United States Office of Research EPA/600/R-06/102 

Environmental Protection and Development September, 2006 

Agency Washington DC 20460 

Performance of 

Stormwater Retention 

Ponds and Constructed 

Wetlands in Reducing 

Microbial Concentrations 

U.S. Environmental Protection Agency 

Office of Research and Development 

National Risk Management Research Laboratory 
 

THIS PAGE INTENTIONALLY LEFT BLANK
EPA/600/R-06/102 

September, 2006

Performance of Stormwater Retention Ponds 

and Constructed Wetlands in Reducing 

Microbial Concentrations

By 

Scott D. Struck 

Ariamalar Selvakumar 

Michael Borst 

Urban Watershed Management Branch 

Water Supply and Water Resources Division 

National Risk Management Research Laboratory 

Edison, NJ 08837 

NATIONAL RISK MANAGEMENT RESEARCH LABORATORY 

OFFICE OF RESEARCH AND DEVELOPMENT 

U.S. ENVIRONMENTAL PROTECTION AGENCY 

CINCINNATI, OH 45268
Notice 

The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development 

performed and managed the research described in this report. It has been subjected to the Agency‘s peer 

and administrative review and has been approved for publication as an EPA document. Any opinions 

expressed in this report are those of the author and do not, necessarily, reflect the official positions and 

policies of the EPA. Any mention of products or trade names does not constitute recommendation for use 

by the EPA. 

ii 
Abstract 

Stormwater runoff can transport high concentrations of pathogens to receiving waters. Bacteria indicator 

organisms, as surrogates for pathogens, are the most often reported cause of receiving water impairments. 

Stormwater best management practices (BMPs) are often considered effective tools to mitigate the effects 

of stormwater pollutants before they appear in receiving waters. However, BMP performance for pathogen 

removal is not well documented. Many questions remain on the transport and fate of indicator bacteria that 

enter and exit stormwater BMPs. 

The National Risk Management Research Laboratory (NRMRL), part of U.S. EPA’s Office of Research and 

Development (ORD) investigated the fate of indicator organisms in the stormwater runoff entering and 

exiting two commonly used BMPs, constructed wetlands and retention ponds. This research used 

controlled-condition, pilot-scale systems that represent larger field-scale systems to determine the dominant 

mechanisms that influence the reduction of indicator organism concentrations. The pilot-scale work was 

supported by bench-scale laboratory experiments investigating the effects of single parameters such as 

temperature, sunlight, and salinity on indicator organism inactivation rates. Presented in this report are the 

results of developing techniques for creating bacterially enriched stormwater, bench-scale studies, and the 

pilot-scale BMP research. Bench-scale study results show that the temperature and sunlight affect the 

inactivation rates significantly. Results from the pilot-scale research suggest that constructed wetlands and 

retention ponds lower microbial concentrations in stormwater runoff. Bacteria inactivation generally 

followed the first-order, K-C* empirical model that acknowledges an irreducible concentration. Factors 

such as sunlight and temperature provide much of the inactivation in indicator bacteria, but other factors 

(e.g., predation, sedimentation, filtration, sorption, pH, and BOD) appear to also influence indicator bacteria 

concentrations. Future research validating results of the pilot-scale systems to field-scale systems should be 

done. 

Developing microbial inactivation models to predict effluent concentrations from BMPs will help reduce 

the uncertainty and improve the capabilities of surface water quality models. First-order models that do not 

consider background concentrations or resuspension, may underestimate actual bacterial concentrations. 

iii 
Foreword 

The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation’s 

land, air, and water resources. Under a mandate of national environmental laws, the Agency strives to 

formulate and implement actions leading to a compatible balance between human activities and the ability 

of natural systems to support and nurture life. To meet this mandate, EPA’s research program is providing 

data and technical support for solving environmental problems today and building a science knowledge 

base necessary to manage our ecological resources wisely, understand how pollutants affect our health, and 

prevent or reduce environmental risks in the future. 

The National Risk Management Research Laboratory (NRMRL) is the Agency’s center for investigation of 

technological and management approaches for preventing and reducing risks from pollution that threaten 

human health and the environment. The focus of the Laboratory’s research program is on methods and 

their cost-effectiveness for prevention and control of pollution to air, land, water, and subsurface resources; 

protection of water quality in public water systems; remediation of contaminated sites, sediments and 

ground water; prevention and control of indoor air pollution; and restoration of ecosystems. NRMRL 

collaborates with both public and private sector partners to foster technologies that reduce the cost of 

compliance and to anticipate emerging problems. NRMRL’s research provides solutions to environmental 

problems by: developing and promoting technologies that protect and improve the environment; advancing 

scientific and engineering information to support regulatory and policy decisions; and providing the 

technical support and information transfer to ensure implementation of environmental regulations and 

strategies at the national, state, and community levels. 

This publication has been produced as part of the Laboratory’s strategic long-term research plan. It is 

published and made available by EPA’s Office of Research and Development to assist the user community 

and to link researchers with their clients. 

 

 Sally C. Gutierrez, Director 

 National Risk Management Research Laboratory 

iv 
Contents 

Notice........................................................................................................................ ii 

Abstract......................................................................................................................iii 

Foreword...................................................................................................................iv 

Contents.................................................................................................................... v 

List of Figures.............................................................................................................vii 

List of Tables..............................................................................................................viii 

Acronyms and Abbreviations........................................................................................ .ix 

Acknowledgements......................................................................................................x 

Executive Summary.......................................................................................................xi 

Chapter 1 Introduction............................................................................................................................... 1-1

Urbanization..............................................................................................................................................1-1

Indicator Bacteria ......................................................................................................................................1-2

Stormwater BMPs.....................................................................................................................................1-4

Pollutant Attenuation .......................................................................................................................1-5

Chapter 2 Factors Affecting Microbial Indicator Concentrations......................................................... 2-1

Temperature ..............................................................................................................................................2-1

Sunlight .....................................................................................................................................................2-2

Physical Processes (Sedimentation, Sorption, and Filtration)...................................................................2-3

Salinity ......................................................................................................................................................2-4

Predation....................................................................................................................................................2-4

Other Potential Factors..............................................................................................................................2-5

Summary ...................................................................................................................................................2-5

Chapter 3 Bench-Scale Research............................................................................................................... 3-1

Purpose......................................................................................................................................................3-1

Inactivation Rate Models...........................................................................................................................3-1

Temperature .....................................................................................................................................3-2

Salinity.............................................................................................................................................3-2

Light.................................................................................................................................................3-3

Materials and Methods..............................................................................................................................3-3

Sample Collection............................................................................................................................3-3

Experimental Methods.....................................................................................................................3-4

Temperature Study...........................................................................................................................3-5

Salinity Study...................................................................................................................................3-5

Light Study ......................................................................................................................................3-5

Analysis of Indicator Organisms .....................................................................................................3-6

Data Analysis/Reduction .................................................................................................................3-7

Results.......................................................................................................................................................3-7

Time and Temperature.....................................................................................................................3-7

Light.................................................................................................................................................3-9

v 
Salinity...........................................................................................................................................3-11

Summary .................................................................................................................................................3-12

Chapter 4 Pilot-Scale Research ................................................................................................................. 4-1

Study Site and Experimental Design.........................................................................................................4-1

Creating Bacterially Loaded Stormwater.........................................................................................4-2

Storm Event Simulation...................................................................................................................4-3

Water Quality Monitoring, Solids, Light and Bacterial Indicator Sampling.............................................4-3

Sediment Sampling Procedures.................................................................................................................4-5

Predation Sampling Procedures.................................................................................................................4-5

Data Management .....................................................................................................................................4-6

Statistical Analyses ...................................................................................................................................4-6

Results.......................................................................................................................................................4-6

Scaling Consideration .............................................................................................................................4-16

Summary .................................................................................................................................................4-16

Chapter 5 Discussion of Results and Conclusions.................................................................................... 5-1

Effects of Temperature..............................................................................................................................5-1

Effect of Sunlight/Light Intensity..............................................................................................................5-2

Effects of Sedimentation, Sorption, and Filtration....................................................................................5-2

Effect of Salinity .......................................................................................................................................5-3

Effect of Predation ....................................................................................................................................5-3

Effect of Other Potential Factors...............................................................................................................5-4

Inactivation Rates Due to Collective Environmental Factors ...................................................................5-4

Evaluation of the First-Order Decay Equation..........................................................................................5-5

Conclusions...............................................................................................................................................5-9

Chapter 6 References.................................................................................................................................. 6-1

Appendix A Growth of Indicator Bacteria for Pilot-Scale Research ...................................................... 1

Introduction .................................................................................................................................................. 1

Methods........................................................................................................................................................ 1

Results and Discussion................................................................................................................................. 2

Conclusion.................................................................................................................................................... 3

vi 
 List of Figures 

Figure 1-1. Number of reported surface water quality impairments (top 7) since January 1, 

1996.......................................................................................................................................... 1-3

Figure 1-2. Number of approved TMDLs by pollutant (Top 7) since January 1, 1996....................... 1-4

Figure 3-1. Drainage area (A) and outfall location (B) of the study in Edison, New Jersey. ............. 3-4

Figure 4-1. Pictures of the retention pond (A) constructed wetland (B) treatment systems. .............. 4-2

Figure 4-2. Mean turbidity in the retention pond and constructed wetland in all storm events 

except October 2005. Whiskers indicate 95% confidence intervals. ................................ 4-7

Figure 4-3. Effluent concentration of indicator organisms with conductivity and dissolved 

oxygen.................................................................................................................................... 4-11

Figure 4-4. Effluent concentrations of indicator organisms with ORP and pH................................. 4-12

Figure 4-5. Effluent concentrations of indicator organisms with temperature.................................. 4-13

Figure 4-6. Effluent indicator bacteria concentrations with in-situ turbidity.................................... 4-13

Figure A-1. Mean indicator bacteria concentrations by day. Vertical bars denote 95% 

confidence intervals............................................................................................................... A-2

vii 
List of Tables 

Table 1-1. Range of Concentrations of Indicator Organisms Found in Varying Waterbodies ....................1-3

Table 2-1. Some Reported Die-off Rates (K) for Indicator Organisms .......................................................2-2

Table 3-1. Light Intensities Corresponding to the Height of Light Source above the Water Surface .........3-6

Table 3-2. Summary Table of Analytical Procedures..................................................................................3-6

Table 3-3. Inactivation Rate Constants for Each Indicator Organism at Tested Temperatures in the 

Isothermal Experiments ................................................................................................................................3-8

Table 3-4. Organism-Specific Reference Temperature Rate Constant and Temperature Coefficient 

Determined Using Salt-Free Dark Experimental Results .............................................................................3-9

Table 3-5. Regression-Estimated Values of Inactivation Rate Constants at 26.17oC for Experimental Light 

Intensities ......................................................................................................................................................3-9

Table 3-6. Regression-Estimated Coefficients from Light Experiment.....................................................3-10

Table 3-7. Regression-Estimated Values of Inactivation Rate Constants Determined for Salinity 

Concentrations ............................................................................................................................................3-11

Table 3-8. Regression-Estimated Coefficients from Salinity Experiment.................................................3-12

Table 4-1. Target Indicator Organism Densities in Mesocosms after Addition of Enriched Stormwater ...4-2

Table 4-2. Effluent Time of Hand Collected and Programmed Autosample Collection .............................4-4

Table 4-3. Expected Beginning Densities After Loading and Expected Dilution Factors...........................4-5

Table 4-4. Average Event in-situ Physical and Chemical Results...............................................................4-8

Table 4-5. Inactivation Rates for the Constructed Wetland, Retention Pond, Dark and Light Controls for all 

Indicator Bacteria Organisms for each Sampling Event.............................................................................4-10

Table 4-6. In-situ Indicator Organisms Average Background Concentrations..........................................4-15

Table 4-7. Sediment Bacteria Indicator Organisms Sampled in November of 2004.................................4-15

Table 4-7. Macroinvertebrate Groups Identified in the Retention Pond and Constructed Wetland ..........4-16

Table 5-1. Inactivation Rate Coefficients from Batch and Field Studies.....................................................5-7

Table 5-2. Retention Pond Overall, Temperature, Sunlight, and Other Factors Rate Coefficients. ............5-7

Table 5-3. Constructed Wetland Overall, Temperature, Sunlight, and Other Factors Rate Coefficients. ...5-8

Table A-1. Growth Rates of Bacterial Indicators Based on Regression Analyses ..................................... A-3

viii 
Acronyms and Abbreviations 

ANOVA Analysis of Variance 

APHA American Public Health Association 

ASCE American Society of Civil Engineers 

ASTM American Standard Testing Methods 

BHI Brain Heart Infusion 

BMP Best Management Practice 

BOD Biochemical Oxygen Demand 

CFU Coliform Forming Unit 

CWA Clean Water Act 

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen 

DQI Data Quality Indicator 

EC Escherichia coli

EMC Event Mean Concentration 

EN or ENT Enterococci 

EPA US Environmental Protection Agency 

FC Fecal Coliforms 

FR Federal Register 

FS Fecal Streptococci 

HDPE High Density Polyethylene 

MDE Maryland Department of the Environment 

MDL Method Detection Limit 

MF Membrane Filtration 

MPN Most Probable Number 

MS4 Municipal Separate Storm Sewer System 

MWLAP Ministry of Water, Land and Air Protection 

NPDES National Pollutant Discharge Elimination System 

NRMRL National Risk Management Research Laboratory 

NSQD National Stormwater Quality Database 

NTU Nephelometric Turbidity Unit 

ORISE Oak Ridge Institute for Science and Education 

ORP Oxygen Reduction Potential 

ppt Parts Per Thousand 

PVC Poly Vinyl Chloride 

RPD Relative Percent Difference 

RSD Relative Standard Deviation 

SM Standard Methods 

SS Suspended Solids 

TC Total Coliforms 

TMDL Total Maximum Daily Loads 

ix 
TNTC Too Numerous To Count 

TOC Total Organic Carbon 

TSS Total Suspended Solids 

QA/QC Quality Assurance/Quality Control 

USEPA US Environmental Protection Agency 

USGS United States Geological Survey 

UV Ultraviolet 

UWMB Urban Watershed Management Branch 

UWRF Urban Watershed Research Facility 

WEF Water Environment Federation 

WQS Water Quality Standards 

YSI Yellow Springs Instruments 

x 
Acknowledgements 

An undertaking of this type requires the dedication and cooperation of many individuals. The technical 

direction and coordination for this project was provided by the technical project team of the Urban 

Watershed Management Branch. Many members of the Branch assisted in making this product available to 

the public. Special recognition is extended to these members of the Branch: 

Anthony N. Tafuri, P.E., Branch Chief 

Thomas P. O’Connor, Environmental Engineer 

Mary Stinson, Chemical Engineer 

Dr. Christopher Nietch, currently with NRMRL, WSWRD, Cincinnati, OH 

Carolyn Esposito, Quality Assurance Officer 

Thomas P. O’ Connor and Mary Stinson performed reviews of this report. Carolyn Esposito reviewed the 

quality assurance project plan and this report. Mano Sivaganesan of Water Supply and Water Resources 

Division, Cincinnati reviewed the statistical part of this report. 

Most monitoring, sampling and laboratory analysis was performed by personnel from US Infrastructure, 

Inc., the operating contractor of the Urban Watershed Research Facility under EPA contract # EP-C-04-064. 

Most notably, Christa Casciolini, Yogesh Parikh, Toby Swetje, and Clarence Smith should be recognized 

for their contribution to the completion of the sampling and analyses. 

xi 
Executive Summary 

USEPA’s 305(b) water quality reports consistently show stormwater runoff as a leading cause of water 

quality impairment in the United States. Water quality standards (WQS) have been developed through 

states and the federal government to improve the condition of our nation’s surface and groundwater. 

When waters of the United States do not meet the WQS, regulations have been put in place to overcome 

impairments. To improve or prevent further degradation of water quality, regulators rely on best 

practicable control technologies currently available to reduce the loading of stressors from point sources 

(and, at times, non-point sources). Microorganisms are a high priority stressor because of the many 

waterbodies that are listed as impaired. 

This report documents the efforts to evaluate simple predictive relationships affecting concentrations of 

indicator organisms in stormwater runoff based on environmental conditions. The report begins by 

describing the breadth of surface water resources affected by bacterial stressors to identify needs for 

continued research on understanding the engineering approaches for management of point- and non-point 

sources associated with this stressor. In Chapter 2 factors from the scientific literature that influence 

bacteria indicator concentrations are reviewed. The subsequent chapters describe bench- and pilot-scale 

experiments that attempt to determine the dominant factors that favor a reduction in indicator bacteria 

concentrations. Finally, Chapter 5 contains synopses of the results of these experiments and an 

assessment of the first-order decay formula’s ability to predict bacterial concentrations based on influent 

concentrations and other environmental factors. Inactivation rates for each bacteria indicator from these 

experiments and coefficients are given. 

Combined in this report are bench- and pilot-scale data to assess first-order equations to better predict the 

performance of constructed wetland and retention pond best management practices (BMPs). By 

measuring varying physical, chemical, and biological parameters that may influence effluent indicator 

organism concentrations and characteristics of other stormwater parameters that are often contained in 

stormwater runoff factors that most influence inactivation rates were determined. The BMPs used in this 

research are small-scale, controlled systems (termed mesocosms) that offer a unique environment for 

investigating many parameters that can affect the reduction of indicator organisms. 

Detailed in Appendix A is the development of methods to grow and harvest bacteria indicators to provide 

an enriched source to increase the concentrations in stormwater for wet weather flow research. The 

mesocosm research necessitated this ancillary research as a technique to establish the desired influent 

bacteria concentrations. The technique may prove useful to others undertaking similar research. 

xii 
Chapter 1 Introduction 

The Clean Water Act (CWA) of 1972 developed an ambient water quality management program to 

measure the condition of a waterbody and determine whether that waterbody meets the criteria associated 

with the designated use. By definition, this process depends on setting appropriate water quality 

standards (WQSs). Realistic standard setting must balance watershed conditions (hydrologic, ecological, 

and land use) against the corresponding need to protect human health, infrastructure, and the 

environment. 

Where waters of the nation are not meeting established WQSs after implementing best practicable control 

technologies currently available to reduce the loading of stressors from point sources or other pollution 

control programs, the CWA requires establishing a Total Maximum Daily Load (TMDL) for each 

pollutant of concern. As part of the 1987 amendments to the CWA, Congress added Section 402(p) to 

cover point-source discharges composed entirely of stormwater. Section 402(p)(2) of the Act requires 

permit coverage for discharges associated with industrial activity and discharges from large (over 250,000 

people) and medium (between 100,000 and 250,000 people) municipal separate storm sewer systems 

(MS4s). These discharges are referred to as Phase I MS4 discharges. 

USEPA issued regulations on December 8, 1999 (64 FR 68722), expanding the National Pollutant 

Discharge Elimination System (NPDES) stormwater program to include discharges from smaller MS4s 

(including all systems within “urbanized areas” and other systems serving populations less than 100,000) 

and stormwater discharges from construction sites that disturb one to five acres, with opportunities for 

area-specific exclusions. This program expansion is referred to as Phase II. 

