Environ. Sci. Technol. 2010, 44, 631–637

and in situ treatment technologies (8, 9). Even with these Ecological Control of Fecal Indicator tools, water-quality managers are often faced with the difficult in an Urban Stream task of developing and implementing total maximum daily loads (TMDLs) for FIB-impaired water bodies in the face of uncertainty about the root causes of FIB impairment and †,‡ CRISTIANE Q. SURBECK, associated human health risks. Documented sources of § SUNNY C. JIANG, AND uncertainty include (a) the many point and nonpoint sources ,‡ STANLEY B. GRANT* of fecal pollution that potentially contribute FIB in a particular Department of Chemical Engineering and Materials Science water body (10); (b) the spatially and temporally variable and Department of Civil and Environmental Engineering, nature of FIB sources and the fate and transport processes Henry Samueli School of Engineering, University of that disperse them (11); (c) growth of FIB in some aquatic California, Irvine 92697-7070 environments (12–15); (d) the existence of FIB in both culturable and nonculturable states (16); and (e) the opera- Received November 18, 2009. Accepted December 2, 2009. tionally defined nature of FIB assays, by which different EPA- approved assays can yield different estimates for FIB concentration in the same sample (17). Against this complex backdrop, there are currently over 10 000 FIB (or “”) Fecal indicator bacteria (FIB) have long been used as a impairments in the U.S., and in southern California, the cost marker of fecal pollution in surface waters subject to point of implementing control measures in even relatively small source and non-point source discharges of treated or untreated (340 km2) catchments could exceed $100 million (18). In this human waste. In this paper, we set out to determine the study, we describe coordinated field, laboratory, and model- source(s) of elevated FIB concentrations in Cucamonga Creek, ing studies aimed at identifying the nonpoint sources of FIB a concrete-lined urban stream in southern California. Flow in impairment in a prototypical concrete-lined urban stream the creek consists primarily of treated and disinfected wastewater in southern California. Description of Field Site. Cucamonga Creek is a concrete- effluent, mixed with relatively smaller but variable flow of lined flood control channel located in a highly urbanized runoff from the surrounding urban landscape. Dry and wet region of San Bernardino County, southern California (Figure weather runoff contributes nearly 100% of FIB loading to 1). The creek is a tributary to the Santa Ana River, which Cucamonga Creek, while treated wastewater contributes drains three of the 12 most populous counties in the United significant loading of nutrients, including dissolved organic States. Flow in the Santa Ana River is a mixture of effluent carbon (DOC), phosphorus, nitrate, and ammonium. FIB from wastewater treatment plants, urban runoff, and ground- concentrations are strongly positively correlated with DOC water and surface water runoff from the undeveloped San concentration in runoff (Spearman’s F g 0.66, P e 0.037), and Gabriel Mountains (19). The Santa Ana River is used both for microcosm studies reveal that the survival of Escherichia recreation and to recharge a groundwater aquifer that serves coli and enterococci bacteria in runoff is strongly dependent as a primary source of potable water for Orange County residents. Both Cucamonga Creek and the Santa Ana River on the concentration of both DOC and phosphorus. Below threshold are on the U.S. EPA’s 303(d) list as impaired for coliform concentrations of 7 and 0.07 mg/L, respectively, FIB die off bacteria and . The reach of Cucamonga Creek -1 exponentially (die-off rate 0.09 h ). Above these thresholds, FIB selected for this study has a drainage area of approximately -1 either grow exponentially (growth rate 0.3 h ) or exhibit a 200 km2, is bounded by a major freeway (I-60) to the north periodic steady-state in which bacterial concentrations fluctuate and the Prado Wetlands to the south, and receives a discharge around some mean value. The periodic steady-state pattern of ∼0.7 m3/s of highly treated and disinfected wastewater is consistent with a Lotka-Volterra predator-prey oscillation effluent from the Inland Empire Utilities Agency Regional model, and the clearance rate (20 µL predator-1 h-1) obtained Plants 1 and 4 (number 3 on the map in Figure 1). by fitting the model to our data is consistent with the hypothesis Downstream of the discharge, during dry weather, flow in that predacious regulate FIB concentrations in the creek consists of approximately 1 part surface runoff to 10 parts treated wastewater. During wet weather, storm runoff runoff at high DOC concentrations. Collectively, these results contributes substantial