CICEET Progress Report for the period 3/16/06 Through 9/15/06

Project Title: Transport and Capture of Pathogens from Urban Stormwater Runoff Using Bioretention
Principal Investigator(s): Allen P. Davis, University of Maryland
Additional Investigator(s): Eric A. Seagren, University of Maryland; Jeffrey S. Karns, USDA
Project Start Date: September 1st, 2005

Figures

Figure 1

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Figure 10

Tables

Table 1

Table 2

Table 3

Table 4

Tasks to meet objectives
Task 1 and Task 4 were scheduled to meet the objectives described above during this project time period.

Progress on Tasks
Task 1- Evaluation of conventional bioretention media for capture and destruction of pathogens using column studies, is nearly completed. Task 4 -“Long-term” sustainability studies, has been set up and is being conducted. The experimental materials and methods are briefly described in the following paragraphs.

1. Bacterial Growth
Pathogenic microorganisms in urban stormwater runoff represent an important and growing water quality problem. The study by Oliveri et al. (1977) indicates large concentrations of fecal coliform and pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus in urban stormwater. The EPA’s Ambient Water Quality Criteria for Bacteria-1986 (USEPA, 1986) recommends the use of Escherichia coli or enterococci as pathogen indicator organisms in fresh waters. In particular, E. coli O157:H7 is one of the major pathogenic waterborne bacteria of concern (USEPA, 2001). A derivative of E. coli O157:H7 strain B6914 that has lost the shiga-toxin genes and has been engineered to contain the green fluorescent protein gene was used as the test indicator organism, thereby allowing controlled monitoring of a specific microbial pathogen in the laboratory studies. The original inoculum of strain B6914 was provided by Dr. J. S. Karns (Environmental Microbial Safety Laboratory, USDA).

To prepare the feed solution for the column studies, a colony of strain B6914 from a LB Lennox agar plate was picked using a sterile inoculating loop and used to aseptically inoculate 5 mL of LB broth (10 g trytone, 5 g yeast extract and 5 g sodium chloride per 1 liter distilled water) with 100 mg/mL ampicillin (Ap) in a sterile centrifuge tube. Subsequently, that tube was incubated at 37°C with shaking (120 rpm) overnight. Next, a 1 mL sample from this liquid culture was aseptically transferred into 50 mL of LB broth (Ap 100 µg/mL) in a sterile 250 mL flask with a cotton plug. This culture was incubated at 37°C with shaking (120 rpm), and then the cells were harvested during the late exponential phase (OD_600nm ~ 0.9). To harvest the cells, the culture broth was centrifuged (4,500 rpm) for 10 min, the cell pellet was washed with phosphate buffered saline (PBS, pH=7.5, Gerhardt et al., 1994) and then centrifuged again. After pouring off the supernatant, the cells were suspended in PBS to a final concentration of approximately 1.1_10⁹ cells per mL as a stock solution for use in the column studies described below. Once prepared, the stock solution was always used within 30 min.

2. Analyses of bacterial physicochemical characterizations
2.1. Cell surface hydrophobicity
Cell surface hydrophobicity was determined by measuring the surface contact angle as described by van Loosdrecht et al. (1987a) and performing the bacterial adherence to hydrocarbons (BATH) test. For measuring the surface contact angle, a 10 mL cell suspension, grown as described above, was filtered through a 0.45 mm-pore-size membrane filter (Pall Corporation, MI). The filter containing a continuous bacterial layer was mounted on glass slide and dried in a desiccator for a half hour to obtain a stable water contact angle. A water droplet (1 µL) was applied on the bacterial layer, and the contact angle was immediately measured with a Goniometer (Model 100, Rame-hart Inc., NJ) at 27 °C. Six replicates were conducted.

Bacterial adherence to hydrocarbons was measured using n-hexadecane, n-octane and o-xylene as the assay hydrocarbons (Rosenberg et al., 1980; Gannon et al., 1991). The cell suspension was prepared as described above, and diluted to a concentration of approximately 1.1_10⁸ cells per mL using PBS. A mixture of 5.0 mL of this cell suspension and 0.00, 0.25, 0.50, 0.75, 1.0, 1.5, or 2.0 mL of the test hydrocarbon (n-hexadecane, n-octane or o-xylene) was added to sterilized 12 mL test tubes and mixed using a vortex mixer for 2 min at 27 °C. After allowing 30 min for the separation of hydrocarbon and aqueous phases, the hydrocarbon phase was carefully removed with a pipette, and then the absorbance of the aqueous phase was determined at 400 nm using a Spectronic 21 spectrophotometer (Bausch & Lomb, U.S.A). A reduction in the absorbance of the aqueous phase was used as the measure of cell surface hydrophobicity. In addition, the lower aqueous phase and the upper "cream" were examined microscopically to ensure that the cells were indeed attached to the hydrocarbon.