Urbanization 

USEPA used urbanized areas and population to define program boundaries because of the increased risk

to human health in these areas through greater potential for exposure from point and non-point sources 

and risks associated with a greater population density. Another component of the selected boundaries is 

that urbanization characteristically results in a larger percentage of impervious areas that lead to larger 

quantities of stormwater runoff that contribute significant amounts of debris and other pollutants (e.g., 

litter, oils, microorganisms, sediments, nutrients, organic matter, and heavy metals) to receiving waters. 

USEPA identified urban stormwater runoff as one of the four leading causes of water quality impairment 

related to human activities in lakes and reservoirs (USEPA, 2002). Poor water quality, especially 

pathogen contaminated water, can cause illnesses such as gastroenteritis (characterized by vomiting, 

diarrhea, abdominal pain or fever) or upper respiratory (ear, nose, and throat) infections to exposed 

swimmers. Highly polluted water can occasionally cause serious diseases such as typhoid fever, 

1-1 
dysentery, hepatitis, and cholera. An epidemiological study conducted in the Santa Monica Bay adjacent 

to Los Angeles County, CA found higher risks of a broad range of symptoms, including upper respiratory 

and gastrointestinal cases for people swimming closer to storm drains, implicating stormwater runoff as 

the source of the illnesses (Haile et al., 1999). Similarly, the Southern California Coastal Water Research 

Project showed that more than half of Southern California’s shoreline (from Santa Barbara to San Diego) 

is unsafe for swimming after rainstorms because of bacteria carried to the ocean by urban runoff (Noble et 

al., 2000). 

 

Indicator Bacteria 

To protect public health, surface waters are tested for indicators that serve as a proxy for harmful

pathogens. Indicator bacteria are used because it is difficult to measure the pathogens themselves. 

Indicator bacteria organisms tested by public health agencies include fecal indicator bacteria such as total 

coliforms, fecal coliforms, fecal streptococci, Escherichia coli (E. coli), and enterococci. The 

concentrations of these indicators are used to determine the potential for fecal contamination and to 

compare to public health-based thresholds. Like the pathogens they represent, fecal indicator bacteria are 

found in feces from both human sources (e.g. sewer discharges, and failing septic systems) and nonhuman sources (e.g. pets, waterfowl, and farm animals). Historically, total and fecal coliforms with fecal 

streptococci have served as the preferred indicators, but there are efforts to substitute enterococci and E. 

coli for water quality monitoring because of higher correlation with gastrointestinal illness (Gray, 2000). 

E. coli and enterococci are more representative of warm blooded animal fecal contamination in water than 

total or fecal coliforms. They both can survive, but generally not grow outside the intestinal tract 

(Ashbolt et al., 2001). In 1976, the USEPA recommended that states adopt a bathing WQS of fecal 

coliforms not to exceed 200 organisms/100 mL (USEPA, 1976). In 1986, based on the higher correlation, 

the USEPA recommended that states revise the recreational water quality microbial criteria to use 

enterococci for marine waters and E. coli or enterococci for freshwaters. Suggested criteria are 35 

enterococci per 100 mL for marine waters and 33 enterococci per 100 mL and 126 E. coli per 100 mL for 

freshwaters (USEPA, 1986). If a single sample exceeds 235 E. coli per 100 mL in freshwater and 104 

enterococci per 100 mL in saltwater, the USEPA recommends that a swimming area be closed, or posted 

until levels are lower. Several states have established policies that advisories are posted at more 

protective levels of indicator bacteria. Although EPA advised the states of the benefits of changing the 

indicators, many states continue to use the traditional indicators for a variety of reasons. 

Some have questioned the relationship between indicator bacteria and human health risks associated with 

pathogen exposure in surface water. Few epidemiological studies have tested the health effects of 

exposure to waters receiving direct and recent stormwater runoff. However, Wade et al. (2003) 

quantitatively analyzed many studies conducted during the past 50 years. The studies, when analyzed 

collectively, provide a weight of evidence showing the regulations suggested by the USEPA for the 

enterococci rule (for marine waters) and E. coli rule (for freshwaters) are protective when considering the 

risks associated with recreational water contact including swimming. 

Stormwater or stormwater-influenced receiving waterbodies can have indicator bacteria concentrations 

that greatly exceed WQS. Because elevated fecal indicator bacteria are often associated with stormwater 

runoff, some state and local agencies close swimming areas preemptively whenever rainfall exceeds a set 

amount based on site-specific studies. In a recent Water Environment Federation (WEF) report (WEF, 

2006), the authors summarized 51 studies from around the world that found ranges in concentrations of 

total coliforms, fecal coliforms, E. coli, fecal streptococci, and enterococci as reported in Table 1-1. 

Sources of these samples included coastal waters, rivers, creeks, drainage canals, and wetlands. 

1-2 
Table 1-1. Range of Concentrations of Indicator Organisms Found in Varying Waterbodies 

Indicator Organism 

Concentration Range 

(per 100 mL) 

Median 

(per 100 mL) 

Total Coliforms 1 x 101 – 2 x 106 1.2 x 104

Fecal Coliforms 1 x 101 – 8 x 106 5.1 x 103

Eschericia coli (E. coli) 1 x 102

 – 2 x 106 1.7 x 103

Fecal Streptococci 1 x 101 – 2 x 104 1.7 x 104

Enterococci 1 x 101 – 8 x 104 ––– 

 Source: WEF, 2006; Maestre and Pitt, 2005 

When looking at impaired waterbodies in the United States, Maestre and Pitt (2005) using data from the 

National Stormwater Quality Database, reported nationwide median concentrations for fecal coliforms, 

fecal streptococci, total coliforms and E. coli that are found in Table 1-1. On the US EPA’s 303(d) list of 

impaired waters, pathogen contamination is the most commonly listed source (7,742 reported 

impairments, Figure 1-1) and second for the number of approved TMDL allocations (2,608 TMDLs, 

Figure 1-2), indicating the prevalence of this stressor (USEPA, 2004). Of the 2,608 approved TMDL 

allocations, more than 84% are for fecal coliforms. 

Figure 1-1. Number of reported surface water quality impairments (top 7) since January 1, 1996. 

1-3 
Figure 1-2. Number of approved TMDLs by pollutant (Top 7) since January 1, 1996. 

Researchers have correlated aquatic microorganism densities with terrestrial watershed factors such as 

land use, density of housing, population, development, percent impervious area, and domestic animal 

density (Young and Thackston, 1999; Mallin, 1998; Glenne, 1984; Francy et al., 2000; Selvakumar and 

Borst, 2006). Surface runoff samples from more densely populated, sewered areas generally showed 

higher bacterial counts than runoff from less developed areas serviced by septic tanks (Young and 

Thackston, 1999). Selvakumar and Borst (2006) found microorganism concentrations from high-density 

residential areas were higher than those associated with low-density residential and landscaped 

commercial areas. 

Stormwater BMPs 

A stormwater best management practice (BMP) is a technique, measure, or structural control that is used 

to manage the quantity and improve the quality of stormwater runoff in the most cost-effective manner. 

The USEPA (1999) defines BMPs as "schedules of activities, prohibitions of practices, maintenance 

procedures, and other management practices to prevent or reduce the pollution of waters of the United 

States.” There are two general types of BMPs used to reduce the threat of stormwater runoff pollution 

from urbanizing areas: (i) nonstructural or source control BMPs; and (ii) structural or treatment BMPs 

(USEPA, 1993). 

Nonstructural BMPs refer to those stormwater runoff management techniques that use natural measures to 

reduce pollution levels, do not require extensive construction efforts, and either limit the generation of 

stormwater runoff, or reduce the amounts of pollutants contained in the runoff. They do not involve 

fixed, permanent facilities and they usually work by changing behavior through government regulation 

(e.g., planning and environmental laws), persuasion, and economic instruments (Taylor and Wong, 2002). 

These BMPs include institutional, educational, or pollution prevention practices. 

Structural BMPs are engineered systems and methods designed to provide temporary storage and 

treatment of stormwater runoff for the removal of pollutants (MWLAP, 1992; MDE, 2000; Clar et al., 

2003). Structural BMPs improve the quality; control the quantity of stormwater runoff or both. The 

1-4 
USEPA recommends installing stormwater BMPs within the landscape to influence runoff rates and 

reduce stressor levels in the stormwater runoff before it reaches the receiving water. 

Stormwater runoff is not identical in each location. The character of the drainage area strongly influences 

not only the runoff volume and rate from a given rain event but also the stressor concentrations. 

Regardless of the landscape where the BMP is installed, the same potential processes occur within the 

structure to mitigate the stressors and flow. These processes control the effluent rates and stressor levels 

regardless of the designated use of the receiving water. Fundamentally, a constructed wetland or 

retention pond of given characteristics attenuates the stressor load runoff regardless of land use or the 

receiving waters. The BMP’s capabilities are established by their design, construction, and maintenance, 

and not whether the device installed is part of the source water protection strategy or is a means to protect 

recreational water, e.g., as part of a TMDL strategy. 

If it is determined that a BMP approach (including an iterative BMP approach or treatment train) is

appropriate to meet the stormwater component of a TMDL, USEPA recommends that the regulatory 

language within the TMDL reflect this. Reductions in concentrations in effluents reaching the 

recreational waters depend on BMP performance. To estimate the reduction in stormwater pollutant 

concentrations passing through BMPs for developing TMDL allocations, the performance of each BMP 

must be well established. Much of the existing information on BMP performance comes from current 

literature and the American Society of Civil Engineers (ASCE) International BMP Database 

(www.bmpdatabase.org). This database, although one of the largest collections of data on BMP 

performance, has a paucity of information to adequately assess the performance of many stormwater 

BMPs (Andrews et al., 2004). 

Pollutant Attenuation 

BMPs are generally passive tools that use physical, chemical, and biological processes that promote 

natural microbiological inactivation to reduce this and other stressor concentrations in the effluent. These 

systems are not a means of chemical disinfection as in wastewater treatment. Determining the dominant 

mechanisms of stormwater bacterial and pathogen removal by these devices is an important step in 

predicting trends in effluent concentrations to meet state and federal WQSs and for developing TMDLs. 

Few quantitative studies have been carried out to determine the relative importance of various removal 

mechanisms by constructed wetlands and retention ponds for indicator bacteria, consequently the ability 

of these BMP treatments to reduce concentrations in stormwater runoff before reaching receiving waters 

where WQS must be met, are poorly understood. 

Two commonly-used structural BMPs for controlling pollutants in stormwater are constructed wetlands 

and retention ponds. Treatment and therefore design within these two systems rely predominately on 

slowing water transport time that provides increased settling. Other environmental factors that contribute 

to the natural decay process (referred to here as inactivation) in these management practices include 

irradiance (sunlight), temperature, turbidity, salinity, toxic substances, and predation. A simple first-order 

decay model: Kot C Ce t o

− = (3-1) 

 

Where: Ct = concentration of organism at time t (CFU/100 mL); Co = concentration of organism at time 

zero (CFU/100 mL); Ko = overall inactivation rate constant at the environmental conditions (h-1); and t = 

elapsed time since time zero (h); is commonly used to predict the effluent concentrations in these systems. 

The literatures report a wide range of values for K, however. Those values are typically based on a single 

source making extrapolation to different conditions difficult. 

1-5 
The primary objectives of this study were to: 

(1) determine, using bench-scale studies, the factors most important to evaluate commonly used 

inactivation models for indicator bacteria; 

(2) document the effects of two types of structural best management practices (retention pond and 

constructed wetland) on the removal efficiencies of indicator organisms (total coliforms, fecal 

coliforms, E. coli, and enterococci) in stormwater; 

(3) evaluate the applicability of first-order decay model; 

(4) evaluate the effluent concentrations of indicator organisms in stormwater runoff as they flow 

from constructed wetland and retention pond BMPs to determine overall inactivation rates from 

these systems for various indicator organisms; 

(5) record physical, chemical and biological parameters to determine any correlation with effluent 

indicator bacteria concentrations; and 

(6) develop relationships to serve as predictors for concentrations of indicator bacteria in the effluent 

of selected BMPs. 

Investigating the inactivation of indicator organisms from stormwater runoff that passes through retention

ponds and constructed wetlands is a complex undertaking. This project involves the analysis of various 

types of environmental and biological factors and multiple laboratory methodologies. A combination of 

bench- and pilot-scale studies were selected to take advantage of controls and conditions each scale has to 

offer. Bench-scale work was done to identify timing of samples and gain an understanding of the 

magnitude select factors would have on bacteria inactivation rates. It was also recognized early that there 

was little chance of obtaining constant conditions in a pilot-scale study subject to many environmental 

influences. Likewise, the replication of all potential environmental factors and their combinations within 

the laboratory was not feasible. Studies of both scales proved to provide the necessary conditions and 

controls to complete the project. The following chapter provides, in detail, the primary factors and 

supporting literature considered when considering the affect environmental factors have on indicator 

organism inactivation. 

1-6 
Chapter 2 Factors Affecting Microbial Indicator Concentrations 

Stormwater runoff often accounts for a large fraction of total microbial loading to many receiving waters 

(Jamieson et al., 2004; Crabill et al., 1998; Nix, 1994; Qureshi and Dutka, 1979; Olivieri et al., 1977; 

Wanielista, 1977; Geldreich et al., 1968; Weibel et al., 1964) with the potential to adversely impact 

drinking water sources, contact recreation areas, and protection and propagation of aquatic life (Sunen 

and Sobsey, 1999; Haile et al., 1999). Studies have also identified potential links between stormwater 

runoff and waterborne disease outbreaks in human populations (Currieo et al., 2001; Rose et al., 2001). 

Once introduced into the environment, microorganisms are affected by various environmental factors. 

There are known effects from chemical, physical, and biological sources that influence indicator bacterial 

growth, die-off, and inactivation. This chapter reviews some of these factors. While not exhaustive in its 

coverage, it covers the factors believed to be the most pertinent to retention ponds and constructed 

wetlands. 

Temperature 

Temperature plays an important role in microorganism die-off and has often been cited as the most 

important environmental factor. In general, microorganism=s survival is prolonged at lower temperatures 

(Ferguson et al., 2003). Experiments conducted by Selvakumar et al. (2004) showed that growth rates of 

indicator organisms are greatly reduced at 4o

C. Geldreich et al. (1968) noted that organism persistence 

remained higher at 10o

C than similar samples at 20oC. 

In the natural environment, several studies reported different die-off rates for various microorganisms in 

surface water (Table 2-1). Medema et al. (1997) found that the die-off of E. coli and enterococci were 

approximately ten times faster than die-off of Cryptosporidium parvum oocysts, but die-off rates of 

Clostridium perfringens were slower than those of oocysts. They also noted that die-off of these 

indicators was faster at 15o

C than at 5oC. Dutka and Kwan (1980) reported that E. coli, Streptococcus 

faecalis, and Salmonella thompson could survive in 17-18o

C waters for at least 28 days and E. coli was 

found in greater concentrations than Streptococcus faecalis. Baudisova (1997) reported that the die-off 

rate of E. coli is greater than that of total and fecal coliforms. Canteras et al. (1995) noted a clear 

negative relationship between die-off and temperature. At 10o

C, 36 h was necessary to reduce the 

population of E. coli to 10% of the initial concentration compared to 8.4 h at 42o

C. Greater reduction of 

the die-off rate was noticed in the range between 10 and 18o

C than between 18 and 42oC. 

2-1 
Table 2-1. Some Reported Die-off Rates (K) for Indicator Organisms 

Indicator 

Organism

K (h-1) Condition Reference 

0.042-0.229 

0.033 

0.058 (0.029-0.125)

Freshwater (20o

C) 

Average freshwater (20o

C) 

Seawater (20o

C) 

Total Coliforms Thomann and Mueller (1987) 

0.018 River water (12 d) Baudisova (1997) 

Total or Fecal 

Coliforms 

0-0.1 

0.104-0.254 

New York Harbor, salinity 

2-18 ppt, 

dark samples 

New York Harbor, salinity 

15 ppt and sunlight 

Thomann and Mueller (1987) 

1.542-4.583 Seawater and sunlight Fecal Coliforms Thomann and Mueller (1987) 

0.021 River water (12 d) Baudisova (1997) 

Enterococci 0.003 

0.009 

River water (5o

C); 42 d 

River water (15o

C); 0-14 d 

Thomann and Mueller (1987) 

0.003-0.083 Seawater, salinity 10-30 ppt Thomann and Mueller (1987) 

0.022 River water (12 d) Baudisova (1997) 

E. coli

0.004 

0.008 

River water (5o

C); 42 d 

River water (15o

C); 0-14 d 

Medema et al. (1997)

Fecal Streptococci 0.75-2.292 Seawater and sunlight Thomann and Mueller (1987) 

Much of the earliest work on bacterial removal assumed that temperature was the most important factor 

controlling the removal mechanism, as described by the first-order equation developed by Marais and 

Shaw (1961). Studies, such as Klock (1971) and Ferrara and Harleman (1981) also emphasized on first 

order concentration reductions with temperature-dependent rate constants. 

Recent investigations considered bacterial removal as a more complex mechanism involving interactions 

between the physical, chemical and biological systems present in wetlands and retention ponds, although 

temperature clearly remains an important parameter. For example, Polprasert et al. (1983), Pearson et al.

(1987a, b), Barzily and Kott (1991), Mara et al. (1992a, b), and Mezrioui et al. (1995a, b) all found that 

removal rates of fecal coliforms increased with increasing temperature. No matter which indicator 

organism is tested, temperature clearly affects indicator bacteria. 

 

Sunlight 

Numerous studies have shown sunlight as an important factor in microorganism die-off though it is 

difficult to separate effects from other factors entirely. Sinton et al. (1994) studied inactivation in 

sunlight of fecal coliforms and enterococci from sewage and meat works effluent concluding that die-off 

rate of fecal coliforms was 2-4 times that of enterococci and inactivation is generally slower at lower light 

intensities. Alkan et al. (1995) found that variability of enteric bacteria (i.e., enterococci and E. coli) dieoff due to the effect of sunlight depends on the variability of the intensity of light and other small scale 

environmental factors such as turbidity, sewage content, and degree of mixing. Importantly, they further 

2-2 
reported that the die-off rates of E. coli and enterococci from exposure to light were similar. Canteras et 

al. (1995) reported that sunlight was the most important factor affecting die-off of E. coli with 90% 

concentration reductions within about 1 h (i.e., inactivation rate of about 0.89 h-1) at 18o

C and 8.5% of 

salinity when light radiation was greater than 120 W/m2

 (12 mW/cm2). Yukselen et al. (2003) studied the 

effects of solar radiation and temperature on bacterial die-off rates in Black Sea coastal waters and found 

that solar radiation was the most significant factor affecting the mortality of coliform bacteria. No 

significant effect of temperature was observed in the presence of solar radiation. However, the effect of 

temperature is significant in dark experiments with die-off taking approximately 20 times longer to reach 

90% concentration reductions compared to values in the light. Davies-Colley et al. (1999) reported that 

sunlight is the main factor causing natural attenuation in waste stabilization ponds, although dissolved 

oxygen (DO) and pH can also influence the rate of disinfection. Die-off studies on E. coli and Salmonella

were conducted in two different ecosystems: Morlaix estuary in English Channel and Bay of Toulon on 

Mediterranean Sea. In the Morlaix estuary, most of the bacteria were mixed with turbid waters and were 

able to survive a long time as light penetration was prevented by suspended matter, lowering the effect of 

sunlight. On the contrary, through lack of nutrients and very high sunlight intensity, die-off rates in 

Mediterranean waters were high with 90% mortality within 2 h near the water surfaces, and several hours 

in deep waters (Pommepuy et al., 1992). 