flow in the creek, upward of 2 m3/s. indicate that FIB impairment of Cucamonga Creek is best viewed as an ecological phenomenon characterized by both bottom- Methods and Materials up and top-down control. Creek Sampling. Referring to the map in Figure 1, sampling sites on Cucamonga Creek were selected to provide infor- Introduction mation on the quality of source waters (runoff in the channel at site A, runoff from the storm drain at site E, and tertiary Pathogens and fecal indicator bacteria (FIB) are the most treated wastewater at site B), and downstream mixtures of frequent causes of surface water impairment in the U.S. after sources waters (sites C, D, F, G, and H). Field sampling metals (1). Reflecting the global dimension of this problem occurred during seven events between July 2005 and October (2), an array of new tools have recently been developed to 2006, capturing a range of air temperatures (daily mean values help assess and manage FIB impairments, including mi- from9to22°C) and antecedent dry periods (from 0 to 60 crobial source tracking methods (3), predictive models (4–7), days) (Figure 1). Depending on flow conditions in the channel, safety concerns did not allow all sites to be sampled during * Corresponding author telephone: 949-824-8277; fax: 949-824- every event (Table S1, Supporting Information). Creek 2541; e-mail: [email protected]. sampling included in situ measurements of dissolved oxygen, † Current address: Department of Civil Engineering, The University of Mississippi, University, MS, 38677-1848. salinity, conductivity, pH, and temperature (Horiba U-10 ‡ Department of Chemical Engineering and Materials Science. hand-held meter, Horiba, Ltd., Kyoto, Japan) and collection § Department of Civil and Environmental Engineering. of grab samples analyzed for the FIB (EC)

10.1021/es903496m  2010 American Chemical Society VOL. 44, NO. 2, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 631 Published on Web 12/22/2009 FIGURE 1. Map of Cucamonga Creek sampling sites (left panel) and weather conditions during the seven sampling events (indicated by vertical dashed lines) including precipitation and daily minimum, mean, and maximum air temperature (right panel). and enterococci bacteria (ENT) using chromogenic substrate soy broth, 0.3% yeast extract, and 1.0% lactose in sterile water) assays known commercially as Colilert-18 and Enterolert, (21). On this single occasion, aliquots from the microcosm implemented in a 97-well Quanti-tray format (20). Grab were analyzed for ENT using both culture-dependent (En- samples were also collected for measurement of dissolved terolert) and culture-independent (Q-PCR analysis for 23SrD- organic carbon (DOC), soluble phosphorus, nitrate and NA specific to ENT) assays. The protocol for the culture- ammonium. The volumetric discharge of treated wastewater independentassayispresentedintheSupportingInformation. to Cucamonga Creek (at site C) and the stream discharge in Statistical Analyses. Spearman’s rank correlation (F) for Cucamonga Creek downstream of the confluence with treated nonparametric data was used to quantify the covariation wastewater (at site 6) were recorded during the sampling between measured variables (SPSS v. 15, Chicago, IL). FIB events, as were measurements of air temperature and rate constants were calculated by linear regression of the precipitation. Information on sampling protocols, analysis natural logarithm of the FIB concentrations against time and procedures, and flow and meteorological data acquisition equating the slope of the line to -k or µ (for die-off or growth, are reported in the Supporting Information. respectively). Microcosm Experiments. Coincident with six of the seven sampling events described above (all except 7/6/05), large Results volume (ca., 4 L) samples of runoff (from sites A and/or E) Nutrient and FIB Measurements in Cucamonga Creek. and treated wastewater (site B) were collected. Splits of the Spatial and Temporal Patterns. FIB concentrations were near water samples were filter sterilized by passage through a 0.2 or below the detection limit (10 or 100 MPN/100 mL) in all µm filter (Fisher Scientific, Waltham, MA), and mixtures of water samples collected from treated wastewater and were unfiltered and filter-sterilized waters were prepared in 500 significantly higher (ranging from 100 to over 40 000 MPN/ mL Erlenmeyer flasks (see Table S2 of the Supporting 100 mL) in water samples collected from surface runoff Information for the set of mixtures prepared on each sampling (Figure 2, left panel). The log of FIB concentrations in runoff date). Microcosms were incubated in the dark for at least collected from sites A and E rise and fall synchronously 12 h in a constant temperature incubator (model 2325, (Spearman’s F)0.64, P ) 0.086 and F)0.88, P ) 0.0094, Sheldon