2.2. Electrophoretic mobility
The electrophoretic mobility of strain B6914 was measured with a Zetasizer 3000 (Malvern Instruments Ltd., Malvern, UK), which can be converted to zeta potential by the method of Helmhotz-Smoluchowski (Hiemenz, 1986). The cell suspensions, grown as described above, were diluted in synthetic runoff to a concentration of approximately 1.2_10⁸ cells per mL before determination of the zeta potential at 25°C. The potential difference between the electrodes was 200V. The zeta potentials of cells suspended in synthetic runoff were determined. Four replicates were conducted.

3. Preparation of conventional bioretention media
Conventional bioretention media is composed of sandy loam soil, sand and mulch. The sandy loam soil was provided by Construction Service Division, Department of Public Works and Transportation of Prince George’s County, Maryland. The sandy loam soil was collected from the soil layer at a depth of 15-35 cm, 900 m east of Reid La, Tantallon, Maryland. Soils were mixed in a large container and dried at room temperature for 2 days, and then were crushed to pass a 1.18 mm sieve. The physical and chemical properties of the soil (S1) were determined by the University of Delaware Soil Testing Program (See Table 1). The sand (US Silica Company, Berkeley Springs, WV) was obtained from a local home supply store. The mulch was obtained from the College Park Department of Public Works (MD). It was produced through composting municipal yard wastes (leaves and grass clippings). The sand and mulch also were sieved through a 1.18 mm sieve, and then the sand, soil and mulch were mixed at a volume ratio of 5:3:2. This ratio is typical for use in bioretention facilities (MDE, 2000; DER, 2001). The particle-size distributions of these media were analyzed using the dry-sieving technique in the Geotechnical Engineering Laboratory, University of Maryland, College Park (Das, 1992). The results of these analyses are presented in Table 2.

4. Column setup and bromide tracer tests
All column experiments were performed in glass chromatography columns (Kontes, Vineland, NJ) with a 20 µm porosity polyethylene bed support. The inner diameter of these columns is 2.5 cm and the height is 23 cm. The conventional bioretention media was dry-packed into each column through pouring in small batches and tapping to achieve homogeneity. The final depth of the media is about 21.7cm, and the bulk density is approximately 1.34 g/cm³. Prior to initiating a pathogen transport experiment, the column was flushed to reach saturation using synthetic runoff. The content of the synthetic runoff was based on references on urban stormwater runoff chemistry (Davis et al., 2001). The synthetic runoff was made using distilled water with the amendments listed in Table 3. The influent was pumped into the column from the top, and the effluent was collected from the bottom of the column. The column experimental set up is shown in Figure 1. All glassware (reservoir, vials, etc.) and tubing were sterilized by autoclaving prior to use to avoid introduction of exogenous microbes.

During most pathogen transport experiments, bromide tracer tests were used to support the microbial transport and capture studies by estimating the velocity and dispersion of water flow in the columns. Sodium bromide was dissolved in the synthetic runoff to obtain a bromide concentration of 200 mg/L, and the solution was then pumped into the column at a flow rate of 40 mL/hr (equal to an approach velocity of 8 cm/hr) for 6 hrs. This hydraulic loading represents a common storm (0.4 cm/hr; return period < 1 year) concentrated by a factor of 20 from the drainage area to the treatment facility (DES, 1993; Davis et al., 2001). The effluent samples were collected every quarter-hour or half-hour from the bottom of the column. The bromide concentrations of influent and effluent samples were measured using a Cole-Parmer 27502-04, 05 bromide electrode (Cole-Parmer Instrument Company, Vernon Hills, IL). The detection limit for the bromide probe is 0.01 mg/L in the presence of other ions.