A close relationship was found between the light intensity and the decay rate. Gameson and Gould 

(1975) concluded that about half the lethal effect of light is attributable to wavelengths below 370 nm 

with an additional quarter of the lethal effect attributable to the 370-400 nm and 400-500 nm bands, 

respectively. The effect of longer wavelengths, greater than 500 nm, is negligible. 

The exact mechanism whereby microorganisms become non-viable after sunlight exposure is not entirely 

clear. Photons can excite exogenous or endogenous sensitizers (e.g., humic acid) present in the water that 

damage DNA or other cellular components of the bacteria, directly. Photons can also cause damage 

indirectly by promoting the production of free radicals in the presence of dissolved oxygen and organics. 

Chamberlin and Mitchell (1978) and Eisenstark (1971) noted that the mechanism of light-induced 

bacterial decay depends on the presence of endogeneous sensitizers or chromophores, which adsorb light 

energy and cause cell damage directly or by reaction with oxides to form superoxides, which in turn may 

cause damage to the cells. 

Physical Processes (Sedimentation, Sorption, and Filtration) 

Microbes in the water column may associate with particles or remain in the ‘‘free’’ or unassociated phase. 

This free phase includes organisms that exist individually or as agglomerated groups (aggregates) held 

together by organic and inorganic particles. Microbes associated with particles, particularly denser 

inorganic particles, will tend to settle from the water column more quickly than free organisms or those 

associated with less dense particles that remain more mobile in the environment. It has also been 

observed that microbes associated with particles tend to survive longer in natural waters than free 

microbes (Howell et al., 1996; Sherer et al., 1992; Burton et al., 1987; Gerba and Schaiberger, 1975). 

These particle associations can affect not only microbial fate and transport, but also the time these 

organisms remain a threat to human health. 

Gannon et al. (1983) and Davies and Bavor (2000) assessed the performance of stormwater 

impoundments and constructed wetlands for microbial concentration reductions by measuring inflow and 

outflow, with results suggesting that sedimentation was a primary mechanism of removal. Gannon et al.

(1983) also demonstrated that a significant fraction of fecal coliforms in the water column was retained by 

a 5 mm filter, indicating that some bacteria were attached to particles, but making no distinction regarding 

the nature of the particles. In subsequent work, Schillinger and Gannon (1985) analyzed the partitioning 

2-3 
of several bacterial indicators in samples taken from the water column of a stormwater drain under wet 

weather conditions. Similar filtration experiments revealed that at least half the bacteria in the water 

column passed through a 5 mm filter, but again the nature of the particles was not addressed. 

Characklis et al. (2005) found that substantial fractions of five different microbial organisms, including 

bacterial, protozoan, and viral indicators, were associated with settleable particles in stormwater. The 

results also found partitioning behavior varied by organism and with conditions (e.g., storm vs. 

background). This study attempted to correlate the microbe–particle association with specific 

environmental factors (e.g., TSS, TOC, particle number) but did not yield strong evidence of a 

relationship. However, the results suggest that for some organisms (e.g., fecal coliforms) there may be a 

relationship between the fraction that is particle associated and particle concentration, microbial 

concentration, or both. 

The processes of sedimentation, sorption, and filtration are difficult to separate. In the project discussed

in this report, the physical processes are treated collectively and referred to as sedimentation. Some 

consider sedimentation as the main mechanism of pollutant removal in general in constructed wetlands 

and retention ponds. With the tendency for a significant portion of some stormwater pollutants to bind to 

particulates, sedimentation is often considered as the primary factor when designing treatment practices. 

Longer detention periods in retention ponds promote sedimentation of solids in the course and medium 

size fractions. Similarly, the presence of extensive vegetation in constructed wetlands can encourage 

sedimentation (Pundsack et al., 2001).

Salinity 

Osmotic stress can also play a role in the concentrations of concentrations of indicator bacteria. The dieoff rate is generally much faster in marine and estuarine waters than in freshwater (Thoman and Mueller, 

1987). Yan et al. (2000) found that both light intensity and salinity have significant effects on the 

inactivation of E. coli in wastewater discharged into the ocean through submarine outfall system. Solic 

and Krstulovic (1992) found inactivation rate increased as salinity increased. They also noted that higher 

salinity and high levels of solar radiation combined produced a synergistic effect, resulting in higher 

mortality rates of fecal coliforms. Hanes and Fragala (1967) showed that E. coli, coliforms and 

enterococci had greater inactivation rates with increasing salinities, with 6.2, 4.6, and 1.6 times greater 

death rate, respectively, at 100% seawater compared to 0% seawater. Similarly, Anderson et al. (1979) 

found a decreased survival rate for E. coli with increasing salinity, ranging from 53.5% survival at 10 ppt 

after 8 days of exposure, to 2% survival at 30 ppt for the same period. Fuijoka et al. (1981) found that 

seawater caused rapid inactivation of fecal streptococci and fecal coliform, whereas the organisms 

remained stable for three days in freshwater. Mancini (1978) indicated that components in seawater in 

addition to salt may be responsible for inactivation in seawater. 

Salinity concentration is an important factor in coastal receiving waters such as estuaries or the coastal 

ocean. Although many estuarine and coastal systems receive stormwater runoff, generally stormwater 

retention ponds and constructed wetlands are not constructed in areas that are tidally influenced or are in a 

continuously saline environment. The project discussed in this report only briefly address bacterial 

attenuation from this stressor. 

Predation 

Wetlands and retention ponds can support a diversity of aquatic animals including micro-crustaceans 

(copepods, ostracods, and cladocerans), shrimp, crayfish, insects (dragonfly larvae, water beetles, and 

water boatman), pond snails, tadpoles, frogs, and fish. These organisms are a crucial component of 

2-4 
wetland and shallow open-water ecosystems, providing food-web linkages between plants, 

microorganisms and other animals. Predator–prey relationships are important in the control of 

mosquitoes (Greenway et al., 2003) and may contribute to the control of bacterial populations (Davies 

and Bavor, 2000) in these systems. Green et al. (1997) determined bacterivorous activity was an 

important factor in the removal of bacteria in constructed wetlands treating wastewater. Mandi et al.

(1993) determined predation by nematodes and Decamp and Warren (1998) by ciliates and rotifers were 

significant factors in determining bacterial densities in constructed wetlands although in the former study 

the predation was not quantified. In the latter, Decamp and Warren (1998) determined that ciliates such 

as paramecium ingested as many as 13 fluorescently labeled E. coli per cell per minute. Fernandez et al.

(1992a, b) also concluded that predation and competition were extremely important in the removal of 

fecal coliforms. As part of a large study to model the removal of fecal coliforms, Troussellier et al.

(1986) investigated the effects of grazing by rotifers and of biological oxygen demand (BOD) loading. 

They found rotifers can significantly affect fecal coliform concentrations in aquatic systems. 

Other Potential Factors 

There are many other factors that can affect the densities of indicator organisms. BOD, pH, and DO have 

all previously been mentioned as potential contributing factors to inactivation rates of bacteria. Solic and 

Krstuvolic (1992) noted that fecal coliforms thrived at a pH range of 6-7, declining in numbers outside of 

this range, with greater rate of mortality in acidic environments. Chemical factors include oxidation, 

exposure to biocides excreted by plants, and sorption to organic matter. Additional biological removal 

mechanisms may include antimicrobial activity of root exudates (Kickuth and Kaitzis, 1975; Axelrood et 

al., 1996), activity of lytic bacteria or viruses (Axelrood et al., 1996), retention in biofilms (Brix, 1997), 

and natural die-off (Gersberg et al., 1989a, b). 

Few studies have thoroughly investigated the effect of nutrients (including dissolved organic carbon and 

trace metals) on the inactivation of microorganisms in stormwater in the environment. Thomas et al.

(1999) found bacterium Campylobacter had low survival rates in nutrient-containing microcosms. In 

microcosm studies in fresh and salt waters, Noble et al. (2004) found that nutrient levels had an 

insignificant effect on the persistence of fecal indicator bacteria. 

Summary 

As can be seen, literature values on indicator bacteria inactivation in surface waters are quite variable. 

Much of the literature pertains to wastewater treatment and dairy waste studies. There have been few 

studies conducted investigating the viability of indicator bacteria using stormwater as a medium. Of the 

field studies on surface waters, conditions of the watershed and storm information such as intensity and 

duration are not well documented or are incomplete. Therefore, interpreting data and comparing results 

are often tenuous at best. 

Which of these factors has the greatest influence on indicator bacteria inactivation? Are there 

combinations of factors that have the greatest affect? What conditions affect the contribution from each 

individual or group of factors? The next three chapters detail both bench- and pilot-scale experiments 

conducted for data collection and evaluation of the first-order decay model that uses environmental 

factors to determine the inactivation rate, K, to predict effluent concentrations in BMPs. 

2-5 
 

Chapter 3 Bench-Scale Research 

Purpose 

The bench-scale research tested the proposed descriptive relationships between concentration and 

identified presumptive controlling variables (time, temperature, light intensity, and salinity) outlined in 

the literature and listed below. The work used statistical analysis, specifically nonlinear regression, to 

quantify the organism-specific inactivation rate constants for traditional (fecal streptococci, and total and 

fecal coliforms) and alternate (enterococci and E. coli) microbial indicators in stormwater. The research 

separately assessed the influence of time, temperature, light intensity, and salinity on the inactivation rate 

constants to isolate the effects and maintain the analytical load within laboratory capacity. This approach 

simplifies the analysis but neglects the potential interactions among the independent variables. 

The research exposed stormwater samples to controlled-conditions to measure the change in 

microorganism concentrations after known exposure periods. The experimental design selected the 

controlled independent variables and their ranges based on the broad, literature-reported influence and the 

likelihood of the condition existing in the structural BMPs (e.g., retention ponds and constructed 

wetlands). As widely reported in the literature, time, temperature, and light intensity are important 

environmental variables which determine the rate of change of indicator organism concentrations. 

Salinity was included in this study as it is reported as an environmental factor which influences the 

microbial decay and can be potentially important in some BMPs installed in coastal settings or when the 

stormwater runoff results from areas where communities apply road salt during winter. 

Inactivation Rate Models 

The literature reports indicator organism inactivation rates in various water types (Table 2-1). However, 

information on inactivation rates for indicator organisms in stormwater and effects of natural factors on 

survival rates is limited except for one study by Geldreich et al. (1968). 

Most published studies use first-order decay known as Chick’s Law to describe indicator organism 

inactivation with time. Under this premise, the concentration-time relationship is: 

Kot C Ce t o− = (3-1) 

 

Where: Ct = concentration of organism at time (CFU/100 mL); 

 Co = concentration of organism at time zero (CFU/100 mL); 

 3-1
 

 Ko = overall inactivation rate constant at the environmental conditions (h-1); and 

 t = elapsed time since time zero (h). 

There are several approaches to estimate the effects of environmental variables on the overall rate 

constant. The simplest approach assumes additive effects: 

K0 = + ++ K K KK TSl f (3-2) 

Where: KT = inactivation rate constant due to temperature (h-1); 

 KS = inactivation rate due to salinity (h-1); 

 Kl = inactivation rate constant due to light (h-1); and 

Kf = inactivation rate constant due to other factors such as sorption, filtration, and 

sedimentation (h-1). 

Temperature 

The effect of temperature is often approximated by using the Arrhenius-van’t Hoff equation (Khatiwada 

and Polprasert, 1999): 

( 20)

20

T K K T T− = Φ (3-3) 

Where: KT = inactivation rate constant due to temperature at T = T0

C (h-1); 

 K20 = inactivation rate constant due to temperature at T = 200

C (h-1); 

 T = temperature in 0

C; and 

 ФT = temperature coefficient (dimensionless). 

The selection of the reference temperature (200

C) is somewhat arbitrary. Other temperatures can and 

have been used. As much of the literature uses 200

C as the reference value, this research follows this 

convention. 

Salinity 

The simplest approach to estimating the effect of increasing salinity on the rate constant assumes a linear 

increase with salinity. 

KS S = Φ S

S

 (3-4) 

Canteras et al. (1995) proposed a variation on the additive effects described by equation (3-5). That work 

proposes a combined effect due to the combination of temperature and salinity. 

( 20)

, 20

T K K T S T S− =Φ Φ (3-5) 

Where: KT,S = inactivation rate constant due to temperature and salinity (h-1); 

K20 = inactivation rate constant at dark, 200

C and zero salinity (h-1); 

ФT = temperature coefficient (dimensionless); and 

ФS = salinity coefficient (dimensionless). 

 3-2
 

Light 

The effect of light intensity on the inactivation rate constant is normally expressed as: 

Kl l = Φ ZI (3-6) 

Where: Фl = light proportionality coefficient (cm2

/mW-h); and 

IZ = light intensity at depth Z below the surface (mW/cm2

). 

Unlike temperature and salinity which can be reasonably assumed to be uniform throughout the system, 

light intensity varies with depth below the water surface. The intensity at a given depth, IZ,, decreases 

exponentially with distance (Gameson and Gould, 1975). The value is often estimated as: 

(1 ) O h

Z

I I e

h

τ

τ = − (3-7) 

Where: Io = light intensity at the earth surface (mW/cm2

); 

τ = vertical light extinction coefficient (1/m); and 

Z = depth of water (m). 

The extinction coefficient varies with water proprieties including color and turbidity (Lee and Rast, 

1997). 

Combining equations, the overall equation using the Canteras et al. (1995) assumption for salinity is: 

( 20)

20

T S K K o T S lZI K − = Φ Φ +Φ + f (3-8) 

Materials and Methods 

Sample Collection 

Stormwater was collected from an outfall after that drained a 10-acre portion of the Middlesex County 

College Campus near the USEPA facility in Edison, New Jersey (Figure 3-1). The drained area was 

predominantly campus maintenance buildings and student parking lots. Samples were only collected for 

this work when the rain event met the USEPA monitoring guidance (USEPA, 1992). Generally, the 

project required at least 3 mm (1/8th in.) total rainfall, preceded by at least 72 h without measurable 

precipitation. Automatic samplers (Hach, Loveland, CO) placed in the outfall collected a flow weighted 

composite sample when the flow water depth in the outfall initially reached 2.54 cm (1 in.). Areavelocity flow meters (Hach, Loveland, CO) connected to the automatic samplers triggered the internal 

peristaltic pump to add 1-L aliquots to a 20-L, pre-cleaned container when an incremental specified flow 

volume was measured. The incremental volume was set based on forecasted total rainfall. 

After collection, the samples were transported to the on-site laboratory and allowed to quiescently settle 

for 10 to 20 min at room temperature to allow most settling solids to fall to the container bottom. The 

water from the settled collection container was transferred leaving about 1 in. in the composite container 

bottom to limit the potential effects of settleable particulates on the experiments and avoid interference 

with the enumeration process. While continuously stirring the container holding the decanted supernatant 

(Stir Pak Mixer), a peristaltic pump transferred aliquots to 250-mL pre-cleaned HDPE bottles. All 

subsample bottles were completely filled leaving no headspace. 

 3-3
 

USGS DOQQ, 1998 

Figure 3-1. Drainage area (A) and outfall location (B) of the study in Edison, New Jersey. 

Experimental Methods 

The experiments were conducted by placing the 250-mL HDPE containers in water baths (Precision, A 

Division of Jouan Inc., Winchester, VA) to maintain constant temperature. The temperature of each 

water bath was established at least one day before inserting the bottles. Aluminum foil wrapping on the 

outside of the bottles prevented light exposure for experiments other than those investigating the effects 

of light exposure. The temperature of the water bath and the temperature of an equal volume of deionized 

water in separate containers were monitored using a NIST-traceable digital thermometer and recorded at 

1-min intervals using logging thermisters (Onset Corp, Bourne, MA). The recorded temperatures 

confirmed that the stormwater in the 250-mL containers required from 30 to 340 min to reach the water 

bath temperature. The temperature varied less than 1o

C during the experiment. 

The experiments defined time zero as the time when the sample reached the designated temperature as 

described below. Bottles were removed periodically during the experiment for sampling and analysis. 

The times when bottles were removed from the water bath were established based on the expected 

exponential concentration decline and to collect samples primarily during the normal workday as a cost 

control measure. Samples collected from the bottles were analyzed for five indicator organisms following 

membrane filtration procedure. A set of four samples was collected for the initial time. Subsequent 

sampling collected duplicate samples from the bottle. Time was monitored using commercially-available 

(La Crosse Technology, La Crosse, WI) clocks synchronized to the US Naval Observatory atomic clock. 

The reported elapsed time for removing the bottle from the temperature bath is believed to be accurate to 

within 1 min. DO and pH were monitored daily from independent sample bottles for the duration of the 

 3-4
 

experiment. 

Temperature Study 

 

The temperature-dependence die-off study targeted temperatures of 10o

C, 20oC, and 30oC. Earlier 

USEPA research, measuring the diurnal temperature fluctuation in local BMPs showed that summer 

temperatures reach about 30o

C and fall to less than 5oC in the winter. While extreme water temperatures 

in BMPs are likely to span a slightly wider temperature range; the temperature range used in this study 

should represent the most often encountered temperatures within the BMP. The mean temperatures 

recorded by the data loggers are used in the analysis (usually slightly lower than the targeted 

temperatures), however, for simplicity, the descriptive target temperatures were used. Temperature 

monitoring was included as part of the light and salinity studies. 

Salinity Study 

The salinity-dependence die-off study targeted four salinity levels (0, 10, 20, and 30 ppt). The target 

temperature was set at 25o

C. The salinity range was selected to represent concentrations encountered in 

partially diluted seawater. The salinities were established by adding synthetic sea salt (Instant Ocean, 

Aquatic Systems, Mentor, OH) directly to stormwater while mixing. When the lowest salinity was 

achieved, stormwater was dispensed to the sample bottles. Then, more sea salt was added to achieve the 

next highest salinity level. The salinity of the resulting solution was measured using Hach CO 150 

Conductivity Meter (Loveland, CO). The analytical results showed salinity concentrations of 0.45, 8.1, 

16.1, and 23.5 ppt, while those concentrations for samples other than those in the salinity study were not 

measured. 

Light Study 

The light-dependence study established a target temperature of 25o

C. Samples were exposed to light at 

four intensities including one dark sample. The respective light intensities for different light conditions 

were established using Reptisun 5.0 UVB fluorescent bulbs (Zoo Med Laboratories, Inc., San Luis 

Obispo, CA). The manufacturer reports that the bulbs produce light at UVA (320-400 nm) (30%) and 

UVB (290-320 nm) (6%) wave lengths. Adjusting the distance between the light source and the surface 

of the container controlled light intensities. The distance was maintained at 12.7, 22.9 or 35.6 cm above 

the water surface to have light intensities varying between 20 and 100 mW/cm2

 (200 to 1000 W/m2). 

Average intensity of sunlight is about 120 mW/cm2 (1,200 W/m2

). Light intensities at the sample surface 

were measured using a light meter (International Light IL 1400A with thermopile detector) daily 

throughout the experiments. The measured light intensity varied about 5% during the experiment. Table 

3-1 lists the average light intensities measured throughout the experiment. 

