Menon P, Billen G, Servais P. 2003. Mortality rates of autochthonous and fecal bacteria in 

natural aquatic ecosystems. WATER RESEARCH, 37, 4151-4158.

Nevers, M. N, 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. 

Noble, R. T., Lee, I.M. and Schiff, 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. 

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 stabilization pond effluent in fresh and 

saline waters. Applied and Environmental Microbiology 68:1122-1131. 

Wait, D. A., and M. D. Sobsey, Water Sci. Technol., 43, 139–142, 2001.
6

Walters, S. P.; Yamahara, K. M.; Boehm, A. B., Persistence of nucleic acid markers of healthrelevant organisms in seawater microcosms: Implications for their use in assessing risk in 

recreational waters. Water Research 2009, 43, (19), 4929-4939.