5. Pathogen transport experiments
5.1. Transport and capture of pathogens by conventional bioretention media
Pathogen transport and capture in conventional bioretention media was quantified in duplicate experiments. E. coli O157:H7 strain B6914 in PBS stock solution was diluted in sterile synthetic runoff to a final concentration of 1.1 + 0.3 _10⁶ CFU/mL. The synthetic runoff was stirred and continuously pumped into a column from the top using a Masterplex® L/S peristaltic pump (Cole-parmer Instrument Company, Catalog No. 7553-30) at a flow rate of 40 mL/hr for 6 hrs. The influent samples were collected aseptically in autoclaved vials every one hour, and the effluent samples were collected every quarter- hour or half- hour from the bottom of the column. Strain B6914 cells in the influent and effluent samples were enumerated by dilution in PBS and plating on LB agar containing 100 mg/mL ampicillin using the method of heterotrophic spread-plate counts adapted from Standard Method 9215 (APHA et al., 1995). The plates were incubated at 37°C for one day, and then the colonies were counted. B6914 colonies were confirmed by checking fluorescence under long wave UV (365 nm) and disregarding any colonies that did not glow green. Pathogen transport experiments in the same column were conducted twice at an interval of two weeks.

5.2. Comparing transport and capture of pathogens in laboratory media and media from an established cell
Pathogen capture in the freshly packed laboratory column was compared to that in column packed with media collected from an established bioretention facility. Pathogen transport experiments were conducted using two column packing procedures: (1) the laboratory bioretention media, as described above, was packed into the glass column to achieve a bulk density similar to that of newly-installed bioretention systems, and (2) intact soil cores were obtained from an established bioretention facility and packed into the glass column. The intact soil cores were collected from a bioretention facility near parking lot FF at the University of Maryland, College Park. After the surface plants in the sampling area were removed, topsoil samples to a depth of 21.7 cm were collected using a core sampler with the same diameter as the glass column. The location of sampling is shown in Figure 2, and the characteristics of the soil (S2) are presented in Table 1.

5.3. The effect of ionic strength on pathogen transport and capture
The ionic strength of synthetic runoff containing 200 mg/L sodium bromide was adjusted in the range of 5.8 mM to 50 mM by adding different CaCl₂ concentrations. First, 5.8 mM synthetic runoff with a suspension of B6914 was loaded into a column with conventional bioretetion media to observe the transport and capture of B6914 following the same procedures as described above. Following the 6hr-loading, the media in the column was flushing using synthetic runoff without bromide and B6914 for 14 hr, after which the column was allowed to sit stagnant for two weeks. Then, 50 mM synthetic runoff with a suspension of B6914 was loaded into the column to observe the transport and capture of B6914.

5.4. Pathogen survival in conventional bioretention media
Pathogen survival in conventional bioretention media was evaluated in three separate column experiments. In all three experiments, after the 6 hr stimulated rainfall application, the columns were gravity drained at room temperature (21 + 1 ° C). Subsequently, the columns were allowed to sit stagnant for 0.5, 1.5 or 6.5 days, after which soil samples from seven different depths were aseptically removed and transferred to seven sterile aluminum dishes. A 12 g soil sample from each dish was then diluted at a ratio of 1:10 (by mass) in sterile water and extracted for 2 min by using a Waring blender with a 1-liter glass container. After extraction, the mixtures were allowed to settle for five minutes before dilution and plating from the middle fraction in the blender container (Gagliardi and Karns, 2000). The remaining soil samples in the dishes were dried at 105 °C in an oven for one day to determine soil moisture content (Black, 1965).

To investigate the impact of native microbes on pathogen die-off, the experiments described above for evaluating the survival of strain B6914 over time are being conducted in parallel non-sterile columns and columns sterilized by g-irradiation. The sterile columns were packed with dry conventional bioretention media as described in sections 3 and 4, and then they were gamma-irradiated with a dose of 30 kGy from a Co⁶⁰ source in the Department of Materials Science and Engineering, University of Maryland, College Park.

6. Long-term Sustainability Studies
To evaluate the long-term transport, capture and survival of pathogens in conventional bioretion media, five columns were dry-packed using conventional bioretention media and were set up as described above in sections 3 and 4. These reactors are being loaded once every two weeks at the flow rate of 40 mL/hr with a suspension of B6914 in synthetic runoff. During each 6-hr loading, B6914 numbers in the influent and the effluent are enumerated each hour to evaluate bacterial capture in the columns. In addition, at durations of 2, 5, 9, 13 and 18 months, one of the columns is sacrificed, and the B6914 cells attached to the porous media are enumerated using the extraction method as described in section 5.4 to evaluate pathogen distribution and survival in the conventional bioretention media. At this point, the 2 month columns have been sacrificed and analyzed.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal?
The project objectives have not been changed.

Dissemination activities during this reporting period
An abstract entitled “Column studies on transport and capture of pathogens from urban stormwater runoff using conventional bioretention media” was submitted to the 2nd National Low Impact Development Conference (Wilmington, NC, March, 2007).