The container used to hold the sample in the light study was 250-mL thin, flat sided polystyrene flasks 

with canted neck and plug seal cap (BD Biosciences, Bedford, MA). This container was selected because 

it did not have any effect on the light attenuation and would reduce the depth of water assuring a more 

homogenous light dose. 

 3-5
 

Table 3-1. Light Intensities Corresponding to the Height of Light Source above the Water 

Surface 

Distance (cm) Light Intensity 

(mW/cm2

) 

Average Light Intensity 

(mW/cm2) 

12.7 89.0 – 97.8 94.70 

22.9 50.2 – 58.4 55.23 

35.6 19.7 – 21.9 20.86 

Analysis of Indicator Organisms 

All samples were analyzed for five indicator organisms (total coliforms, fecal coliforms, fecal 

streptococci, enterococci, and E. coli) using membrane filtration methods following Standard Methods for 

the Examination of Water and Wastewater (APHA et al., 1998) (Table 3-2) and described below. 

Table 3-2. Summary Table of Analytical Procedures 

Indicator Organism Method Number Method Title

Total Coliforms SM 9222B Membrane Filter Procedure 

Fecal Coliforms SM 9222D Membrane Filter Procedure 

E. coli SM 9222G Membrane Filter Procedure 

Fecal Streptococci SM 9230C Membrane Filter Procedure 

Enterococci SM 9230C Membrane Filter Procedure 

Total coliforms were determined by incubation on M-Endo agar (24 h at 35o

C) and confirmed by gas 

formation in lauryl tryptose broth and brilliant green lactose broth. Fecal coliforms were incubated on MFC agar (24 h at 44.5o

C) and were confirmed by gas formation in lauryl tryptose broth and EC broth. E. 

coli levels were measured by transferring the membrane from the Endo-type medium to a nutrient agar 

containing 4-methylumbelliferyl-β-D-glucuronide (NA-MUG) and incubating 4 h at 35o

C. Production of 

blue fluorescence on the periphery of colonies under long wavelength UV indicated E. coli. Fecal 

streptococci were determined by incubation on m-Enterococcus agar (48 h at 35o

C). Colonies were 

transferred to brain heart infusion (BHI) agar. Transfers were made to BHI broth and incubated at 35o

C 

for 24 h, with confirmations made by retransfer to bile esculin agar, BHI broth incubated at 45o

C, and 

BHI with 6.5% NaCl. Growth on bile esculin agar BHI broth verifies that the colony is of the fecal 

streptococci group. Growth at 45o

C and in BHI with 6.5% NaCl indicates that the colony belongs to the 

enterococci group. 

Samples were sequentially diluted with sterile buffered water using three dilution factors based on 

previous analyses of similar samples. Dilution factors were estimated to obtain the method-recommended 

colony count on at least one dilution set. Sequential dilutions usually used at least 10 mL aliquots and 

always used at least 5 mL. All results were volume normalized to give concentrations in colony forming 

units (CFU) per 100 mL. 

 

Each analytical batch included laboratory blanks and positive controls. Blanks were run before and after 

each analytical set. Verification was performed on ten colonies for each organism according to the 

procedures listed in Standard Methods (APHA et al., 1998). After incubation, the plates were manually 

 3-6
 

enumerated. Positive controls showed the growth of particular indicator organisms. 

Data Analysis/Reduction 

The data analysis used all incubated plates with colonies in the countable range. The count from each 

incubated plate was normalized to the source concentration using the dilution factor and volume filtered. 

The uncertainty in each sample was estimated as the propagated error using the methods outlined by 

Taylor (1997). The dilution is assumed to be error free. The uncertainty in the filtered volume is 

estimated as ±0.4 mL, the tolerance of the ASTM class A graduated cylinders used in this study. The 

uncertainty in the number of counted colonies is estimated as 10% of the count with a minimum of 1 

colony. 

The sample weighted-average concentration and associated uncertainty was calculated using the 

uncertainty in the individual estimates as the weighting factor. This approach reduces the multiple (5 to 

12) results from the samples and dilutions associated with the original source (bottle) to a single 

concentration estimate for the organism representing the experimental result resulting from the 

environmental condition. Alternate pooling strategies for combining the multiple analyses can be 

developed. For example, the multiple dilutions from each sample could be pooled using the same 

weighting strategy to obtain either four or two estimates for each elapsed time. 

This data reduction relies on regression analysis of the concentration time series developed under the 

established experimental conditions. The approach estimates the effect of the various conditions 

(treatments). The treatments identified were based on the breadth of earlier-published research. No 

attempt was made to establish why the environmental exposure reduces the measured concentration, e.g., 

cellular wall degradation or DNA damage. Because of the reliance on the statistical techniques, it is 

important to prevent artificially increasing the degrees of freedom associated with the analysis that would 

suggest greater confidence in the results. The pooling approach described accomplishes the objective of 

not artificially increasing the degrees of freedom and placing greater emphasis on results with less 

uncertainty. Generally, the concentration time series developed for the individual experiments showed 

undetectably low concentrations for the final analyses. 

The weighted-average concentration for each organism was regressed on the independent variables using 

nonlinear least-squares regression techniques. The nonlinear regression method used the LevenbergMarquardt technique (a modified algorithm of the Gauss-Newton least-squares technique) in Statistica 

software package (version 7.1, Statsoft, Inc.). All regressions are run at the 95% level of confidence 

("=0.05). The reported uncertainty in the calculated coefficients is the confidence interval reported by 

the Statistica software package. After the regression was complete, an Analysis of Variance (ANOVA) 

was run to test the significance of the proposed model. 

Results 

Time and Temperature 

 

The regression used the proposed time-dependent decay function, known as Chick’s Law (equation (3-1). 

The concentrations at each elapsed time for a given temperature were used to estimate the inactivation 

rate constant for the indicator organism at that temperature. Table 3-3 lists the regression results from the 

temperature study with the 25o

C results from the light and salinity studies (discussed below). These 

experiments were also conducted in salt-free, dark, isothermal conditions and represent data that was 

included in the analysis. 

 3-7
 

Table 3-3. Inactivation Rate Constants for Each Indicator Organism at Tested 

Temperatures in the Isothermal Experiments 

Temperature 

(

o

C) 

Total 

Coliforms 

Fecal 

Coliforms E. coli Fecal Streptococci Enterococci 

Inactivation Rate Constants (h-1) 

9.07 0.007±0.010 0.03±0.025*∆ 0.027±0.015* 0.027±0.022*∆ 0.021±0.014 

19.87 0.017±0.035 0.01±0.140 0.085±0.033* 0.076±0.077 0.095±0.038* 

29.32 0.016±0.045 0.76±0.510* 0.136±0.072* 0.100±1.300◊ 0.150±0.420 

26.17 1 0.013±0.020 0.07±0.060* 0.019±0.072◊ 0.039±0.055 0.027±0.015* 

24.74 2 0.0131±0.37* 0.10±0.180 0.029±0.048 0.056±0.029* 0.044±0.038 

* Indicates KT value is statistically significant at α = 0.05 

Δ The first concentration is omitted from the analysis as an apparent outlier 

◊ ANOVA shows regression model is not significant 

1

Data from light experiment 

2

Data from salinity experiment 

In all but two cases, the post-regression ANOVA confirmed the model significance; however, in about 

half the cases the temperature-specific inactivation rate constant was not significant. This is taken to 

mean that these results do not provide a reason to reject the first-order decay model but that the data set is 

often not numerically sufficient to obtain quantitative estimates of the temperature-specific inactivation 

rate constant that excludes zero at the established level of confidence. This result generally occurred 

when the time series included few data triplets (time, temperature, concentration) leading to high 

uncertainty in the numeric values calculated. 

The results listed in Table 3-3 for the portion of the study emphasizing temperature generally demonstrate 

an increase in the calculated decay constant with increasing temperature. Completing the same analysis 

on the results from the subsequent investigations for the light and salinity studies at roughly 25o

C do not 

produce rate constants expected by interpolating between the bounding temperatures of 10 and 20o

C. The 

calculated values (Table 3-1) are not consistent for all organisms although the temperatures were within 

about 1.5o

C. These later two experiments used stormwater collected at the same outfall but at a different 

time. 

The experimental design planned to pool the results of the temperature study data across the experimental 

temperatures. The weighted-average concentration results were regressed on elapsed time using the same 

nonlinear procedures to test the model proposed by equation (3-3). As discussed above, the temperature 

study used a common stormwater source for the samples exposed to the selected temperature conditions. 

These samples had uniform starting concentrations allowing for the time delays in reaching time zero. 

The studies for light and salinity effects used different stormwater samples with dramatically different 

initial concentrations as would be expected from samples collected at different times of the year. To pool 

the data into a common set, the results were normalized to the estimated initial concentration using the 

calculated value of the initial concentration, Ĉo for the specific source calculated above. After 

transforming the data to Ct/Ĉo the above-described procedures were applied to estimate the reference 

temperature decay constant and the temperature coefficient using equation (3-9): 

20 20

ˆ

T K T t t

o

C

e

C

− − Φ = (3-9) 

Table 3-4 lists the regression coefficients. In all cases the temperature coefficient (ФT) and the reference 

decay constant at 20o

C (K20) are statistically significant. The post-regression ANOVA confirms the 

 3-8
 

significance of the regressions and significantly there is consistency of the individual coefficients across 

organisms. 

Table 3-4. Organism-Specific Reference Temperature Rate Constant and Temperature 

Coefficient Determined Using Salt-Free Dark Experimental Results 

Indicator Organism Reference Temperature 

Rate Constant (K20) (h-1) 

Temperature Coefficient

(ФT) 

Total Coliforms 0.016±0.009* 1.057±0.085*

Fecal Coliforms 0.042±0.030* 1.090±0.110*

E. coli 0.036±0.019* 1.023±0.072*

Fecal Streptococci 0.047±0.031* 1.044±0.040*

Enterococci 0.042±0.014* 1.057±0.045*

* Coefficient is statistically significant at α=0.05. 

The ФT values for all the organisms range between 1.02 and 1.09, which is consistent with the values 

reported in the literature. Mancini (1978) and Khatiwada and Polprasert (1999) suggested a value of 1.07 

for fecal coliforms which agrees with the calculated value in this work of 1.09±0.11. 

The DO monitoring showed a steady decline with time. The rate of decrease increased with temperature. 

The DO declined at 0.4, 1.4, and 1.7 mg/L/day at 10, 20, and 30o

C, respectively. At 30oC, the DO was 

nearly depleted after 60 h. At 20o

C, the DO was depleted after 70 h. At 10oC, DO was at 2.75 mg/L after 

72 h. Except for total coliforms, the plates produced non-quantitative counts within 23 h at 30o

C. This 

suggests the die-off is not due to depleted DO, but due to the combined effects of time and temperature. 

The pH of the samples varied slightly, but remained within the near- neutral range (6.5 to 7.0) throughout 

the experiment. Solic and Krstuvolic (1992) noted that fecal coliforms survived within the pH range of 6 

to 7, and declined outside of this range, with greater rate or mortality in acidic environments. The 

average TSS in the sample was 41 mg/L. These water quality indicators are within the range reported in 

the NSQD (Maestre and Pitt, 2005). 

Light 

The analysis assumes the light intensity measured at the sample surface is representative of the exposure 

throughout the container. The limited water depth in the selected container bottles supports this 

assumption. The analysis of the results of the light exposure experiments were examined in a two-step 

process. The weighted-average concentration was first used to calculate the overall coefficient under the 

established condition of light and temperature (KT=26.17,l) for each indicator at each exposure level using 

the model in equation (3-10). 

Kl T, t C Ce l o− = (3-10) 

Table 3-5 lists the regression-estimated values of KT=26.17,l for each indicator organism at each light level. 

The statistical quality of the fecal coliform and E. coli results is generally poor. The increased light 

intensity affects the calculated decay constant confirming that light influences the decay process. The 

calculated decay constant values increase with increasing light intensity. These results support the 

presumptive additive effect of light on the overall rate constant, i.e., KT,l = KT + Фll. 

Table 3-5. Regression-Estimated Values of Inactivation Rate Constants at 26.17oC for 

 3-9
 

Experimental Light Intensities 

Indicator Light Intensity (mW/cm2) 

Organism 0 20.86 55.23 94.7 

Total Coliforms 0.131±0.037* 0.21±0.16* 0.23±0.053* 0.32±0.12*

Fecal Coliforms 0.10±0.18 0.07±0.17◊ 0.08±0.25◊ 0.20±0.24 

E. coli 0.029±0.048 0.14±0.16 0.16±0.23 0.26±0.20*

Fecal Streptococci 0.056±0.029* 0.205±0.063* 0.254±0.022* 0.469±0.025*

Enterococci 0.044±0.038* 0.202±0.070* 0.19±0.15* 0.96±0.14*

* Coefficient is statistically significant 

◊ Regression result is not statistically significant at α=0.05 

The pooled data were then used to estimate the coefficients in the presumptive relationship: 

26.17 ( )

0

KT L I t C Ce t− +Φ = = (3-11) 

Table 3-6 lists the estimated values of the constants for each organism. The effect of light on the decay 

coefficient varies by a factor of four across the organisms showing a difference in light sensitivity. As 

expected, the values of KT=26.17,l generally agree with the values listed in Table 3-5 for the dark 

experiments. The values also agree with the expected estimated values using KT = K20Ф(T-20) evaluated at 

26.17o

C. The light-free exposure for this data set is included in the previous analysis. The added light 

has minimal effects on the decay rates for total and fecal coliforms. There is an order of magnitude 

smaller than the effects on the other indicators. 

Table 3-6. Regression-Estimated Coefficients from Light Experiment 

Indicator Organism Inactivation Rate 

Constant (KT=26.17) (h-1) 

Light Proportionality 

Coefficient (ФL) 

(cm2/mW-h) 

Total Coliforms 0.155±0.047* 0.0016±0.0012*

Fecal Coliforms 0.070±0.090 0.0130±0.0027 

E. coli 0.040±0.037* 0.0025±0.0019*

Fecal Streptococci 0.046±0.015* 0.0057±0.0018*

Enterococci 0.034±0.020* 0.0076±0.0038*

* Coefficient is statistically significant at α=0.05 

The intensity of natural sunlight varies during the course of the day. The exposure levels are further 

variable when considering the clouds that produce the rainfall and resulting runoff. The clouds will filter 

or block incident radiation to differing degrees. This work used artificially generated light to maintain 

constant exposure levels. Other researchers reported that sunlight showed the greatest bactericidal effect 

on organisms. Most research identifies UVB (290-320 nm), UVA (320-400 nm) and blue green visible 

light (400-550 nm) as the portion of the solar spectrum responsible for inactivating microorganisms. The 

UVB portion of the solar spectrum is believed to be the dominant bactericidal agent causing direct DNA 

damage (Sinton et al., 1999). For this reason, UV is used for disinfection in water and wastewater 

treatment processes (Ferguson et al., 2003; Giese and Darby, 2000). 

 

The pH of the samples varied between 6.5 and 7.5. Dissolved oxygen content of the samples varied 

between 8.2 and 12.3 mg/L. The difference in DO from the temperature study is noteworthy. The decline 

 3-10
 

observed in the temperature was not observed in this effort. The light exposure experiments lasted nearly 

50 h. Total suspended solids concentration in the sample was measured at 85 mg/L. While about double 

the concentration recorded in the temperature study, this is still in the typically reported range. 

Salinity 

The data analysis to establish the effects of salinity parallels the approach used to evaluate light effects. 

The individual time series were analyzed for each organism at each salinity level to obtain a preliminary 

assessment of the salinity effect. Table 3-7 lists the values of KT,S determined for each indicator organism 

at each salinity level. 

The effect of salinity is not consistent across indicator organisms. The rate constant for fecal streptococci 

is not significant for any of the salinity levels evaluated. The results for other organisms, e.g., fecal 

coliforms and enterococci, are generally significant. 

 

The results listed in Table 3-7 do not show a clear pattern for the salinity effect on the calculated decay

rate. Applying the technique proposed by Canteras et al. (1995) requires fitting the concentrations to the 

form: 

0

S KT s t C Ce t

− Φ = (3-12) 

Table 3-7. Regression-Estimated Values of Inactivation Rate Constants Determined for 

Salinity Concentrations 

Salinity (ppt) Indicator Organism 0 8 16 24 

Total Coliforms 0.013±0.020 0.039±0.022* 0.020±0.035 0.044±0.038* 

Fecal Colifroms 0.065±0.061* 0.031±0.020* 0.058±0.099 0.417±0.094* 

E. coli 0.020±0.072◊ 0.079±0.079* 0.010±0.110◊ 0.012±0.042 

Fecal Streptococci 0.039±0.054 0.016±0.079◊ 0.027±0.098◊ 0.027±0.089◊

Enterococci 0.027±0.015* 0.035±0.019* 0.005±0.011 0.019±0.008* 

* Coefficient is statistically significant at α =0.05*

◊ Regression result is not statistically significant at α=0.05 

Table 3-8 lists the regression results using the proposed relationship. Overall, the effect of increased 

salinity at the tested concentrations is small. The calculated value of ФS is not generally distinguishable 

from unity for organisms other than total and fecal coliforms. This suggests that, for the span of salinity 

values studied, the added salt had little effect on the decay rate constant. This supports the results 

reported by Canteras et al. (1995) who found largest salinity effect occurs only when the salinity values 

were over 35 ppt. Thoman and Mueller (1987) reported that the inactivation of fecal coliforms is 

generally much faster in marine and estuarine waters than in freshwater. Mancini (1978) also indicated 

that components in seawater in addition to salt may be responsible for greater inactivation effect in 

seawater. 

The pH of the samples varied between 7.0 and 8.0. Dissolved oxygen content of the samples varied 

between 8.3 and 12.2 mg/L. Total suspended solids in the sample were measured at 44 mg/L. 

 3-11
 

Table 3-8. Regression-Estimated Coefficients from Salinity Experiment 

Calculated Coefficients Indicator Organism KS (h –1) ФS

Total Coliforms 0.011±0.011 1.073±0.061*

Fecal Coliforms 0.035±0.023* 1.095±0.060*

E. coli 0.024±0.041 0.990±0.100*

Fecal Streptococci 0.025±0.032 0.998±0.086*

Enterococci 0.032±0.016* 0.957±0.036*

* Coefficient is statistically significant at α =0.05 

Summary 

The results of this experiment demonstrated that the concentration of the tested indicator organisms

decrease exponentially with time. The first-order decay process reasonably models the concentration time 

series for the durations tested. The analysis of the weighted-average concentrations enabled developing 

organism-specific inactivation rate constants in stormwater assuming time, temperature, light intensity 

and salinity are the most significant parameters. 

The temperature study indicated that the indicator organisms persisted at higher concentrations at lower 

temperatures. The inactivation rates increased with increasing light intensity. The added light has 

minimal effects on the inactivation rates for total and fecal coliforms. Different indicator organisms 

exhibited different trends with salinity. Taken as a whole, the results indicate that salinity had little or no 

effect on inactivation rates for these indicator organisms for the salinities tested. 

The difference in temperature results from the temperature study suggests that differences in the 

stormwater influence the reference decay rate. The major measured difference in the characterization of 

the stormwater was the initial concentration of indicator organisms. This further suggests that the 

constants measured in the bench-scale experiments must be viewed as the rate for the specific stormwater 

sample evaluated and cannot be extrapolated to all stormwater sources. The variability of the constants 

between sources, if any, cannot be estimated from these data. 