Manufacturing, Cornelius, OR) held at 30 °C, typical for EC and ENT, respectively), even though these two of summertime conditions. Aliquots collected from the locations drain different subdrainages within the Cucamonga microcosms during the incubation period were analyzed for watershed. During the first two sampling events (July and EC and ENT using Colilert-18 and Enterolert. Two additional October 2005), FIB concentrations measured in the down- microcosms were prepared on 5/06/06 to determine if stream reach of Cucamonga Creek were similar to, or higher chlorine-injured ENT cells in the treated wastewater could than, FIB concentrations measured in runoff (compare black be resuscitated after exposure to either filter-sterilized runoff and blue curves, Figure 2). After January 2006, FIB concen- or excess nutrients in the form of nutrient broth (5.5% tryptic trations at downstream sites were intermediate between

632 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 2, 2010 FIGURE 2. (Left panel) Time series plots of FIB and nutrient concentrations in samples of runoff (sites A and E, blue curves), treated wastewater effluent (site B, red curves), and downstream of the confluence with runoff and treated wastewater (sites G and H, black curves). (Top right panel) Flow circuit used to solve for the pollutant loading in Cucamonga Creek attributable to runoff from sites A and E. Symbols are defined in the text. (Bottom right panel) Estimated fractional runoff loading for selected parameters.

treated wastewater and runoff (EC) or more similar to Creek attributable to sources of surface runoff, fL,SR (derivation wastewater (ENT). in Supporting Information). Nutrients and other water-quality parameters exhibit C (C - C ) analyte-specific temporal and spatial variability (left panel, ) SR DS TWW fL,SR - (1) Figure 2). Phosphorus and nitrate concentrations in the CDS(CSR CTWW) downstream reach of Cucamonga Creek are similar to concentrations measured in treated wastewater. For the other The fraction of pollutant loading attributable to waste- analytes, parameter concentrations measured at downstream water is the complement of eq 1, fL,TWW ) 1 - fL,SR.Ineq1, sites are higher than both surface runoff and treated C represents pollutant concentration, and the subscripts wastewater (oxygen) or exhibit a mixed pattern (ammonium, denote different sampling locations within the flow circuit, conductivity). Time series measurements of oxygen, con- including surface runoff (“SR”, consisting of runoff, “RO”, ductivity, temperature, and pH are plotted in Figure S1 from site A and storm drain flow, “SD”, from site E), treated (Supporting Information). wastewater (“TWW”, site B), and downstream of the con- Loading Analysis. Given the data presented above, together fluence of runoff and treated wastewater (“DS”, sites G and with flow data from the wastewater treatment plant at site H). Equation 1 was applied after separating pollutant B and streamflow at site 6, it should be straightforward to measurements into dry weather (7/6/05, 12/14/05, 10/12/ estimate nutrient and FIB loading to Cucamonga Creek from 06) and wet weather (10/17/05, 2/17/06, 3/1/06, 5/24/06) treated wastewater and runoff. However, the flow data did periods. The assignment of a sampling date as “dry” and not comport with field observationssin particular, during “wet” was based on antecedent dry period (>5 and <5 days) dry weather the observed ratio of runoff to wastewater in the and accumulated precipitation in the previous two days (0 stream was approximately 1:10, while the flow data yielded and >0 cm). a ratio closer to 1:1ssuggesting that one or both flow Fractional runoff loading estimated from eq 1 is plotted measurements were inaccurate. Instead, we estimated the for FIB and nutrients in Figure 2 (lower right panel). The contribution of runoff and wastewater to pollutant loading calculation indicates that ∼100% of wet and dry weather FIB directly from measurements of pollutant concentration in loading is attributable to runoff, while treated wastewater the creek and source waters. This was accomplished by accounts for a larger portion of creek loading for nitrate (100% performing a steady-state mass and volume balance over and 71%), DOC (80 and 72%), and phosphorus (65 and 53%). the flow circuit illustrated in Figure 2 (top right panel). The The contribution of runoff to pollutant loading in Cucamonga analysis yields the following equation for the fraction of Creek is consistently higher during wet weather, although pollutant loading in the downstream reach of Cucamonga the large error bars imply considerable variability within wet

VOL. 44, NO. 2, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 633 TABLE 1. Microcosms Prepared from Surface Water Runoffa

Escherichia coli Enterococcus spp.