Difficulties
To be able to quantitatively separate the bacteria that were added into the column from the native microorganisms present in the conventional bioretention media used, it was necessary to set the concentration of E. coli O157:H7 strain B6914 in the synthetic runoff influent at a concentration of approximately 1.1_10⁶ cells per mL. However, although this concentration facilitated experimentation, it is higher than the concentration of fecal coliform bacteria typically noted in urban stormwater runoff. Average concentrations of fecal coliform bacteria and Escherichia coli bacteria in urban stormwater runoff are reported as 3600 MPN/100 ml and 1450 MPN/100 ml, respectively at typical site (MDE, 2000). This consideration will be discussed as data are evaluated.

Data Generated to date
1. Bacterial physicochemical characterizations
The surface contact angle of strain B6914 was found to be 12+1°, which indicates that strain B6914 is poorly hydrophobic. This finding is consistent with previous studies by Gilber et al. (1991) and van Loosdrecht et al. (1987b) who reported that E. coli NCTC 9002 and E. coli ATCC 8793 respectively, also have low surface contact angles. Thus, it can be concluded that E. coli is a more hydrophilic than a hydrophobic organism (Foppen and Schijven, 2006). Consistent with that observation, the results of the BATH experiment illustrated no significant affinity of strain B6914 towards n-hexadecane or n-octane with only approximately 25% of strain B6914 removed from the aqueous phase by o-xylene (See Figure 3). The use of different volumes of test hydrocarbons did not seem to significantly affect the results. After the hydrocarbon was mixed with the cell suspensions for 2 min, a creamy upper layer was formed. Microscopic examination of the upper layer revealed that the emulsion of o-xylene droplets contained rod-shaped bacteria, while there was no bacteria in the emulsion of n-hexadecane or n-octane droplets. Thus, the results of the BATH experiment further verified that the cell surface of strain B6914, at least as grown for these studies, has a low degree of hydrophobicity. Finally, strain B6914 in synthetic runoff (pH = 7) displayed a negative zeta potential of -2.6 + 0.1 mV, which indicates that this bacterium possesses a weak negative charge.

2. Stability of strain B6914 in the synthetic runoff
Strain B6914 in the synthetic runoff decays very quickly with apparently first-order kinetics and a half-life of approximately 8 hr (See Figure 4). This decay may be attributed to the scarcity of nutrients and toxicity from the used oil or heavy metals in the synthetic runoff. While not surprising, this observation translated into difficulties in maintaining constant cell concentrations in the influent solution for duration of the column experiments. Therefore, to help maintain relatively stable concentrations of strain B6914 in the influent during the 6-hr stimulated rainfall event, new synthetic runoff with a suspension of fresh strain B6914 cells is made and applied to the column every three hours.

3. Pathogen transport and capture
3.1. Pathogen transport and capture by conventional bioretention media
Pathogen transport and capture in the conventional bioretention media was quantified through enumerating B6914 in the influent and effluent of pathogen transport experiments in the same column that were conducted twice at an interval of two weeks. The results of these experiments showed that the breakthrough curves of strain B6914 were very similar (See Figure 5), which indicates that pathogen transport and capture characteristics by conventional bioretention has good reproducibility in the same experiment system.

The transport and capture of B6914 is also being evaluated in the long-term experiments, which have now been conducted for three months, with the columns having been loaded with a strain B6914 suspension in synthetic runoff seven times each. Consistent with the results described above, each of the five columns has demonstrated a stable efficiency for pathogens removal over time. However, when the experiment results for different columns are compared, the breakthrough curves are somewhat different as shown in Figure 6. The removal efficiency of pathogens in the five columns has ranged from 85% to 93%, which may be due to heterogeneities in the physical, chemical, or microbial properties of the mulch or soil, or non-homogenous dry-packing. Nevertheless, in all cases, when strain B6914 in the influent is maintained at a concentration of 1.1 + 0.3 _10⁶ CFU/mL, the conventional biorerention media can achieve 80% or higher pathogen removal in these columns under the tested conditions.

3.2. Comparing transport and capture of pathogens in laboratory bioretention media and media from an established cell
The breakthrough curves of strain B6914 were very close in column studies using the laboratory media and the media from a two-year old bioretention cell at the University of Maryland, College Park (See Figure 7). The high pathogen removal efficiency of both media indicates that pathogen capture by conventional bioretention media is effective, even with the changes in soil structure that may occur over time.