 3-12
 

Chapter 4 Pilot-Scale Research 

This project was designed to determine the factors that influence the rate of microbial inactivation as 

urban stormwater passes through retention ponds and constructed wetlands. Research on constructed 

wetlands inactivation of fecal indicators in wastewater is well documented (Bavor et al., 1987; Gersberg 

et al., 1987; Ottová et al., 1997). Removals of fecal streptococci and coliforms generally exceeded 80% 

and 90%, respectively (Kadlec and Knight, 1996). Gersberg et al. (1987) and Garcia and Bécares (1997) 

concluded that extensively vegetated systems remove indicator bacteria at significantly higher rates from 

wastewater than unvegetated systems. However, because of the potentially high indicator bacteria 

concentrations in stormwater runoff, the remaining 10 - 20% (assuming a performance of 80-90% is 

achieved) in the effluent may increase receiving water concentrations beyond WQS. This is in contrast to 

sanitary and combined stormwater and sanitary systems which, other than during sewer overflow, 

chemically treat the wastewater routed to treatment plants. 

This pilot-scale project intended to build on the bench-scale studies to evaluate the variation of rates of 

inactivation using first-order decay with changes in environmental conditions. The mesocosms designed 

and constructed for this project offer a unique environment where many parameters concerning 

stormwater characterization and flow can be held constant (i.e., characteristics of influent, residence time, 

and pollutant loading). By varying testing dates with typical climatic conditions experienced throughout 

the year, an assessment of the impact of environmental change on bacterial inactivation rates can be 

assessed. A comparison of rates of inactivation with seasonal wet weather events can indicate whether 

water quality managers can rely on model predictions to be accurate during periods when loading may be 

greatest. More information is needed to determine whether models that use first-order decay functions 

when predicting bacteria effluent concentrations from field BMPs (usually as a point source) are 

accurately providing effluent concentration predictions and concomitant loads. This can directly impact 

the loading allowed when determining TMDLs for meeting WQS. 

Study Site and Experimental Design 

A pair of rectangular mesocosms of the same size with two different stormwater BMP treatments 

(constructed wetland and retention pond) were constructed at the Urban Watershed Research Facility 

(UWRF) in Edison, New Jersey (see Figure 4-1). Mesocosm housings were purchased fiberglass 

aquaculture tanks. Mesocosms were constructed by placing a perforated PVC under drain with valve, 

about 4 cm of pea gravel, 2.5 cm of sand, and 25 cm of topsoil. Local cattail plants were transplanted into 

constructed wetland mesocosms. The retention ponds were constructed similarly with like layers and 

depths of gravel, sand, and topsoil, but with no plantings. Tanks had a length, width, and depth of 1.78 

 4-1
 

meters, 0.74 m, and 0.65 m, respectively with a stormwater volume of approximately 227 L. Both 

systems were constructed in August of 2002. 

Creating Bacterially Loaded Stormwater 

Feed concentrations in the stormwater runoff collected on site were low (101-103 CFU/100 mL) compared 

to many urban watersheds. To achieve a higher loading concentration (104

-106 CFU/100 mL) for this 

study, a 500 mL aliquot of stormwater runoff was collected from on-site runoff as described in Chapter 3 

and placed in growth media to encourage growth of the desired bacteria, consequently producing higher 

densities of bacteria in the stormwater. Complete method development is described in Appendix A. 

(A) 

Figure 4-1. Pictures of the retention pond (A) constructed wetland (B) treatment systems. 

(B) 

Target indicator bacteria concentrations in mesocosms following addition of the enriched stormwater are 

found in Table 4-1. The cultured stormwater was introduced over a 30-45 min time period (in an attempt 

to limit thermal shock) to a common supply tank that contained approximately 1000 L of recently 

captured stormwater and mixed for 30 min. Constructed wetland and retention pond mesocosms were 

filled from this same supply source. 

Table 4-1. Target Indicator Organism Densities in Mesocosms after Addition of Enriched 

Stormwater 

Target Concentration Indicator Organism (CFU/100 mL)

Total Coliforms 106 – 107

Fecal Coliforms 106 – 107

E. coli 104 – 105

Enterococci 104 – 105

 4-2
 

It is recognized that the relative proportion of indicator bacteria in the bacterially enriched stormwater 

will change from the original stormwater and may be less representative of the true indicator bacteria 

community. Another recognized difference in the bacterially enriched stormwater is the particle 

association of the indicator bacteria is suspected to be different than the source with less bacteria 

associated with larger particles and more bacteria associated with finer particles or unattached. Although 

this was not measured, it is believed that this property results in a more conservative estimate for the 

indicator organisms when using bacterially enriched stormwater for indicator bacterial loading 

experiments. The thought is fewer colonies are expected to settle out of the water column through 

attachment to larger particles resulting in increased effluent concentrations compared to the stormwater 

source. 

Storm Event Simulation 

Runoff events were simulated by distributing 220 L of collected stormwater runoff from a common 

supply tank that was mixed for 30 min using four vortex mixing eductors to each mesocosm. Additions 

were made sequentially at an average rate of 0.75 L/s measured using a Seametrics WT-P turbine meter 

(SeaMetrics, Kent, WA). At this stormwater discharge rate, it took approximately 5 min to load the BMP 

mesocosms. Using the same method and equal volume as in the mesocosms, cultured stormwater was 

distributed to a 227 L container placed near the mesocosms serving as a control tank. This tank, based on 

proximity, was expected to have the same ambient environmental conditions as the pilot-scale tanks. 

Effluent flow rates and hence detention times in the mesocosms were regulated by the effluent pipe 

orifice (diameter 2 mm) simulating a riser flow control structure as in field BMPs. Mean effluent flow 

rates ranged from 57 – 76 L/hr during the loading event until levels return to their pre-event (static) 

levels. Generally, it took between 20-22 h for water levels to return to their original pre-event level. 

Between simulated storm events, and after drawdown of stormwater to a static pre-event level during 

simulated storm events, mesocosm water levels were maintained through semi-continuous flow from a 

nearby water supply regulated by a float valve positioned opposite and slightly above the effluent orifice. 

Residence times of the mesocosms were previously determined by Struck et al. (2004) using conservative 

dye tracer tests to determine the residence time distribution with Rhodamine WT dye distributed to each 

mesocosm. Tracer concentration in effluents were measured with a YSI Rhodamine WT and verified 

with a Turner Designs 10 AU field flourometer (Sunnyvale, CA). Exponential curves were fit to tracer 

concentration data to calculate the measured mean residence time (Levenspiel, 1999). 

Storm events were planned over a two-year period with three simulated storm events each year. No event 

was to occur within the same month. The dates selected represented typical climatic conditions during 

that time to incorporate changing environmental conditions as a factor in bacterial inactivation. 

Water Quality Monitoring, Solids, Light, and Bacterial Indicator Sampling 

The constructed wetland, retention pond, and control tank each had a YSI water quality sonde placed on 

the sediment surface at a depth of 4 cm in the constructed wetland and 25 cm in the retention pond near 

the overflow orifice. These sondes recorded in-situ temperature, DO, pH, conductivity, and turbidity 

averaged over 10-minute intervals. 

Light intensity was measured using an on-site weather station (Onset Corporation, Bourne, MA). Grab

samples of light were also recorded on six separate occasions between 12:00pm and 3:00pm at the water 

surface in the retention pond and constructed wetland using a hand held light photometer (IL1400, 

International Light Inc., Newburyport, MA). 

 4-3
 

Effluent from the riser pipe of the retention pond and constructed wetland was collected in pre-washed 1-

L HDPE bottles placed at the effluent drainpipe (to collect enough volume for the sample but subject to 

continuous replacement). Microbiological and TSS samples were collected from these 1-L containers 

using automatic samplers (Hach Company, Loveland, CO) and placed into cooled pre-washed 1-L HDPE 

bottles within the sampling device at elapsed times found in Table 4-2. Timed samples were also 

collected at a depth of 5 cm below the water surface in the control tank and labeled as “light controls”. If 

grab samples were necessary due to autosampler failure discrete samples using pre-cleaned PVC bottles 

were collected to obtain the desired sample. 

Six 500-mL bottles were wrapped in aluminum foil filled with 400 mL of inoculated stormwater, sealed, 

and placed in the control container to duplicate environmental conditions (other than sunlight) in the 

mesocosms and control tank. These samples were collected daily and with the extended times (beyond 90 

h) as noted in Table 4-2, as “dark controls” along with the “light” control samples collected from the 

control container. 

These samples separate light and dark affects on the microbial population in the control groups. It should 

be noted that sample #1 in Table 4-2 (time 0) was collected 30 min before stormwater addition for the 

constructed wetland and retention pond recording antecedent baseline conditions. Stormwater controls 

were then loaded and sampled with sample #1 (time 0) representing the influent concentrations and 

conditions of the supply tank and the dark and light controls. 

 

Table 4-2. Effluent Time of Hand Collected and Programmed Autosample Collection 

Sample # h Sample Collected 

1 0 influent, light control, in-situ (background) 

2 5 timed effluent 

3 10 timed effluent 

4 15 timed effluent 

5 21 timed effluent 

6 27 timed effluent, light control, dark control 

7 33 timed effluent 

8 39 timed effluent 

9 45 timed effluent, light control, dark control 

10 51 timed effluent 

11 60 timed effluent, light control, dark control 

12 69 timed effluent 

13 78 timed effluent 

14 90 timed effluent, light control, dark control 

15 114 timed effluent, light control, dark control 

16 150 timed effluent, light control, dark control 

All samples were transferred to appropriate sized plastic pre-cleaned containers for analyses depending on 

the type of analyses and the source of the sample. Samples were transported to the laboratory for 

splitting, filtering, and preservation as necessary within specified holding times. If storage was necessary, 

samples were stored in a refrigerator at 4oC. 

Bacteria indicator samples were analyzed for four indicator organisms (total and fecal coliforms, 

enterococci, and E. coli) following the standard procedures listed in Chapter 3 (Table 3-2). Fecal 

streptococcus analyses were not done because the increased quantity of samples this indicator added was 

beyond laboratory capabilities. 

 4-4
 

The expected indicator concentration in the effluent for total and fecal coliform, enterococci, and E. coli

was 104

 to 106 CFU/100 mL during the first sampling period. Each timed sample was mixed and split 

into three equal volumes of sample for each organism. Three dilutions of each split sample were analyzed 

to assure at least one dilution provided concentrations between 5 and 200 colonies per plate. These 

ranges were expected to shift as the concentrations of indicator bacteria decrease with time (Table 4-3). 

Automatic samplers were programmed to collect two additional 1-L samples 15-20 min following the first 

timed sample, allowing enough time for refilling of the effluent collection bottle. These samples served 

as additional samples for TSS analysis and secondary indicator bacteria samples in the event of an error in 

the first programmed sample collection. 

Table 4-3. Expected Beginning Densities After Loading and Expected Dilution Factors 

Indicator Organism Expected Density 

(CFU/100 mL)

Dilution

Retention Pond Mesocosm 

Total Coliforms 103 – 105 102,103,104

Fecal Coliforms 103 – 105 102,103,104

E. coli 102 – 104 101

,102,103

Enterococci 102 – 104 101,102,103

Constructed Wetland Mesocosm

Total Coliforms 104 – 106 103

,104,105

Fecal Coliforms 104 – 106 103,104,105

E. coli 104 – 106 103

,104,105

Enterococci 103 – 105 102,103,104

Sediment Sampling Procedures 

Mesocosms were divided into four spatial areas to randomize sampling location and avoid spatial biases. 

Four samples were collected from each section in the retention pond and constructed wetland systems. 

Sediments were collected using a clear cylinder (modified syringe) inserted about 5 cm into the 

undisturbed sediments and capped at one end. After removing the sediment core from the mesocosm, the 

core was transferred to a centrifuge tube. Sediment samples were mixed using a sterile spatula. Samples 

were then analyzed using multiple-well fermentation tests for enterococci and E. coli (and total coliforms)

using Enterolert® and Colilert®, respectively, manufactured by IDEXX Laboratories (Westbrook, ME). 

This method was selected instead of membrane filtration because of the difficulty in counting colonies 

with obstructing sediment and due to the potential of growth inhibition by accumulated sediments on the 

filter paper. 

Predation Sampling Procedures 

Laboratory experiments have suggested that ingestion rates should increase with food concentration and 

 4-5
 

that feeding preference should be shown for the most abundant of available foods. Analysis of larger 

macroinvertebrates was the most practical method to determine the potential of predation on bacteria 

populations, due to the time and cost of other methods. Identification and enumeration of 

macroinvertebrates as small as 0.5 mm were done on 5 samples each from the retention pond and 

constructed wetland to determine the populations of either bacteriovores (bacteria eating predators) or the 

presence of macroinvertebrates. The guides Freshwater Macroinvertebrates of Northeastern North 

America (Peckarsky et al., 1990) and An Image-Based Key to the Zooplankton of the Northeast 

(University if New Hampshire, 2005) were used for identification of invertebrates. A top-down approach, 

looking at the densities of macroinvertebrates including those from a higher trophic level (especially 

bacteriovores), was used to qualitatively assess the bacteria concentrations based on the presence and 

quantity of bacteria-eating organisms. The assumption is that an increase in the abundance of bacteria 

would also lead to an increase in the predators that use bacteria as a major food source. Ideally, when 

using this monitoring approach, one would take samples before, during, and after each storm event. 

However, because of the enormous analyses requirements to identify and enumerate the 

macroinvertebrates, as well as notice of recruitment of macroinvertebrates midway through the study, 

macroinvertebrate data are limited to a single sampling event. 

Data Management 

The volume of data for each simulated storm event was large. Much of the continuous data was placed 

into spreadsheets for geometric means and statistical calculations based on time and storm event. For the 

purposes of comparing the physical and environmental characteristics data was averaged over the time 

between each sample collection as noted in Table 4-2. Bacteria indicator organism concentration data 

required some synthesis to calculate sample time averages and inactivation rates as noted in Chapter 3. 

Since indicator bacteria samples were split into three samples and then three separate dilutions, it was 

possible to have up to 12 indicator concentrations with each organism for each timed sample. This was 

usually not the case as often one or more of the three dilutions would result in either values of zero or 

TNTC (plates containing over 200 colonies) as designed to bracket the actual culturable colony-forming 

units. All plates were enumerated, with the exception of those designated as TNTC. For each split 

sample, dilutions with plates containing 1-200 organisms were used for data analysis. Each split sample 

was averaged across dilutions. The three split samples for each sample collection time (total of 16 times) 

were log transformed and regressed with time. Uncertainty was calculated as in Chapter 3. Each 

simulated storm event produced more than 192 data points for indicator bacteria indicators. 

Statistical Analyses 

Sample sizes were large enough to perform statistical comparisons (ANOVA and correlation analyses) 

between constructed wetland, retention pond treatment practices, dark and light control inactivation rates, 

and physical and chemical characteristics. Likewise, sample size allowed for regression analyses of the 

bacterial indicator concentrations on elapsed time between the constructed wetland, retention pond, and 

dark and light controls for the each stormwater event. Slopes of the regressions gave inactivation rate 

constants, K, for each event. Chemical parameters were compared between the treatments and controls 

using correlation matrices. All statistical analyses that have significant p-values and α = 0.05 were noted. 

Results 

Physical and Chemical Properties of the Pilot-scale Systems

Physical and chemical parameters measured in the study are listed in Table 4-4. Water temperatures 

averaged 2.15oC less in the constructed wetland compared to the retention pond. This difference was 

 4-6
 

likely due to shading from the macrophytic vegetation (Typha latifolia, average stem density = 39.3 

stems/m2

). This temperature difference was more notable in the warmer sampling events (difference of 

3.08oC and 1.82oC, respectively) compared to the colder sampling events (difference of 0.46oC). 

DO was higher in the retention pond compared to the constructed wetland; the May, July, and September 

events had the highest values. The process of decomposition of organic matter in the constructed wetland 

can consume some of the DO, causing low concentrations (sometimes near 0 mg/L) during periods of 

increased decomposition. Increases in decomposition are temperature-related with a positive correlation 

between the two. Also, diurnal fluctuations in DO and temperature were reduced during initial storm 

event loading. Values for these parameters did not generally reach pre-event diurnal fluctuations until 

after 48 h of detention for most events. Conductivity was nearly the same in the two systems while pH 

was circumneutral to alkaline in the retention pond but tended to be acidic in the constructed wetland. 

This pattern was noted by Mitch and Gosselink (2000) in constructed wetlands with mineral soils and 

lake sediments by Stumm and Morgan (1996). These differences were attributed to the organic matter 

build-up in sediments and decomposition which tends to make the pH less than 7. The ORP was much 

less (and often negative) in the retention pond compared to the constructed wetland system. The depth of 

inundation of the free water in the retention pond was generally three times that of the constructed 

wetland. This would substantially increase the potential for more reducing conditions through both 

reduced oxygen diffusion with water depth and the lack of photosynthetic oxygen production with 

absence of macrophytic vegetation, resulting in lower ORP values in the retention pond. Light intensity 

in the constructed wetland was consistently 9-10% of that measured in the retention pond. The difference 

in recorded hand-held light intensity for each event was used to adjust the measured irradiance for the 

light control and retention pond to calculate a corrected irradiance expected at the surface of the 

constructed wetland to make irradiance values between pilot-scale systems comparable. 

Most storm events had maximum initial TSS and turbidity values of less than 100 mg/L and 150 NTUs, 

respectively, after stormwater loading to the retention pond and constructed wetland. As expected, 

turbidity and TSS values decreased with time in each system. Geometric mean turbidity values for 

sampling events before October 2005 are shown in Figure 4-2. Turbidity values were averaged for each 

time step and then over each sampling event. 

Retention Pond Constructed Wetland 

Figure 4-2. Mean turbidity in the retention pond and constructed wetland in all storm 

events except October 2005. Whiskers indicate 95% confidence intervals. 