DOC P ln(Co, µ or k ln(Co, µ or k date mixture (mg/L) (mg/L) MPN/100 mL) (h-1) remarks MPN/100 mL) (h-1) remarks 10/17/05 RO (100%) 14 0.32 9.4 (0.1) 0.2 (0.03) strong growth 9.9 (0.2) ND periodic ss SD (100%) 11 0.26 9.3 (0.1) 0.3 (0.03) strong growth 9.4 (0.2) ND periodic ss RO (50%) + TWWfs (50%) 11 0.25 8.6 (0.2) 0.2 (0.04) strong growth 9.3 (0.3) ND periodic ss RO (33%) + TWWfs (33%) + SDfs (33%) 12 0.25 7.9 (0.3) 0.3 (0.06) strong growth 9.0 (0.2) ND periodic ss SD (33%) + TWWfs (33%) + ROfs (33%) 12 0.25 8.3 (0.2) 0.3 (0.03) strong growth 8.1 (0.3) ND periodic ss 12/14/05 RO (100%) 7.1 0.54 8.3 (0.05) ND periodic ss 5.3 (0.2) ND periodic ss RO (10%) + TWWfs (90%) 7.4 0.70 6.0 (0.28) ND periodic ss <4.6 - below detect 2/17/06 RO (100%) 7.9 0.060 7.1 (0.2) ND periodic ss 6.6 (0.3) ND periodic ss RO (75%) + TWWfs (25%) 7.5 0.051 6.6 (0.2) ND periodic ss 6.5 (0.2) ND periodic ss RO (50%) + TWWfs (50%) 6.8 0.043 6.7 (0.2) ND periodic ss 5.4 (0.3) ND periodic ss RO (25%) + TWWfs (75%) 7.2 0.034 5.9 (0.3) ND periodic ss 4.9 (0.2) ND periodic ss 3/1/06 RO (100%) 4.5 0.025 7.7 (0.1) 0.09 (0.01) decay 7.0 (0.2) 0.1 (0.02) decay RO (75%) + TWWfs (25%) 6.8 0.036 7.3 (0.2) 0.1 (0.02) decay 6.7 (0.4) 0.3 (0.1) decay RO (50%) + TWWfs (50%) 6.7 0.048 6.9 (0.2) 0.09 (0.03) decay 5.6 (0.7) 0.08 (0.2) decay RO (25%) + TWWfs (75%) 6.0 0.059 6.3 (0.2) 0.08 (0.02) decay 5.3 (0.3) 0.1 (0.09) decay 5/24/06 TWW (50%)+ NB (50%) 9500 N/A <2.3 N.D. no change 0.7 (0.5) 0.4 (0.03) growth 10/12/06 RO (100%) 7.8 0.23 10 (0.2) 0.15 (0.03) strong growth 8.3 (0.2) ND periodic ss RO (50%) + TWWfs (50%) 6.5 0.23 10 (0.2) 0.2 (0.04) strong growth 7.4 (0.2) ND periodic ss RO (50%) + TWW (50%) 6.5 0.23 9.8 (0.2) 0.3 (0.05) strong growth 7.5 (0.2) ND periodic ss a RO ) runoff from Cucamonga Creek, site A; SD ) runoff from storm drain, site E; TWW ) treated wastewater, site B; fs ) filter-sterilized; DOC ) dissolved organic carbon; P ) phosphorus; ln(Co) ) natural log of initial concentration; µ ) kinetic growth constant; k ) kinetic die-off constant; ss ) steady state; N.D. ) not determined. Standard deviations are indicated in parentheses. and dry categories. Oxygen and ammonium were excluded lert) and culture-independent (qPCR) methods (Figures S10 from the loading analysis because their concentrations at and S12, Supporting Information). As expected, ENT con- downstream sites (CDS) were frequently not bracketed by centrations measured with the culture-independent method source water concentrations (CSR and CTWW)(Figures 2 and were 100-1000 times higher than concentrations measured S1). Assumptions and limitations associated with eq 1 are with the culture-dependent method. discussed in the Supporting Information. Microcosms Prepared from Runoff. Eighteen micro- Covariation of FIB and Nutrients in Runoff. Pairwise cosms were prepared with unfiltered runoff (either RO or correlations between FIB and nutrient concentrations in SD), of which six consisted of unfiltered runoff alone (either surface water runoff from the Cucamonga Creek watershed RO or SD), nine were binary mixtures of unfiltered runoff were computed to identify nutrients that may promote the (either RO or SD) and filtered treated wastewater (TWWfs), growth of FIB. The only Spearman’s rank correlation that one was a binary mixture of RO and TWW, and two were reached a significance level of P < 0.05 was between log- ternary mixtures of unfiltered water from one runoff source transformed FIB and DOC (F)0.66, P ) 0.020 and F)0.61, (RO or SD), filtered water from the other runoff source (either P ) 0.037 for EC and ENT, respectively). SDfs or ROfs), and filtered treated wastewater (TWWfs) (Tables Microcosm Studies. A total of 57 microcosms were 1 and S2, Supporting