3.3. The effect of ionic strength on pathogen transport and capture
The ionic strength of synthetic stormwater runoff significantly affects the capture of pathogens by the media as expected. Specifically, when the ionic strength in the synthetic runoff was increased from 5.8 mM to 50 mM, removal efficiency of B6914 increased from 84% to 97% (See Figure 8). This can be explained by the concept that the attraction of bacteria to the media surface depends on the thickness of the diffuse electric double layer. Increasing ionic strength leads to a decrease in the thickness of the double layer. As a result, the bacterial cells may be able to come close enough to the media surface for the Van del Waals attraction energies to overcome the repulsion barrier, thus causing more bacteria to be trapped in the column (Stevik et al., 2004).

4. Pathogen survival in conventional bioretention media
While the initial capture of pathogens from stormwater runoff represents the immediate treatment issue, the subsequent pathogen destruction is also critical for making the system sustainable. Therefore, this study has further monitored survival of the trapped pathogens in the media between simulated storm events. The results show that the total number of strain B6914 cells attached to the non-sterile conventional media in the column decreased about 88% after gravity drainage overnight (12 h) (See Table 4). Furthermore, after gravity drainage for 6.5 days, the number of strain B6914 cells attached to the non-sterile conventional media was far below our detection limit (300 CFU/g dry media), indicating a nearly 100% die-off. Similarly, for the two-month column sacrificed in the long-term experiment, the total amount of B6914 cells attached to the non-sterile conventional media in the column decreased approximately 90% after gravity drainage overnight. The rapid decline in the number of surviving trapped strain B6914 cells with time may be attributed to the scarcity of nutrients and predation by native microbes, which further verify the effective sustainability of conventional biorention media for pathogen capture. Additionally, the number of strain B6914 dramatically decreased with respect to depth from the top of the column (See Table 4). One possible explanation for this observation is that the moisture content rapidly increased with depth, while moisture content above a certain optimum level has been shown to result in a decrease in microbial cells in natural soil (Postma and Veen, 1990).

To investigate the impact of native microbes on pathogen die-off, the studies of the survival of strain B6914 over time are being conducted in parallel non-sterile columns and columns sterilized by g-irradiation. The breakthrough curves of B6914 indicated that the non-sterile conventional bioretention media provided better removal efficiency for strain B6914 than the sterile media (See Figure 9). Importantly, the results of the bromide tracer experiments showed that gamma irradiation had no significant effects on porosity and dispersion coefficient of media in the column. This suggests that the difference in the breakthrough curves is not due to changes in the transport properties of the sterilized column. Rather, trapped strain B6914 in the sterile media apparently can survive longer as compared to those in the non-sterile media. A possible reason for higher pathogen survival in the sterile column is that gamma irradiation killed the antagonistic native microorganisms that can predate on E. coli or the microorganisms that compete with E. coli for the scarce nutrients in the media. Additionally, though gamma irradiation of dry soil is expected to have no significant effect on soil pH and pore volume distribution (Lensi et al., 1991), it has been shown that gamma irradiation can increase the available nutrients in the media, such as soluble organic carbon, ammonium nitrogen, exchangeable sulfur, exchangeable Mn, etc (Salonius et al., 1967; Lensi et al., 1991; McNamara et al., 2003), which also could have contributed to the enhanced survival of B6914 in the sterilized column. The pathogen survival in the sterile media with time is being further investigated.

Project Objectives for Next Reporting Period

Objectives
(1) Evaluating engineered bioretention media (e.g., iron oxide-coated sand) for the capture and destruction of pathogens using column studies; (2) Developing a reengineered media design for maximizing pathogen attenuation, and investigating transport and capture in this bioretention system; (3) Observing the “long-term” sustainability.

Work plan to Meet Objectives
The goal for the next reporting period is to complete the remaining Task 1 and Task 2 components, which entail evaluating conventional bioretention media and engineered bioretention media (e.g., iron oxide-coated sand) for the capture and destruction of pathogens using column studies. Based on these results, a reengineered design for maximizing pathogen attenuation will be subsequently developed and investigated in Task 3.

Also during this time, Task 4, “long-term” sustainability studies, will be continuously conducted.

Dissemination Objectives for next reporting period
A draft manuscript for publication will be prepared. Also, contact will be made with the Jug Bay NERR to present project results.

Overall Project Timeline Update
A revised project timeline is presented in Figure 10. The dark bars represent the original timeline. The light bars are the revised timeline.