0 10 20 30 40 50 60 70 80 90 10

Time (h)

0

0

10

20

30

40

50

60

Turbidity (NTUs)

0 10 20 30 40 50 60 70 80 90 10

Time (h)

0

0

10

20

30

40

50

60

Turbidity (NTUs)

 4-7
 

Table 4-4. Average Event in-situ Physical and Chemical Results 

Retention Pond Constructed Wetland 

Date Parameter 

Valid 

N Mean Minimum Maximum 

Standard 

Deviation 

Standard 

Error 

Valid 

N Mean Minimum Maximum 

Standard 

Deviation 

Standard 

Error 

Temp (oC) 36 27.0 21.9 32.2 2.9 0.5 36 25.5 22.2 29.4 2.2 0.4 

Cond (mS/cm) 36 0.316 0.296 0.345 0.016 0.00261 36 0.284 0.265 0.316 0.014 0.002 

D0 (mg/L) 36 5.8 1.9 11.4 2.6 0.4 36 3.6 0.8 9.2 2.3 0.4 

pH 36 8.0 7.3 9.1 0.6 <0.1 36 6.7 6.6 7.0 0.1 <0.1 

ORP (mV) 36 364 200 465 93 15 36 465 317 529 67 11 

Turbidity (NTU) 36 11.6 5.1 31.1 5.7 0.9 36 10.3 6.5 23.8 4.8 0.8 

Jun-04 

Irradiance (kJ/m2

) 12 43.9 <0.1 139.1 48.9 14.1 12 4.1 <0.1 12.8 4.5 1.3 

Temp (oC) 36 22.8 18.7 27.3 2.8 0.5 42 19.4 16.1 22.1 1.8 0.3 

Cond (mS/cm) 36 0.217 0.196 0.237 0.011 0.002 42 0.204 0.190 0.237 0.013 0.002 

D0 (mg/L) 36 8.5 5.8 11.5 1.9 0.3 42 4.1 0.7 13.4 3.6 0.5 

pH 36 7.9 7.5 8.3 0.2 <0.1 42 6.4 6.3 6.7 0.1 <0.1 

ORP (mV) 36 -288 -354 -15 95 16 42 555 524 591 19 3 

Turbidity (NTU) 36 11.0 7.9 20.7 3.2 0.5 42 6.6 3.7 18.5 3.8 0.6 

Sep-04 

Irradiance (kJ/m2

) 12 31.6 <0.1 125.0 45.1 12.0 14 3.4 <0.1 11.5 4.7 1.3 

Temp (oC) 42 11.1 5.6 16.1 3.3 0.5 42 10.1 4.6 15.9 3.2 0.5 

Cond (mS/cm) 42 0.189 0.159 0.216 0.018 0.003 42 0.179 0.130 0.208 0.021 0.003 

D0 (mg/L) 42 9.5 6.2 13.9 2.0 0.3 42 9.0 2.3 14.3 4.1 0.6 

pH 42 7.2 7.0 7.3 0.1 <0.1 42 6.3 6.1 6.8 0.2 <0.1 

ORP (mV) 42 -285 -322 -211 29 4 42 403 376 452 19 3 

Turbidity (NTU) 42 7.8 4.0 11.5 1.9 0.3 42 8.4 0.4 27.6 6.5 1.0 

Nov-04 

Irradiance (kJ/m2

) 14 19.0 <0.1 84.4 26.5 7.1 14 1.9 0.0 7.8 2.4 0.7 

Temp (oC) 33 19.9 16.6 27.0 3.2 0.6 39 17.1 15.3 22.3 1.9 0.3 

Cond (mS/cm) 33 0.447 0.394 0.555 0.047 0.008 39 0.680 0.585 0.771 0.052 0.008 

D0 (mg/L) 33 10.5 8.5 15.6 1.8 0.3 39 1.4 0.1 4.4 1.2 0.2 

pH 33 8.3 7.3 9.3 0.5 0.1 39 6.8 6.8 7.0 0.1 <0.1 

ORP (mV) Not Recorded 

Turbidity (NTU) 33 1.8 <0.1 11.2 2.8 0.5 39 4.4 2.5 12.0 2.4 0.4 

May-05 

Irradiance (kJ/m2

) 12 26.9 <0.1 119.0 40.2 11.1 13 2.6 <0.1 17.4 5.6 1.6 

Temp (oC) 24 26.7 24.0 31.2 2.2 0.4 24 24.5 23.1 27.1 1.3 0.3 

Cond (mS/cm) 24 0.253 0.235 0.297 0.018 0.004 24 0.259 0.197 0.371 0.054 0.011 

D0 (mg/L) 24 5.5 3.0 9.5 1.9 0.4 24 1.3 0.1 3.7 1.3 0.3 

pH 24 7.5 7.2 8.2 0.3 0.1 24 6.4 6.2 6.6 0.1 <0.1 

ORP (mV) Not Recorded 

Turbidity (NTU) 24 26.0 6.5 92.0 20.2 4.1 24 61.0 13.5 167.2 50.9 10.4 

Jul-05 

Irradiance (kJ/m2

) 8 29.4 <0.1 119.0 40.3 14.3 8 2.7 <0.1 10.9 3.7 1.3 

Temp (oC) 48 14.6 12.6 17.6 1.6 0.2 48 14.7 11.6 18.4 1.7 0.3 

Cond (mS/cm) 48 0.156 0.066 0.240 0.048 0.007 48 0.184 0.092 0.273 0.046 0.007 

D0 (mg/L) 48 3.2 <0.1 6.3 1.6 0.2 48 2.8 <0.1 9.1 3.0 0.4 

pH 48 7.0 6.1 7.5 0.4 0.1 48 6.0 5.8 6.5 0.1 <0.1 

ORP (mV) Not Recorded 

Turbidity (NTU) 48 849.1 <0.1 1236.5 396.9 57.3 48 937.4 11.2 2141.4 782.5 112.9 

Oct-05 

Irradiance (kJ/m2

) 16 18.7 <0.1 135.3 39.8 10.0 16 1.9 <0.1 12.4 3.7 0.9 

 4-8
 

The October 2005 experimental run had starting TSS values averaging 2,999 mg/L, and turbidity values 

averaging 2,173 NTUs, which is near the maximum of the range expected for most stormwater runoff. 

Active construction in the watershed was evident in the stormwater runoff during this sampling event. 

Inclusion of this stormwater runoff greatly increases the variability in solids concentration, overwhelming 

the smaller concentrations found in the previous runoff events. Thus some analyses occurred with the 

exclusion of this event as noted. 

Bacteria Indicator Organisms 

Samples were not analyzed for enterococci in the first experiment (June 2004) but resumed for all 

subsequent events. Graphs showing the relationship of bacterial indicator organism concentrations for the 

constructed wetland and the retention pond with event physical and chemical parameters can be found in 

Figures 4-3 through 4-5. Conductivity and DO did not appear to significantly effect bacterial 

concentrations over the ranges observed (Figure 4-3). ORP may have moderately affected fecal coliforms 

and E. coli concentrations around 200 mV in the retention pond while densities of fecal coliforms, E. coli, 

and enterococci decreased between 500 and 600 mV in the constructed wetland, but only three events 

were monitored for this parameter. Densities of fecal coliforms and E. coli decreased above a pH of 8.5 

in the retention pond but remained unaffected over the range of observed pH values in the constructed 

wetland (Figure 4-4). Temperature appears to affect bacterial indicator organism concentrations in the 

retention pond. The optimal temperature range that resulted in the greatest number of observed bacteria 

colony forming units was between 11oC and 26oC. A similar trend was noticed in the constructed wetland 

for a temperature range between 11o C and 23oC (Figure 4-5). 

There was a distinct relationship with concentration of all indicator organisms with turbidity in this 

experiment. While there was much scatter in concentrations at turbidities less than 20 NTU, at turbidities 

greater than 100 NTUs there was a predictable increase in bacteria organism concentrations with 

increasing turbidity in both the retention pond and constructed wetland (Figure 4-6). Similar results have 

been shown by the United States Geological Survey (USGS) in larger rivers in northern and central 

Virginia as well as by the USEPA in smaller streams in northern Virginia (Hyer and Moyer, 2003; Struck 

et al., 2006). These solids can potentially affect rates of bacteria attenuation as discussed in Chapter 5. 

Overall inactivation rates for all simulated storm events are shown in Table 4-5. Significant differences 

were observed between the constructed wetland and retention pond in eight of the bacteria indicators for 

the six runoff events. The retention pond had six of these with inactivation rates for: total coliforms in 

June and July; E. coli in May; fecal coliforms in July and enterococci in May and November significantly 

larger than in the constructed wetland. However, the constructed wetland had a significantly larger 

inactivation rate compared to the retention pond for fecal coliforms in June and enterococci in July. Both 

treatments had greater inactivation rates compared to light and dark controls in September and November 

for total coliforms, E. coli, and fecal coliforms. Retention pond inactivation rates were also greater than 

controls for total coliforms in June and July, for E. coli in May and July, and fecal coliforms in May, 

June, and July while constructed wetlands rates were greater for E. coli in July, fecal coliforms in May 

and June, and enterococci in July. Light controls were greater than dark controls in 9 instances, including 

May and June for total coliforms and E. coli, July for fecal coliforms, and May, July, September, and 

November for enterococci. This indicates that light does have an impact on bacteria indicator organisms. 

Bacteria indicator concentrations decreased with time for the retention pond and constructed wetland 

(Figure 4-7). The exponential regression coefficients indicate the inactivation rate. A two step process of 

generating an overall inactivation value for each bacterial indicator organism from 0-50 h and from 50-

100 h was used to generate a best fit relationship. This timeframe was determined by maintaining R2

values of regressions greater than 0.70 while varying the time interval between 0 and 100 until the 

difference in slope (inactivation rate) was maximized for the majority of the bacteria indicator organisms. 

 4-9
 

 4-10

Table 4-5. Inactivation Rates for the Constructed Wetland, Retention Pond, and Dark and 

Light Controls for all Indicator Bacteria Organisms for each Sampling Event 

 Retention Pond Constructed Wetland 

Total Fecal Total Fecal 

Month Year Coliforms E. coli ColiformsEnterococci Coliforms E. coli ColiformsEnterococci

 (h-1) (h-1) 

June 2004 0.2419*+ 0.1484 0.1814+ 0.1529 0.1651 0.3277*+

September 2004 0.144+ 0.1164+ 0.1192+ 0.2030 0.1204+ 0.1204+ 0.1515+ 0.1786 

November 2004 0.1653+ 0.1164+ 0.1485+ 0.1730* 0.1235+ 0.1157+ 0.1137+ 0.1245 

May 2005 0.0949 0.3350*+ 0.1417+ 0.1717* 0.1090 0.0919 0.1233+ 0.0852 

July 2005 0.1811*+ 0.1957+ 0.2610*+ 0.1240 0.0733 0.1894+ 0.1025 0.2112*+

October 2005 0.0437 0.0524 0.0566 0.0512 0.0427 0.0597 0.0536 0.0594 

 

Dark Control Light Control 

Total Fecal Total Fecal 

Month Year Coliforms E. coli Coliforms Enterococci Coliforms E. coli Coliforms Enterococci

 (h-1) (h-1) 

June 2004 0.0247 0.0276 0.0249 0.1390♦ 0.1502♦ 0.0242 

September 2004 0.0700 0.0563 0.0527 0.0773 0.0588 0.0789 0.0760 0.2027♦

November 2004 0.0815 0.0480 0.0445 0.0711 0.0658 0.0724 0.0692 0.1787♦

May 2005 0.0258 0.0725 0.0514 0.0351 0.0679♦ 0.1158♦ 0.0828 0.0884♦

July 2005 0.0637 0.0509 0.0619 0.0944 0.0720 0.0712 0.1136♦ 0.1681♦

October 2005 0.0538 0.0605 0.0514 0.0194 0.0676 0.0862 0.0822 0.0316 

* Indicates a significantly higher value between retention pond and constructed wetland 

+ Indicates a significantly higher value between retention pond or constructed wetland values and control values 

♦ Indicates a significantly higher value between light and dark control values

This method used R2 values (Figure 4-7) of the whole timeframe compared to 0-50 and 50-150 h partial 

timeframes. In all instances, the R2 values improved when dividing the duration of the experiment into 

the two timeframes, suggesting that inactivation rates vary as a function of time with greater rates of 

inactivation during the first 50 h timeframe compared to the second 100 h timeframe. 

Fecal coliforms and enterococci in the retention pond were an exception to this generalization. Several of 

the inactivation rates during the 50-150 h timeframe had values nearing zero suggesting that these 

organisms may have reached or nearly reached background concentrations after 50 h. This is supported 

by the average pre-event background concentrations in the retention pond and constructed wetland found 

in Table 4-6. 

Bacteria Concentrations in Sediment 

Results from the sediment bacteria indicator organism concentrations collected and analyzed one day 

before and two days after the November 2004 storm event are shown in Table 4-7. Sediment bacteria 

increased substantially for total coliforms and E. coli after the storm event. However, concentrations of 

enterococci decreased somewhat over the experiment. The bacteria indicator organisms measured before 

the experimental run are considered as background concentrations as the previous input of indicator 

organisms through stormwater runoff was two months prior to this event. 
Retention Pond Constructed Wetlan

 TCConc ECConc FCConc EntConc

0.0 0.1 0.2 0.3 0.4 0.5 0.6

5E+005E+015E+025E+035E+045E+055E+065E+07

0.0 0.1 0.2 0.3 0.4 0.5 0

5E+005E+015E+025E+035E+045E+055E+065E+07

Conductivity (mS/cm) Conductivity (mS/cm) 

Log 10 Concentration (CFU/100 mL) 

 TCConc ECConc FCConc EntConc

0 2 4 6 8 10 12 14 16

5E+005E+015E+025E+035E+045E+055E+065E+07

0 2 4 6 8 10 1

5E+005E+015E+025E+035E+045E+055E+065E+07

Dissolved Oxygen (mg/L) 

Log 10 Concentration (CFU/100 mL) 

Dissolved Oxygen (mg/L

Figure 4-3. Effluent concentration of indicator organisms with conductivity and dissolved oxygen.  4-11
Retention Pond Constructed Wetland 

Log 10 Concentration (CFU/100 mL) TCConc ECConc FCConc

 EntConc

-400 -200 0 200 400 600

5E+005E+015E+025E+035E+045E+055E+065E+07

Log 10 Concentration (CFU/100 mL) 

ORP (mV) 

-400 -200 0 200 

5E+005E+015E+025E+035E+045E+055E+065E+07

ORP (mV) 

pH 

 TCConc ECConc FCConc

EntConc

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

5E+005E+015E+025E+035E+045E+055E+065E+07

pH 

5.5 6.0 6.5 7.0 7.5 8.0 

5E+005E+015E+025E+035E+045E+055E+065E+07

Figure 4-4. Effluent concentrations of indicator organisms with ORP and pH.  4-12
Figure 4-5. Effluent concentrations of indicator organisms with temperature 

 TCConc ECConc FCConc EntConc

200

400

600

800

1000

1200

1400

5E+005E+015E+025E+035E+045E+055E+065E+07

200

400

600

800

1

1

1

000

200

400

1600

5E+005E+015E+025E+035E+045E+055E+065E+07

Log 10 Concentration (CFU/100 mL) 

Turbidity (NTU) Turbidity (NTU)

TCConc = 1946.3288*exp(0.0046*x) ECConc = 377.4225*exp(0.0058*x) FCConc = 873.1594*exp(0.0053*x) EntConc = 2127.9909*exp(0.0058*x) 

TCConc = 23532.7643*exp(0.0018*x) ECConc = 2892.7463*exp(0.0026*x) FCConc = 9072.4396*exp(0.0022*x) EntConc = 7198.5976*exp(0.003*x) 

0 5 10 15 20 2

5E+005E+015E+025E+035E+045E+055E+065E+075E+085E+095E+105E+11

Constructed Wetland 

 TCConc ECConc FCConc EntConc

0 5 10 15 20 25 30 35

5E+005E+015E+025E+035E+045E+055E+065E+075E+08

Retention Pond 

Tem

perature (C°)

Log 10 Concentration (CFU/100 mL) 

Temperature (C°) 

Figure 4-6. Effluent indicator bacteria concentrations with in-situ turbidity.  4-13
Organism 

KT 0-50hr 

(h-1) R

TC -0.1164 0.95EC -0.0853 0.96FC -0.1797 0.38ENT -0.1142 0.82

Organism 

KT 0-50hr 

(h-1) R2

KT 50-150hr (h-1) R2

TC -0.1308 0.895 -0.0044 0.937 EC -0.0534 0.628 -0.0369 0.876 FC -0.0440 0.464 0.0547 0.956 ENT -0.0044 0.765 -0.0672 0.937 

1.E+001.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+08

0 50 100 150

TCECFCENT

Time (h) 

Retention Pond 

Log 10 Concentration (CFU/100 mL) 

1.E+001.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+081.E+091.E+101.E+11

0 50 

Constructed W

Time (h)

Figure 4-7. Indicator organism concentrations with time. Regressions fits are for time = 0-50 h and 5

coefficients (k-values) of the exponent (slope) are shown in the tables. 

4-14
 

Table 4-6. In-situ Indicator Organisms Average Background Concentrations

Indicator 

Organism 

Background Concentration 

(CFU/100 mL ± Standard Error) 

Retention Pond Constructed Wetland

Total Coliforms 

E. coli 

Fecal Coliforms 

Enterococci 

1.39x104 ± 3.85x103 3.37x104 ± 4.04x103

6.42x100 ± 7.22x100 2.55x101 ± 6.21x100

1.02x104 ± 2.55x103 8.09x103 ± 1.12x103

3.70x101 ± 8.29x100 2.01x101 ± 5.46x100

Bacteria indicator organisms present in the sediments have the potential to be resuspended in the water 

column with turbulent flow or disturbance, and may contribute to maintained or increased effluent 

bacteria indicator organism concentrations in the future. 

Predation 

Table 4-8 shows the groups of organisms identified in the samples. The average number of organisms 

enumerated (invertebrate density) in the constructed wetland was 777 and in the retention pond was 442 

organisms. However, due to the high variance in the samples, this difference was not significant. 

The difference in the number of organisms present (invertebrate taxa richness) was significant between 

the constructed wetland and retention pond, averaging 15.75 organisms and 7.5 organisms, respectively. 

These groups often had more than one species in a taxonomic group represented in each system as shown 

in parenthesis. Ostrocods were not speciated below this subclass. 

Table 4-7. Sediment Bacteria Indicator Organisms Sampled in November of 2004 

Indicator Organism Indicator Organism 

Concentrations BEFORE Concentrations AFTER 

Stormwater Loading Stormwater Loading 

(MPN) (MPN) Indicator 

Organism Retention Pond

Total Coliforms 2.25x104 1.94x105

E. coli <MDL 7.41x104

Enterococci 1.62x104 1.88x103

Constructed Wetland

Total Coliforms 3.70x104 >2.41x105

E. coli 2.37x104 >2.41x105

Enterococci 2.02x105 8.67x103

 

4-15
 

Table 4-7. Macroinvertebrate Groups Identified in the Retention Pond and Constructed 

Wetland 

Retention Constructed 

Taxa Pond Wetland 

Oligochaetae 1 231 (3) 

Chironomidae 0 12 

Cladocerans 1479 (3) 190 (2) 

Coleoptera 0 9 

Collembola 0 1591 (2) 

Copepoda 22 (2) 269 (2) 

Ephemeroptera 42 0 

Hemiptera 0 8 (2) 

Hydracnidia 0 9 (2) 

Ostrocoda 186 782 

Rotifera 39 1 

*Parentheses indicate the number of taxa identified in that group 

Cladocerans were the dominant species present both in richness (3 species) and in density (83%) in the 

retention pond. Ostrocods (11%), ephemeroptera (2%), rotifers (2%), copepods (1%), and oligochaetes 

(<1%) made up the rest of the composition within the retention pond. The dominant invertebrate species 

in the constructed wetlands were collembola, ostrocods, copepods, and oligochaetes composing 51%, 

25%, 9%, and 7%, respectively. 

Scaling Consideration 

Mesocosms have a history of use as a research tool for ecological studies of aquatic and terrestrial 

ecosystems (Grice and Reeve, 1982; Odum, 1984; Lalli, 1990; Adey and Loveland, 1991; Beyers and 

Odum, 1993; Kangas and Adey, 1996). They have been used in commercial scale applications, such as in 

wastewater treatment, food production (Kangas and Adey, 1996), and in ecosystem restoration (Callaway 

et al., 1997). Use of mesocosms, particularly in wetland science, has become more common as a research 

tool for use in studies of the fate and effect of pollutants, biogeochemical cycles, and the effects of 

nutrients on ecosystem dynamics. 