Information). prepared from samples of treated wastewater at site B Microcosms prepared from unfiltered runoff exhibited (designated “TWW”), surface water runoff from Cucamonga one of three FIB survival patterns illustrated in Figure 3: (1) Creek at site A (designated “RO”), and surface water runoff exponential growth of FIB characterized by a growth rate µ from the storm drain at site E (designated “SD”). In the results (Figure 3A); (2) exponential decline of FIB characterized by presented below, source waters that have been filter-sterilized die-off rate k (Figure 3B); and (3) a pattern we referred to as are indicated by the subscript “fs”. “periodic steady state” in which FIB concentrations fluctuated Negative Controls. Twelve of the microcosms were around a relatively constant value (Figure 3C). The survival negative controls, carried out to demonstrate that FIB were pattern (growth, die-off, or periodic steady state) observed removed from source waters after passage through a 0.2 µm for each microcosm treatment is summarized in Table 1, filter (Table S2, Supporting Information). As expected, FIB along with estimated values for µ or k. EC grew in microcosms concentrations were below the detection limit (10 or 100 containing RO or SD on 10/17/05 and 10/12/06. EC and ENT MPN/100 mL) in all microcosms containing only ROfs,SDfs, died off in microcosms containing RO on 3/1/06. ENT or TWWfs. exhibited a periodic steady state in RO collected on 10/17/ Microcosms Prepared from Treated Wastewater. Fifteen 05, 12/14/05, 2/17/06, and 10/12/06. EC exhibited a periodic microcosms were prepared with unfiltered TWW, of which steady state in RO collected on 12/14/05 and 2/17/06. four had only TWW, eight were mixtures of TWW and filter- Nutrient Concentrations and FIB Survival Patterns. The sterilized surface water runoff (either ROfs or SDfs), and one three FIB survival patterns noted above appear to be was an equal mixture of TWW and nutrient broth (Table S2, associated with specific concentration ranges of DOC and Supporting Information). All microcosms containing unfil- phosphorus. EC and ENT die off in microcosms containing tered TWW had initial FIB concentrations below the detection low initial concentrations of both DOC and phosphorus (<7 limit (10 or 100 MPN/100 mL). With one exception, FIB mg/L and <0.07, respectively) (black filled circles, Figure 4A). concentrations remained below the detection limit through- ENT exhibits periodic steady state in all microcosms with out the incubation period (12-34 h). In the microcosm initial DOC concentrations >7 mg/L (gray filled and open containing nutrient broth, EC remained below the detection circles, Figure 4A), while EC exhibits periodic steady state limit throughout the incubation period, but ENT increased over a more narrow range of initial DOC concentrations, with time as measured by both culture-dependent (Entero- between 7 and 8 mg/L (gray filled circles, Figure 4A). The

634 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 2, 2010 FIGURE 4. Influence of nutrients on FIB survival patterns FIGURE 3. Three different FIB survival patterns observed in observed in microcosms prepared from unfiltered runoff. (A) microcosms containing unfiltered runoff: (A) exponential growth Relationship between initial DOC and phosphorus concentration of EC in the microcosm containing runoff from site E on 10/17/ and the type of FIB survival pattern observed. (B) Contour plot 05, (B) exponential die-off of EC in the microcosm containing of EC die-off (negative contours )-k) and EC growth (positive runoff from site A on 3/1/06, and (C) periodic steady state of contours ) µ) relative to the initial concentration of ENT in the microcosm containing an equal