The project is somewhat behind the initially projected schedule because the laboratory experiments were not initiated until November 2005, and only the basic research approaches were developed during the first phase. Nevertheless, significant progress was made during this phase, and continued progress is expected during the next phase. Therefore, assuming no major problems ensue, we expect some of the backlog in the schedule to be made up in the next phase.

Expenditures
Expenditures are in the range expected for the work accomplished to date.

End User Advisor Feedback
End User Advisor: Christie L. Minami, P.E.
Organization: Maryland State Highway Administration ­ Highway Hydraulics Division
Location: Baltimore, Maryland
Phone number: 410-545-8412
E-mail: CMinami@sha.state.md.us

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
Potential applications for this research include improving stormwater runoff quality management. Governmental transportation departments as well as site developers could use the technology to design better stormwater management practices. Governmental regulatory agencies could use the technology to improve design guidelines and strengthen regulatory requirements and establish new design criteria. Additionally, at this stage, the research opens more questions that may result in continuing research even further and examining other variables not included but brought into attention in the present research endeavor.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
Key challenges include cost justification (the cost of deployment, implementation, and construction materials acquisition must justify the benefits gained), regulatory aspects (the development of this technology could result in new requirements and design criteria for stormwater management facilities), and social acceptance (the public may interpret stormwater management facilities that use this technology as beneficial since pathogens can be removed and thus provide a health and environmental benefit, or as unhealthy cesspools of pathogens and disease regardless of facts presented to reassure them otherwise).

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
This has been the first progress report submitted. Nothing has changed about the project’s potential applicability.

Questions/comments/ suggestions for the researchers?
The project uses “conventional bioretention media.” How was the determination made regarding the definition of “conventional?”

On page 3, conventional bioretention soil is defined as a mix of soil, sand and mulch at a ratio of 5:3:2. Is this correct? SHA uses a mix ratio of 3:5:2. This may result in different behaviors in pathogen survivability and transport, limiting potential benefit to SHA.

On page 3, mulch actually seems to be compost. The two are not the same and should not be treated as such. Please clarify if mulch or compost is used in the study. SHA, for example, uses 2x shredded hardwood bark mulch, aged at least 6 months. This may result in different behaviors in pathogen survivability and transport, limiting potential benefit to SHA.

On page 3, the report seems to indicate that the testing materials (soil, mulch) were screened to a specific size prior to mixing and placement into columns. Field facilities typically do not use screened materials of this magnitude. Could this influence results and cause significant disparities between laboratory data and field data?

A reference on pathogen type and concentration in typical stormwater runoff was not found in the report. Is there such a reference? Was it examined for this study?

On page 4, it is stated that the hydraulic loading representing a common storm was concentrated by a factor of 20 to represent a drainage area to a facility. What is the equivalent drainage area size and is this considered typical? How was the definition of “typical” determined for this research?

On page 6, it was noted that the study was performed at room temperature. What is this temperature considered to be? Were there any variations in temperature? Might temperature affect survivability of pathogens? In the field, temperatures vary by season and may affect results, with increase pathogen activity during summer months. Might this be examined within the scope of this research?

On page 6, a mistake was noted. Section 5.4, second paragraph, the sentence begins “The sterile columns was packed with dry conventional bioretention…” should be “...columns were packed…”

On page 7, a comment on the concentration of fecal coliform typically noted in urban stormwater runoff was stated, however this concentration and reference was not noted. Please provide a reference and concentration amount. Also, is there a typical concentration in natural soil? Perhaps below detection limits? This should be stated somewhere in the report.

On page 8, it was stated that pathogen decay rates may be a result of the concentration of oil in the synthetic runoff. This could be an avenue of study to truly examine how oil affects pathogens, as well as other microbial activity within bioretention soil.

On page 9, the effect of ionic strength on pathogens was examined. How was ionic strength changed? Is this an accepted method?

On page 10, the first sentence “…indicating a nearly 100% died off” should be “…a nearly 100% die-off” or the “a” should be omitted.

On page 11, section 2 part a: Objectives propose the examination of engineered bioretention media, such as iron oxide-coated sand. Is this worthwhile? How easily available is this material? Is the use of such material cost effective?

On page 11, section 2, part a: Objectives propose developing a re-engineered media design for maximizing pathogen attenuation. Is this worthwhile? So far, the research shows at least 80% removal of pathogens. Is this sufficient? With maximized pathogen attenuation, how might this affect plant survivability or other pollutant removal efficiencies?