A given condition when considering comparing mesocosms natural ecosystems is that ecological 

complexity is to some degree reduced or lost in microcosm or mesocosm studies depending on the size of 

the mesocosms being used relative to large ecosystem-scale research. Scale can change nutrient cycling, 

the number of trophic levels, number of species within trophic levels, habitat types, and connectivity 

between habitats (Beyers and Odum, 1993). Because of this, some caution needs to be used when 

extrapolating mesocosm results to larger systems. Once models created using mesocosms are validated in 

the field, application of model results at a larger scale can be made. 

Summary 

The results highlight the varying influence environmental factors have on the inactivation of indicator 

bacteria routed into constructed wetland and retention pond systems. The differences between these 

treatment systems and light and dark controls are statistically measurable implying some variables may 

have a greater influence than others. A detailed discussion of these results and their relation to the benchscale study are considered in Chapter 5. 

4-16
 

Chapter 5 Discussion of Results and Conclusions 

Microbial contamination from fecal origins in stormwater runoff poses a risk to human health through the 

consumption of drinking water and recreational and bathing contact with surface waters. Indicator 

bacteria serve as the regulatory meter by which water quality is measured and standards must be met. 

This chapter discusses the results of the bench and pilot-scale studies to evaluate the use of the first-order 

decay function for predicting indicator bacteria concentrations in BMP effluent. It compares the two 

studies to determine similarities and differences in inactivation rate constants, coefficients, and affects of 

environmental conditions on bacterial indicators. 

A primary factor affecting indicator bacteria, often understated in microorganism studies, is time. Time is 

incorporated into every inactivation rate. By definition the inactivation rate is the change in 

concentrations of indicator bacteria (presumably decrease) for a designated period. In both the benchand pilot-scale experiments, time was always a significant variable when evaluating the parameters 

affecting indicator bacteria concentrations. Many types of BMPs increase the storage time and decrease 

flow velocities as a primary mechanism of operation. 

Results from the bench-scale (Chapter 3) and pilot-scale (Chapter 4) studies show environmental 

conditions affect indicator bacteria concentrations in retention ponds and constructed wetlands. 

Temperature, sunlight, and salinity, were investigated in these studies. Other environmental factors 

typical of constructed wetlands and retention ponds were also considered. 

Effects of Temperature 

Generally, the results from the bench-scale and pilot-scale experiments agree with the literature (e.g., 

Easton et al., 2005; Ferguson et al., 2003; Geldreich et al., 1968; Medema et al., 1997; and Canteras et 

al., 1995) emphasizing temperature generally demonstrates an increase in the calculated inactivation rate 

constant with increasing temperature. Similarly, inactivation rates are lower at lower temperatures. This 

trend was most notable in the pilot-study during the October 2005 sampling event. Selvakumar et al.

(2004) noted that concentrations of organisms did not change significantly when the samples were stored 

at 4o

C beyond the standard holding time of 24 h. Geldreich et al. (1968) noted that organism persistence 

remained at higher levels at 10o

C compared to 20oC. In the pilot-scale experiment the optimal 

temperature range for growth (as indicated by overall indicator bacteria concentrations) was similar to 

values reported in the literature with indicator concentrations increasing with temperature, reaching a 

maximum concentration from 20o

-25oC in both the retention pond and constructed wetland. Medema et 

al. (1997) found that inactivation was faster at 15o

C than at 5oC. Canteras et al. (1995) noted a clear 

positive correlation between inactivation and temperature. In their study, when test conditions were at 

10o

C, 36 h was necessary to reduce the population of E. coli to 10% of the original as opposed to 8.4 h at 

5-1
 

42oC. Greater inactivation was also noticed in the range between 10 and 18oC than between 18 and 42oC. 

The pilot-scale study found indicator organism concentrations were much greater over the first 50 h as 

compared to the following 100 h. Although over a longer period of time, Easton et al. (2005) reported 

that the 0-7 day inactivation rates were much larger than the 7-21 day rates. It may be possible that 

during the 0-7 days studied by Easton et al. (2005) there have been varying rates of die-off. In the pilotscale study, by increasing the temporal resolution of the first 150 h compared to seven days (168 h), the 

changes in bacteria inactivation could be more easily observed. 

Effect of Sunlight/Light Intensity 

Many studies have shown that sunlight is an important factor in bacteria indicator inactivation (Sinton et 

al., 1994; Canteras et al., 1995). The bench-scale study supported this by showing that the effect of light 

on the overall decay coefficient was substantial, especially for non-coliform bacteria. If, for example, 

ambient light levels are 100 mW/cm2, then the light increases the E. coli inactivation rate constant by 0.25 

h-1, which is a six-fold increase over the dark value. Similarly, the pilot-scale study shows statistically 

lower inactivation rate constants in the dark control compared to the light control for total coliforms and 

E. coli for the months of May and June (Table 4-5). According to Table 4-4, June had the greatest 

irradiance of any of the dates sampled while May ranked fourth in light intensity (because of cloudy 

conditions during the experiment). Enterococci showed the greatest difference in inactivation rate 

constants between light and dark controls followed by total coliforms and E .coli. The primary difference 

between these controls was the exposure to sunlight. These differences in rate constants, up to 0.12 h-1

for enterococci and E. coli, are substantial and are also supported by the inactivation rates observed in the 

bench-scale study for total coliforms, fecal coliforms, fecal streptococci, enterococci, and E. coli indicator 

bacteria exposed to the highest light intensity. 

Effects of Sedimentation, Sorption, and Filtration 

Sedimentation, sorption, and filtration processes are generally accepted as the dominant mechanism for 

the removal of solids and other sediment-related stressors such as heavy metals. The settling velocity has 

been used as an approximation of the overall rate constant due to these factors in stormwater treatment 

systems (Wong and Geiger, 1997). Because the particle settling velocities are related to the grading, 

shape, and density of the particles entering the system, settling velocities measured in the laboratory can 

only serve as an indicator of the rate constant for sedimentation. Other environmental factors such as 

non-ideal flow conditions would be expected to increase solids in the water column through resuspension, 

while some posit that higher density vegetation can increase the rate of the settling constant (Wong and 

Geiger, 1997). 

The pilot-scale experiment compared the effluent concentration of a constructed wetland and a retention 

pond. While treatment volume was quite different between the two systems, the major difference 

between these systems was the presence of vegetation. Results comparing the TSS and turbidity in the 

two systems indicate that the constructed wetland and retention pond showed little difference in turbidity 

and effluent TSS. However, settling velocity appears to be greater in the constructed wetland under the 

higher (>100 NTUs) sediment loading observed in October 2005. 

The difference in indicator bacteria concentrations and the inactivation rate constants between the 

constructed wetland and retention pond in our study support settling as a contributing but not primary 

factor in bacterial inactivation. With the overall differences in turbidity and TSS between the constructed 

wetland and retention pond, relatively small for most of the simulated storm events (Figure 4-2), it is 

probable a large portion of the influent may have been unassociated (free) or associated with very fine 

particles. Similarly, the effects of settling may be artificially small as an artifact of the manner in which 

5-2
 

the enriched stormwater is created (described in Chapter 4). The smaller particles would result in a longer 

time necessary to settle in these systems and was likely to occur within the duration of the experiment. 

Another possibility is that the increased solids characterized in the influent (especially for October 2005) 

may reduce the effect of other environmental variables. Increased particulates may occlude light 

penetration or prevent predation by bacteriovores through limiting access and harboring the indicator 

bacteria that are agglomerated to these solids. 

Wong and Geiger (1997) suggest, when selecting an appropriate K value for sedimentation, filtration, and 

sorption using the settling velocity of the fiftieth percentile sediment grade with adjustments for increased 

effectiveness for wetlands having higher vegetation density. However, as experienced in this study, this 

may not adequately predict the effluent concentrations of stormwater runoff passing through passive 

treatment systems if bacteria are either unassociated with settleable particles or if they are associated with 

the fine particle fraction, i.e., less than 2 um in size (Davies and Bavor, 2000). 

Effect of Salinity 

Salinity was only assessed in the bench-scale study and was not included in the pilot-scale study. The 

bench-scale study results indicate that different organisms exhibited different trends at varying salinity 

concentrations. Overall, the effect of increased salinity at the tested concentrations was small. The 

calculated value of ФS was not generally significant from non-saline controls. This suggests that, for the 

span of salinity values studied, the added salt has little effect on the inactivation rate constant and 

supports the results reported by Canteras et al. (1995) who found the largest salinity effect occurs when 

the salinity values were over 35 ppt. Thoman and Mueller (1987) reported that the inactivation of fecal 

coliforms is generally much faster in marine and estuarine waters than in freshwater. Mancini (1978) 

indicated that components in seawater in addition to salt may be responsible for inactivation in seawater.

Salinity is less often a factor in most BMPs but it is a consideration when stormwater controls are to be 

placed in the coastal and estuarine environments, or when a BMP receives runoff from areas treated with 

road salts. 

Effect of Predation 

Previous research has suggested bacterivory can significantly reduce indicator bacteria organism 

concentrations (Green et al., 1997; Mandi et al., 1993; Decamp and Warren, 1998; Pretorius, 1962; 

Fernandez et al., 1992a; and Troussellier et al., 1986). There are a variety of invertebrates present in the 

constructed wetland and retention ponds in this study. While the retention pond and constructed wetland 

had different taxa represented in each of these systems, dominant invertebrates in both systems have been 

shown to consume large quantities of indicator bacteria depending on the population size of both 

predators and prey. The major difference in species richness between the systems was the retention pond 

was dominated by cladocerans while the constructed wetland had populations of oligochaete, collembola, 

copepod, and ostrocod invertebrates. The difference in predatory effects of the dominant species on 

indicator bacteria concentrations in each system is not known. Characteristics of the constructed wetland 

and retention pond do suggest why there may be different invertebrate communities between the different 

systems. Constructed wetlands generally had taxa that are associated with greater organic matter (derived 

from the macrophytic vegetation) (i.e., oligochaetes, collembola, copepods, and ostrocods) (Peckarsky et 

al., 1990). Collembola and ostrocods are reported to feed on detritus algae, fungi, and dead animal matter 

with collembolan having special mouthparts for consuming the surface film or underlying bacterial 

populations (Peckarsky et al., 1990). Therefore, their numerical importance in the constructed wetlands 

was not surprising. Cladocerans and copepods can affect bacterial populations in both wetlands and open 

water systems. Both taxa have been shown to consume greater than 25% of the bacterial populations in 

near shore areas of lakes (Heath et al., 1999). The association of collembolans with detrital organic 

5-3
 

matter and cladocerans with more open water habitat may explain the relative densities of these 

invertebrates in both the retention pond and constructed wetland environments. 

The identification and enumeration of the taxa did not provide quantifiable results to determine the 

predatory contribution of invertebrates on indicator bacteria concentrations. However it does provide 

anecdotal evidence that invertebrates may contribute to the reduction of indicator bacteria in natural 

systems. The pilot-scale study, while not directly measuring bacteria indicator inactivation rates due to 

predation, included the overall effects of predation by incorporating this effect into the cumulative 

inactivation rate as discussed in the section addressing collective environmental factors below. 

Effect of Other Potential Factors 

The many other factors (i.e., DO, pH, conductivity, oxidation reduction potential) that can contribute to 

inactivation rates of indicator bacteria were not directly assessed in this report. The bench-scale study 

addressed DO in a peripheral manner while the pilot-scale study did not address other potential factors 

individually but grouped their effects into the overall inactivation rates discussed as collective 

environmental factors in the next section. 

Inactivation Rates Due to Collective Environmental Factors 

The data on inactivation rates for microorganisms in stormwater and effects of natural factors on survival 

rates are limited except for one study by Geldreich et al. (1968). They reported a decay rate of 0.061 h-1

at 20o

C for fecal coliforms. The inactivation rate constant of fecal coliforms at 20oC obtained in the 

bench-scale study (0.047±0.031 h-1) was similar to Geldreich et al. (1968) and the 0-50 h and 50-150 h 

rates for fecal coliforms observed in the retention pond in the pilot-scale study. 

The bench-scale results observed showed a good relationship with the first-order equation. In this portion 

of the study, total coliforms had a much slower inactivation rate than other indicator organisms. 

Traditional indicators (total coliforms and fecal coliforms) had lower inactivation rates than the alternate 

indicators (E. coli and enterococci) suggesting that use of traditional indicators may tend to predict higher 

concentrations compared to the alternate indicators. Depending on the stressor(s) for which the BMP is 

designed, this could affect the necessary retention time calculated when designing BMPs.

When correlation analysis was done for chemical and physical parameters with overall inactivation rates 

in the pilot-scale study, conductivity with E. coli and enterococci with DO were significantly correlated in 

the retention pond. The constructed wetland had no significant physical or chemical correlations with 

inactivation rates. Conductivity of the in situ water may be a surrogate for total dissolved solids. 

However, standard methods suggest the relationship is not constant (APHA et al., 1998). The 

relationship between total dissolved solids and conductivity is a function of the type and nature of the 

dissolved cations and anions in the water (i.e., the ability of the water to carry a charge). Some total 

dissolved solids measuring devices measure the conductivity of the water with the assumption that the 

primary dissolved minerals are either a combination of NaCl or KCl. Other anions and cations, such as 

sodium sulfate, sodium bicarbonate, or possibly some organic molecules with ionic and cationic charges 

can contribute to the conductivity in water samples suggesting total dissolved solids, while not directly 

measured in the experiment, may be correlated with E. coli concentrations in the retention pond if other 

mineral or organic compounds are present. 

The pilot-scale study generally followed the first-order rate equation. A jackknife relationship showing a 

different rate constant for the first 50 h compared to longer periods, as in Figure 4-7, was appropriate for 

some indicator bacteria. This relationship was also observed by Thomann and Mueller (1987) for bacteria 

5-4
 

distributions in rivers with resistant strains. In addition, with indicator species concentrations (Figure 4-

7) having an average predicted background concentration of 101

–104 organisms/100 mL and in situ

background concentrations ranging from 6.42x100

-3.37x104 organisms/100 mL, there is reasonable 

support for changes to the first-order rate equation in wetland and retention pond BMPs. Kadlec and 

Knight (1996) suggest that because of residual indicator bacteria populations present in wetlands, bacteria 

removal efficiency is a function of the inflow bacteria concentrations. Removal efficiency typically is 

higher at high inflow concentrations, but declines to low or negative values when inflow concentrations 

are lower than the in situ bacteria production rates. However, during periods when influent flow rates are 

turbulent, causing resuspension of the previously settled solids, removal efficiency may not depend on 

influent concentrations alone. Because these settled sediments are associated with in situ bacteria 

populations, there may be an increase in effluent concentration of indicator bacteria with turbulent or high 

flow or when sediments are disturbed by other means (i.e., waterfowl, muskrats, etc.) compared to the 

influent concentration. Similarly, sediment resuspension may be more likely to occur in wetland and 

retention pond BMPs that are poorly designed, have reached the design life, or are not maintained and 

may contribute to lower or negative indicator bacteria inactivation rates (and removal efficiencies). 

Evaluation of the First-Order Decay Equation 

Recalling that one of the primary objectives of this research was to evaluate the first-order decay equation 

for predicting bacteria indicator concentrations affected by environmental conditions, a bench-scale study 

to look at selected variables identified in the literature as important to inactivation rates was developed. 

With the bench-scale information as the primer, the pilot-scale experiments utilized controlled 

mesocosms to further develop the possible effects of typical environmental conditions (similar to 

expected field BMP conditions) have on indicator bacteria concentrations in BMP effluent. Both the 

pilot-scale study and the bench-scale study demonstrated the first-first order decay function adequately 

models indicator bacteria concentrations in the short term. However, during longer periods, the firstorder decay equation may not apply to effluent from these types of BMPs. Literature has reported that the 

assumptions for a first-order decay function (i.e., steady flow conditions) may seldom be met in studies 

concerning stormwater runoff in constructed wetlands and retention ponds (Wong and Geiger, 1997). 

Other researchers have suggested using surface area based models for wetlands constructed for the 

treatment of wastewater (Kadlec and Knight, 1996). One of these models is known as the K-C* model 

which incorporates a concentration term, C*, that represents the background concentration often present 

in natural systems. The formula is: 

( *)

( *)

out K

in

C C

e

C C

− − = − (5-1)

Where: Cout = effluent concentration; Cin = influent concentration; C* = background concentration; K = 

rate constant for the water quality parameter being treated based on time of detention. 

However, Wong and Geiger (1997) point out that the stochastic nature of stormwater-related systems 

introduces significantly different system functions compared to wastewater treatment. These authors 

formulate a procedure that incorporates the use of the K-C* model and the interaction between the 

requirements for wetland storage for inflow stochasticity and stormwater treatment. 

They recommend an adaptation of the Kadlec and Knight’s K-C* model with the formula: 

/ * ( *) KA Q C C C Ce out in− =+− (5-2)

5-5
 

Where: Cout = effluent water quality target; Cin = influent event mean concentration; C* = background 

concentration; K = rate constant for the water quality parameter being treated; A = constructed wetland or 

retention pond area; and Q = steady state flow. 

It should be noted that equations 5-1 and 5-2 have attempted to incorporate conditions that meet the 

assumptions for the first-order decay equation or include environmental realities such as background 

concentrations of indicator organisms (C*) to improve prediction of this stressor. The rate constant K,

which governs inactivation rate determinations in the first-order decay equation, is the only means of 

incorporating environmental variables to better predict effluent concentrations in surface water models. 

The bench and pilot studies discussed in this report have estimated inactivation rate constants for 

indicator bacteria. Further, constant coefficients have also been estimated to predict the effect that 

environmental factors have on overall indicator bacteria inactivation rates. 

Returning to Khatiwada and Polprasert (1999) and Canteras et al. (1995) equations from Chapter 3, the 

following formula for overall inactivation rate constant is proposed: 

20

20 T

T K K IK overall l f p K − = Φ +Φ + + (5-3) 

 

Where: Koverall = overall inactivation rate constant; K20 = inactivation rate constant due to temperature at 

20o

C; ФT = temperature coefficient; Фl = light proportionality coefficient; l = light intensity (mW/cm2); Kf 

= inactivation rate constant due to other factors such as sorption, filtration, and sedimentation; and Kp = 

inactivation rate constant due to predation. 

Temperature and light could be quantified through the combination of both studies in this report. Salinity 

was found to have little effect on bacteria indicators in the constructed wetland and retention pond 

systems used in this study. Due to the inability to separate sorption, sedimentation, predation, and other 

environmental factors in the study, substituting the variable Kother instead of Kf and Kp to include these 

(and other) processes in one variable is proposed. As a result, the inactivation rate formula from above 

can be written as: 

20

20

T K K overall T l other I K − = Φ +Φ + (5-4) 

Where definitions are as above and Kother = inactivation rate constant due to other factors such as sorption, 

filtration, sedimentation, predation, pH, DO, conductivity, oxidation reduction potential, etc. 