End User Advisor: Neil Weinstein
Organization: The Low Impact Development Center, Inc.
Location: 4600 Powder Mill Road, Suite 200 Beltsville MD 20705
Phone number: (301) 982 5559
E-mail: nweinstein@lowimpactdevelopment.org

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
The technology has widespread application due the need for low cost and highly effective retrofit technology to address pathogens in non-point stormwater for resource/health protection and regulatory compliance

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
The accumulation of pollutants in the media, the breakthrough points, the effectiveness of the technology when compared to other structural (i.e. proprietary devices) and non-structural (i.e. filter strips) approaches. The need for carefully manufactured media may also be a challenge. There will need to be many more similar studies in various climactic regions and settings to make the results and projections from the research statistically valid for use in resource management and regulatory programs. Field conditions are incredibly variable so there needs to be a significant amount of work done to validate the approach.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
Significant progress has been made and the results are very promising.

Questions/comments/ suggestions for the researchers?
What are effects, if any, of other chemicals or constituents from runoff that may influence results. This includes thermal loading, high and low volumes of runoff, and saturation of media. Discussion of implications for management issues, such as health limits would be useful.

PI Response to End User Advisor Feedback
We appreciate our End User Advisors’ review of our report. Based on these comments, some corrections have been made and some literature citations have been added in the report. Responses to the comments and questions are presented below.

1. The project uses “conventional bioretention media.” How was the determination made regarding the definition of “conventional?”
The bioretention soil mixture, as defined by the Prince George’s County DER (2001), is composed of 50-60% sand, 20-30% leaf compost, and 20-30% top soil (sandy loam, loamy sand, or loam texture per USDA textural triangle) by volume. Conventional bioretention media used in this project is a mixture of sand, sandy loam soil and mulch (leaf compost) at a volume ratio of 5:3:2.

2. On page 3, conventional bioretention soil is defined as a mix of soil, sand and mulch at a ratio of 5:3:2. Is this correct? SHA uses a mix ratio of 3:5:2. This may result in different behaviors in pathogen survivability and transport, limiting potential benefit to SHA.
Conventional bioretention media used in this project is a mixture of sand, sandy loam soil and mulch at a volume ratio of 5:3:2. The text has been corrected in the report.

3. On page 3, mulch actually seems to be compost. The two are not the same and should not be treated as such. Please clarify if mulch or compost is used in the study. SHA, for example, uses 2x shredded hardwood bark mulch, aged at least 6 months. This may result in different behaviors in pathogen survivability and transport, limiting potential benefit to SHA.
Leaf compost was used in the laboratory column experiments for our project because hardwood bark mulch pieces would be too large and problematic for the columns. We do not expect this variation to significantly impact our pathogen performance results.

4. On page 3, the report seems to indicate that the testing materials (soil, mulch) were screened to a specific size prior to mixing and placement into columns. Field facilities typically do not use screened materials of this magnitude. Could this influence results and cause significant disparities between laboratory data and field data?
The study of hydrodynamic dispersion by Klotz and Moser (1974) indicated that the ratio of column diameter to average media diameter (d₅₀) must be greater than 25 in order to neglect boundary disturbances. Due to the small size of columns (diameter 2.5 cm) used in this project, the sandy loam soil, sand and mulch were sieved through a 1.18 mm sieve prior to mixing the conventional bioretention media. The d₅₀ of the mixture is 0.74 mm. Thus, the ratio of column diameter to average media diameter (d₅₀) is 34, which is bigger than 25. This issue will be examined in our upcoming field studies.
Klotz, D., Moser, H. 1974. Hydrodynamic dispersion as aquifer characteristic: model experiments with radioactive tracers, pp. 341-355. In: Isotope techniques in groundwater hydrology, Vol. 2. International Atomic Energy Agency, Vienna.

5. A reference on pathogen type and concentration in typical stormwater runoff was not found in the report. Is there such a reference? Was it examined for this study?
The bacteria type in stormwater runoff and the reason that E. coli O157:H7 strain B6914 is a good choice are presented on Pages 1 & 2. Average concentrations of fecal coliform bacteria and Escherichia coli bacteria in urban stormwater runoff are listed on Page 7. Additional data will be reported from the upcoming field tests.

6. On page 4, it is stated that the hydraulic loading representing a common storm was concentrated by a factor of 20 to represent a drainage area to a facility. What is the equivalent drainage area size and is this considered typical? How was the definition of “typical” determined for this research?
References about this concern are provided on Page 4,

7. On page 6, it was noted that the study was performed at room temperature. What is this temperature considered to be? Were there any variations in temperature? Might temperature affect survivability of pathogens? In the field, temperatures vary by season and may affect results, with increase pathogen activity during summer months. Might this be examined within the scope of this research?
The study was performed at room temperature (21 + 1 ° C). The effects of temperature and moisture content on pathogen capture and transport will be investigated in Task 3.