Table 5-1 lists coefficients for light and temperature from the bench-scale study and the light and dark 

controls of the field-scale study. Inactivation rates at 20o

C (K20) obtained in the field study are larger than 

the values obtained from bench-scale experiments except for E. coli. Temperature coefficients (ФT) 

obtained from both bench- and pilot-scale studies are not statistically significant different. Light 

proportionality coefficients (Фl) obtained from pilot-scale study are much larger than the values obtained 

from bench-scale study supporting the conclusion that natural sunlight has a much larger effect on 

inactivation rates compared to artificially induced light. 

Using the light and dark control inactivation rates, the inactivation rate due to other parameters was 

calculated as the measured Klight +temperature value. Subtracting the Ktemperature value from the Klight +temperature, 

results in a calculated Klight value. The Kother value was then calculated by subtracting Klight and Ktemperature

from the Koverall value that was measured for the retention pond and constructed wetland. All K values for 

the retention pond and constructed wetlands can be found in Tables 5-2 and 5-3, respectively. 

5-6
 

Table 5-1. Inactivation Rate Coefficients from Batch and Field Studies 

Indicator 

Organism 

Batch Study Field Study 

K20 (h-1) ФT

ФL (cm2/

mW-h) K20 (h-1) ФT

ФL (cm2/

mW-h)

TC 0.016±0.009* 1.057±0.085* 0.0016 0.066±0.007* 1.005±0.011 0.0092 

FC 0.042±0.030* 1.090±0.110* 0.0130 0.053±0.006* 1.017±0.004* 0.0047 

EC 0.036±0.018* 1.023±0.072* 0.0025 0.057±0.008* 1.013±0.002 0.0022 

ENT 0.042±0.014* 1.057±0.045* 0.0076 0.054±0.006* 1.050±0.014 0.0070 

* Coefficient is statistically significant at α=0.05. 

Table 5-2. Retention Pond Overall, Temperature, Sunlight, and Other Factors Rate Coefficients

Indicator Month/Year Koverall Ktemp Klight Kothers 

Organism (measured) (measured) (calculated) (calculated)

(h-1 )

TC June 2004 0.242 0.025 0.114 0.103 

 September 2004 0.144 0.070* -0.011* 0.085 

 November 2004 0.165* 0.082* -0.016* 0.010 

 May 2005 0.095 0.026 0.042* 0.027 

 July 2005 0.181 0.064* 0.008* 0.109 

 October 2005 0.044* 0.054* 0.014* -0.024 

 Annual Average 0.145 0.054 0.025 0.052 

FC June 2004 0.181 0.025 -0.001 0.157 

 September 2004 0.119 0.053* 0.023* 0.043 

 November 2004 0.149* 0.045* 0.024* 0.079 

 May 2005 0.142 0.051* 0.033 0.059 

 July 2005 0.261 0.062* 0.052* 0.147 

 October 2005 0.057* 0.051* 0.031* -0.025 

 Annual Average 0.152 0.048 0.027 0.077 

EC June 2004 0.148 0.028 0.122 -0.002 

 September 2004 0.116 0.056* 0.023* 0.038 

 November 2004 0.116* 0.048* 0.024* 0.044 

 May 2005 0.335* 0.073 0.043 0.219 

 July 2005 0.196 0.051* 0.020* 0.125 

 October 2005 0.052* 0.061* 0.025* -0.034 

 Annual Average 0.161 0.053 0.043 0.065 

ENT September 2004 0.203 0.077* 0.126* 0.001 

 November 2004 0.173* 0.071* 0.108* -0.006 

 May 2005 0.172 0.035* 0.053* 0.083 

 July 2005 0.124 0.094* 0.074* -0.044 

 October 2005 0.051 0.019* 0.013 0.019 

 Annual Average 0.145 0.059 0.075 0.011 

* Coefficient is statistically significant at α=0.05 

The Klight +temperature value for the constructed wetland was not directly measured but could be calculated. 

To calculate a the Klight +temperature value for the constructed wetland the Klight value from the light control 

was multiplied by the weighted average of light intensity expected at the surface of the constructed 

5-7
 

wetland. The weighted average was calculated as 10% of the light that reached the retention pond surface 

(based on light meter measurements) for six hours out of 24 hours of effective light exposure, multiplied 

by 18 hours out of 24 hours in which the exposure was relatively the same as in the retention pond. This 

resulted in a multiplication factor of 0.775 •Klight of the retention pond. All negative calculated values 

were assumed to be a propagation of error and are therefore expected to be within the range of error for 

the respective inactivation rate constant. 

Table 5-3. Constructed Wetland Overall, Temperature, Sunlight, and Other Factors Rate 

Coefficients

Indicator 

Organism Month/Year Koverall 

(measured) 

Ktemp 

(measured) 

Klight 

(calculated) 

Kothers 

(calculated) 

TC June 2004 

(h-1)

0.153 0.025 0.088 0.040 

September 2004 0.120 0.070* -0.009* 0.059 

November 2004 0.124* 0.083* -0.013* 0.054 

May 2005 0.109 0.026 0.033* 0.050 

July 2005 0.073 0.064* 0.006* 0.003 

FC 

October 2005 0.043* 0.054* 0.011* -0.022 

Annual Average 0.104 0.054 0.019 0.031 

June 2004 0.328 0.025 -0.001 0.304 

September 2004 0.152 0.053* 0.018* 0.081 

November 2004 0.114* 0.045* 0.019* 0.050 

May 2005 0.123 0.051* 0.025 0.047 

July 2005 0.103 0.062* 0.040* 0.001 

EC 

October 2005 0.054* 0.051* 0.024* -0.021 

Annual Average 0.146 0.048 0.021 0.077 

June 2004 0.165 0.028 0.095 0.042 

September 2004 0.120* 0.056* 0.018* 0.046 

November 2004 0.116* 0.048* 0.019* 0.049 

May 2005 0.092* 0.073 0.033 -0.014 

July 2005 0.189* 0.051* 0.016* 0.123 

ENT 

October 2005 0.060* 0.061* 0.019* -0.020 

Annual Average 0.124 0.053 0.033 0.038 

September 2004 0.179* 0.077* 0.098* 0.004 

November 2004 0.125* 0.071* 0.084* -0.030 

May 2005 0.085* 0.035* 0.041* 0.009 

July 2005 0.211* 0.094* 0.057* 0.060 

October 2005 0.059* 0.019* 0.010 0.030 

Annual Average 0.132 0.059 0.058 0.015 

* Coefficient is statistically significant at α=0.05 

Inactivation rate constants vary throughout the year based on different affects of the environmental 

factors. In general, the combination of other factors had the greatest effect on inactivation rates in the 

retention pond for the indicator bacteria evaluated in this study. Enterococci were an exception to this. 

Temperature was the found to be more important than light, however light is still a significant factor and 

should be considered when using the first-order equation. In the constructed wetland, temperature had the 

greatest effect on inactivation rates for the selected indicator bacteria. Other factors had a greater 

influence on inactivation rates for all organisms except for enterococci, where light appeared to be as 

5-8
 

important as temperature. Inactivation rates for fecal coliforms were most affected by other factors 

followed by temperature and then light.

Application of the inactivation rate constants found in Tables 5-2 and 5-3 can provide an overall 

inactivation rate constant incorporating temperature, light intensity, and a lumped factor for other 

environmental variables. The overall inactivation rate constant can be applied to equation 5-2 to 

determine the required area necessary to achieve WQSs when designing constructed wetland or retention 

ponds. If a background concentration is determined, the overall rate constant can also be applied to 

equations 5-1 and 5-2 to predict effluent concentrations. The first-order decay equation is most accurate 

when used with the inactivation rates and uncertainties in short-term models to predict stormwater runoff 

effluent quality from constructed wetland and retention pond BMPs to improve or prevent further 

degradation of water quality. Longer-term modeling would benefit from applying separate inactivation 

rates for periods immediately following stormwater runoff and periods unaffected by stormwater runoff. 

Conclusions 

The studies in this report demonstrated that the concentration of the tested indicator organisms decrease 

exponentially with time. The first-order decay process reasonably models the concentration time series 

for shorter durations. The first-order decay model is a simple and efficient means of predicting indicator 

bacteria concentrations in stormwater runoff effluent from BMPs such as retention ponds and constructed 

wetlands. Results from the studies discussed in this report provide new data on inactivation rate constants 

coefficients, and uncertainties used in this equation. The factors of light, time, and temperature influence 

processes in all retention ponds and wetlands constructed to mitigate the effects of stormwater runoff on 

the receiving waters. A combination of other factors (e.g., predation, sedimentation, sorption, filtration, 

pH, BOD, pH, and DO) can also contribute to the inactivation of indicator bacteria in constructed wetland 

and retention pond BMPs. Reliable rates, coefficients, and the uncertainties expected in the reported 

values will add to the accuracy of surface water quality models. Water quality models are a primary tool 

for evaluating permit applications (e.g., NPDES) and have an important regulatory role in developing 

TMDL allocations. These models should incorporate the affects of BMPs to better model their potential 

for improving water quality in the watershed. The incorporation of simple reliable models is an important 

step in assuring that the models used in determining bacterial TMDL loading and allocations meet the 

state of the science. 

BMPs were originally designed to control runoff volumes and rates by attenuating the flow. The 

attenuation increases the time between the rainfall-generated runoff and the water reaching the receiving 

water. The time lag serves to reduce the concentration of these indicator organisms. Structural BMPs 

then can be effective in reducing indicator bacteria concentrations contained in stormwater runoff. Low 

inactivation rates may occur in BMPs where inflow bacterial concentrations are lower than the in situ

bacteria productions rates, or turbulent flow through the BMPs causes resuspension of sediments. 

Quantitative microbial partitioning estimates can represent critical inputs in areas where sedimentation is 

a primary mode of indicator organism inactivation when modeling the location and severity of impaired 

waters. The lack of reliable partitioning information currently leads most surface water modeling efforts 

to assume that microbes exist in the free phase. The presumption of only free-phase organisms biases 

model results to greater dispersion and shorter microbial longevity. However, from the results obtained 

from this project, factors such as temperature and light intensity have been shown to be as, or more 

important to, indicator bacteria inactivation rates. This would suggest that when attempting to mitigate 

bacteria in runoff, watershed managers should construct BMPs to maximize the temperature increase 

from solar exposure. Similarly, the added effects of light, even at constant temperature, can increase 

inactivation rates, improving BMP performance. The extent to which shading in constructed wetlands, 

due to vegetation or the deeper water of retention ponds, attenuates the effect of incident light will vary

5-9
 

with runoff and in situ water properties (e.g., turbidity, color) in the BMP. It is also important to 

recognize that bacteria loading seldom acts as a single environmental stressor of concern. The watershed 

manager must consider the effects of the increased effluent temperature on the receiving waters, 

particularly when the receiving water is a low-order cold water stream. Also, the results from this study 

suggest that the regulatory indicator selected can influence BMP design. The apparent insensitivity of 

coliforms to light levels suggests that the shading effects may be reduced when this is selected as the 

water quality indicator. When the monitored indicator organism is E. coli or enterococci, the effect of 

light would be expected to be greater than for coliforms.

It is accepted that placement of appropriate BMPs in watersheds can lead to improvements in receiving 

water quality by reducing the overall load of pollutants to receiving waters. If watershed managers can 

reduce microbial loads in waterbodies using the range of possible BMPs, verification of these stormwater 

management tools will help MS4 Phase I and Phase II communities reduce microbial loadings and meet 

requirements set out by the TMDL process. Long-term microbial load reductions will improve the overall 

water quality and could potentially lead to increased consumption of fish and shellfish, increased use of 

recreational waters, reduced beach closures, and increased protection of source water used as drinking 

water sources. 

The limitations of BMP effectiveness in reducing bacterial loading to WQS must be recognized. In most 

natural treatment systems there will be an irreducible concentration that is often maintained in system 

sediments. Dilution of BMP effluent likely plays a significant role in attaining WQS in receiving water. 

However, elimination of bacteria indicators may require chemical treatment. In addition, overall 

effectiveness and efficiencies of BMPs hinge on proper design and maintenance of these systems.

5-10
 

Chapter 6 References 

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Appendix A Growth of Indicator Bacteria for Pilot-Scale Research 

Introduction 

To overcome the stochastic nature in stormwater runoff, researchers often employ the use of “synthetic 

stormwater”, a laboratory-produced stormwater matrix of known pollutant concentrations, in their 

research. Literature indicates synthetic stormwater has been used most often for evaluating BMP 

performance for the removal of heavy metals and total organic carbon (TOC) (Driscoll et al., 1990; Liu et 

al. 2001; Liu et al. 2001) but has also been used in nutrient (nitrogen and phosphorus) (Davis et al., 1998; 

Kim et al, 2003), chemical oxygen demand (potassium salt), and total organic carbon (motor oil) 

(Fassman and Yu, 2001; Hong et al. 2006) studies, as well. However, the authors could not find any wet 

weather flow studies that used bacterial indicator-based synthetic stormwater to evaluate the performance 

of BMPs. Consequently, the researchers developed a method for creating a stormwater source that has a 

known concentration of common indicator organisms (i.e., total coliforms, fecal coliforms, E. coli, and 

Enterococci) and contains genotypic representation (assumed, not measured) typical of bacterial 

indicators found in urban stormwater for use in evaluating pilot-scale stormwater BMPs. 

Methods 

Stormwater runoff from the Middlesex County College (9.75 acres) campus was collected in a 11,300 L 

tank from an outfall near the Urban Watershed Research Facility (UWRF) in Edison, NJ and stored on 

site (Figure 3-1). After thorough mixing, a 60 mL aliquot of stormwater was collected for each sampling 

event with 30 mL each placed in a flask containing 1L of tryptic soy (TS) broth (for coliforms and E. coli) 

and 1L of brain heart infusion (BHI) broth (for enterococci). A stir bar was added to each flask. The TS 

broth flask was then placed on a stir plate in a 37oC incubator while the BHI flask was placed on a stir 

plate in a 41oC incubator. Concentrations of bacteria in the inoculated stormwater (broth-stormwater 

mixture) were measured daily via membrane filtration using Standard Methods 9222B, 9222D, 9222G, 

and 9230C as described in Chapter 3. Methods for positive controls followed manufacturer’s 

specifications (Becton Dickinson and Company, Sparks, MD). 

Trend analysis of the concentration results from five- and seven-day sampling periods determined the 

growth curves of indicator bacteria populations. Regression analyses of the growth phases for each 

organism were used to determine the rate of growth. Rate of growth, in general, was grouped by days of 

positive growth versus days of no or negative growth. Results allowed for future prediction of bacterial 

concentrations so that experimental use of indicator bacteria could occur at the time of greatest bacterial 

concentration. A measured volume of inoculated stormwater could then be combined with the desired 

A- 1
 

volume of original stormwater runoff for use in BMP performance studies. The inoculated aliquot and 

stormwater was mixed, forming the “synthetic stormwater”, and distributed to pilot scale BMPs for 

performance evaluation. 

Results and Discussion 

Average starting concentrations on day zero of total coliforms, fecal coliforms, E. coli, and enterococci 

were 2.3 x 105

, 1.7 x 104, 5.8 x 103, 3.1 x 103 CFU/100 mL, respectively. All indicator organism 

concentrations reached a maximum on day two and then declined (Figure A-2). Enterococci 

concentrations declined at a much slower rate with day two and day five concentrations very similar 

(Figure A-2; Table A-1). Starting concentrations of fecal coliform were one order of magnitude lower 

and E. coli and enterococci starting concentrations were two orders of magnitude lower than the 

concentrations of total coliforms. After the second day all indicator bacteria concentrations except for 

enterococci were within the same order of magnitude (~109

 CFU/100 mL). Enterococci concentrations 

remained greater than 109

 CFU/100 mL until the seventh (last) day of the experiment. 

 TC

01234567

Day

 FC

01234567

Day

2

3

4

5

6

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Figure A-1. Mean indicator bacteria concentrations by day. Vertical bars denote 95% 

confidence intervals. 

A- 2
 

Table A-1. Growth Rates of Bacterial Indicators Based on Regression Analyses 

Growth Rate: Growth Rate: 

Indicator Organism Days 0-2 (R2) Days 2-7 (R2) 

Total Coliforms 2.565 log (0.922) -0.297 log (0.903) 

Fecal Coliforms 3.106 log (0.916) -0.373 log (0.877) 

E. coli 3.356 log (0.913) -0.434 log (0.920) 

Enterococci 3.321 log (0.704) -0.087 log (0.823) 

Conclusion 

The methods for developing indicator bacteria-rich synthetic stormwater in this laboratory experiment can 

be useful for applications that specifically require stormwater that is characterized by abundant bacterial 

indicator concentrations. Bacterial loading can be closely predicted using growth rate curves. A wide 

range of bacterial loading capabilities can be achieved by using this approach to stormwater research. 

One must recognize that the relative proportion of indicator bacteria will change from the original 

stormwater and may be less representative of the true microbial community structure. Likewise, the 

particle association of the indicator bacteria is suspected to be different with less bacteria associated with 

larger particles and more bacteria associated with finer particles or unattached as opposed to the initial 

stormwater runoff. However, it is believed that this property results in a more conservative estimate for 

the indicator organisms when using synthetic stormwater for indicator bacterial loading experiments as 

fewer colonies settle out of the water column. 

Based on indicator bacteria concentrations from the first sample collection after spiking, there is some 

bacterial die-off that occurs when diluting original stormwater with the inoculated stormwater. A 

potential cause of this die-off may be thermal shock, although equilibration procedures attempt to 

minimize the temperature changes between the collected stormwater and the laboratory grown inoculated 

stormwater. 

The synthetic stormwater runoff produced in this project has been effectively used in pilot studies for 

assessing the performance of small scale controlled stormwater wetland and retention pond BMPs. 

Citations 

Bavor, H.J., D.J. Roser, and S. McKersie. 1987. Nutrient removal using shallow lagoon solid matrix

macrophyte systems. In: Aquatic Plants for Water Treatment and Resource Recovery, Reddy, K.R. and 

W.H. Smith, (editors). Magnolia Publishing, Orlando, FL. pp. 227-235. 

Driscoll, E.D., P.E. Shelley, and E.W. Strecker. 1988. Pollutant Loadings and Impacts from Highway 

Stormwater Runoff, FHWA-RD-88-006, -008. 

Fassman, E. A. and S. L. Yu. 2001. “Comparison of Pollutant Removal Performance of Wetland 

Vegetation.” In American Society of Civil Engineers EWRI World Water and Environmental Resources 

Congress in Orlando, FL. Washington, DC. 

Gersberg, R.M., R. Brenner, S.R. Lyon, and B.V. Elkins. 1987. Survival of bacteria and viruses in 

municipal wastewater applied to artificial wetlands. In: Aquatic Plants for Water Treatment and Resource 

A- 3
 

Recovery, Reddy, K.R. and W.H. Smith, (editors). Magnolia Publishing, Orlando, FL. pp. 227-235.

Hong, E., E.A. Seagren, and A.P. Davis. 2006. Sustainable Oil and Grease Removal from Synthetic 

Stormwater Runoff Using Bench-Scale Bioretention Studies. Water Environment Research, Vol. 78, No. 

2. pp. 141-155. 

Kim, H., E.A. Seagren, and A.P. Davis. 2003. "Engineered bioretention for removal of nitrate from 

stormwater runoff." Water Environment Research, Vol. 75, No. 4, pp. 355-367. 

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