8. On page 6, a mistake was noted. Section 5.4, second paragraph, the sentence begins “The sterile columns was packed with dry conventional bioretention…” should be “...columns were packed…”
This typing mistake has been corrected.

9. On page 7, a comment on the concentration of fecal coliform typically noted in urban stormwater runoff was stated, however this concentration and reference was not noted. Please provide a reference and concentration amount. Also, is there a typical concentration in natural soil? Perhaps below detection limits? This should be stated somewhere in the report.
The concentrations of fecal coliform bacteria and Escherichia coli bacteria in typical urban stormwater runoff have been added on Page 7.

10. On page 8, it was stated that pathogen decay rates may be a result of the concentration of oil in the synthetic runoff. This could be an avenue of study to truly examine how oil affects pathogens, as well as other microbial activity within bioretention soil.
This is true, but it is beyond the scope of this study.

11.On page 9, the effect of ionic strength on pathogens was examined. How was ionic strength changed? Is this an accepted method?
The ionic strength of synthetic runoff was adjusted by adding different CaCl₂ concentrations. The method has been presented on Pages 5 & 6.
Some salts (such as NaCl, CaCl₂, KCl, etc.) are commonly used to adjust ionic strengthen for the study of bacteria transport and deposition (Martin et al., 1992; Powelson and Mills, 2001). Calcium is the dominant cation in the synthetic runoff (See Table 1), thus CaCl₂ was chosen to adjust ionic strength of synthetic runoff in this study.

Martin, R.E., Bouwer, E.J., Hanna, L.M. 1992. Application of clean-bed filtration theory to bacterial deposition in porous media. Environ. Sci. Technol. 26, 1053-1058.
Powelson, D.K., Mills, A.L. 2001. Transport of Escherichia coli in sand columns with constant and changing water contens. J. Environ. Q. 30, 238-245.

12. On page 10, the first sentence “…indicating a nearly 100% died off” should be “…a nearly 100% die-off” or the “a” should be omitted. The reviewer is correct and the text has been edited.

13. On page 11, section 2 part a: Objectives propose the examination of engineered bioretention media, such as iron oxide-coated sand. Is this worthwhile? How easily available is this material? Is the use of such material cost effective?
Iron oxide-coated sand (IOCS) has been found to be very effective in capturing bacteria (Mills et al., 1994; Knapp et al., 1998). The removal coefficient and practical applicability of employing this material in bioretention will be investigated in subsequent experiments.

Knapp, E.P., Herman, J.S., Hornberger, G.M., Mills, A.L. 1998. The effect of distribution of iron-oxyhydroxide grain coatings on the transport of bacterial cells in porous media. Environ. Geol. 33, 243-248.
Mills, A. L., Herman, J.S., Hornberger, G.M., DEJESÚS, T.H. 1994. Effect of solution ionic strength and iron coatings on mineral grains on the sorption of bacterial cells to quartz sand. Appl. Environ. Microbiol. 60, 3300-3306.

14. On page 11, section 2, part a: Objectives propose developing a re-engineered media design for maximizing pathogen attenuation. Is this worthwhile? So far, the research shows at least 80% removal of pathogens. Is this sufficient? With maximized pathogen attenuation, how might this affect plant survivability or other pollutant removal efficiencies?
Our results to date find that conventional bioretention media can achieve greater than 80% removal of pathogens. However, if pathogen concentrations in the influent are too high (such as during individual storms or for stormwater hotspots), pathogen concentrations in the effluent may still be higher than water quality standards for indicator bacteria. Thus, evaluating the engineered media for the transport and destruction of pathogens and developing a re-engineered media are worthwhile.
The impact of adding engineered media on plant survivability and other pollutant removal efficiencies will be considered during final project evaluations.

15. What are effects, if any, of other chemicals or constituents from runoff that may influence results. This includes thermal loading, high and low volumes of runoff, and saturation of media. Discussion of implications for management issues, such as health limits would be useful.
The effect of ionic strength on transport and capture of pathogens has been investigated. The used oil or heavy metals in the synthetic runoff may be toxic to E. coli O157:H7 strain B6914 and make it decay quickly. However, studies on the effect of heavy metals and used oil are beyond the scope of this study. Some management issues (such as health limits) will be discussed based on the data of field tests.

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