mixture of runoff phosphorus and DOC in the microcosm. (C) Abrupt transition from site A and filter-sterilized treated wastewater from site B from die-off to growth that occurs as DOC concentrations are on 10/17/05. All logarithms are base e. increased beyond 7 mg/L. Note that, to emphasize the change from die-off to growth along arrow A, the two highest initial phosphorus concentrations are not included in panel B. growth rate of EC appears to be affected by both initial DOC and phosphorus concentrations, as documented in the ) ( -1 contour plot in Figure 4B. EC transitions from die-off to (µmax 0.75 0.24 h ) obtained by averaging 17 independent growth with increasing initial phosphorus concentration at estimates of µmax for EC growth on glucose in the laboratory fixed initial DOC concentration (arrow A, Figure 4B). The (22). growth rate of EC approximately doubles (from 0.15 to 0.3 h-1) with increasing initial DOC concentration at fixed initial Discussion phosphorus concentration (arrow B, Figure 4B). The abrupt There is no question that FIB are a useful sentinel for fecal transition between die-off and growth at an initial DOC pollution in surface waters impacted by point sources, such concentration of ∼7 mg/L is illustrated for EC in Figure 4C. as outfalls of untreated or partially treated sewage (23). Their For DOC < 7 mg/L, EC die off with time at a rate of utility as a marker for fecal pollution is more uncertain in approximately 0.09 h-1. For DOC > 7 mg/L EC grow with settings that lack an obvious point source of sewage. On the time at a rate of approximately 0.3 h-1. This latter growth one hand, a recent study conducted in Melbourne, Australia rate is approximately one-half the maximum growth rate found that EC concentration in tributaries of the Yarra River

VOL. 44, NO. 2, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 635 was a marker for nonpoint sources of sewage from septic population model that assumes bacterial growth is balanced tank leach fields located near the river bank (24). On the by consumption of bacteria by a predator. The Lotka-Volterra other hand, numerous studies document the environmental predator-prey oscillation model (34) was fit to ENT con- growth of FIB in fresh and marine waters and sediments centrations measured in the microcosm consisting of a 1:1 (12–14, 25–30). To the extent that FIB grow in the environ- mixture of unfiltered runoff from site A and filter-sterilized ment, they will likely have little value as a sentinel for fecal treated wastewater from site B (see the Supporting Informa- pollution or as a predictor of recreational waterborne illness tion). Plots of the model-predicted prey (solid red line) and (31). In part to address these concerns, the U.S. Environ- predator (dashed red line) concentrations are superimposed mental Protection Agency is currently conducting epide- on measurements of ENT in Figure 3C. The prey predictions miologic studies to assess the human health effects of correctly capture the period and magnitude of ENT cycles recreating in surface waters contaminated by nonpoint source (compare data points with red solid line). The model derived pollution (32). These new epidemiologic data are expected clearance ratesdefined as the volume of water from which to inform the development of new ambient water quality bacterial cells are completely removed by a predator per criteria which should be released in 2011. unit of timesof ∼20 µL grazer-1 h-1 is similar to clearance The field and laboratory data presented in this study were rates (∼10 µL grazer-1 h-1) reported for rotifers and other focused around identifying sources of FIB contamination in protists that graze on heterotrophic bacteria in aquatic Cucamonga Creek, a concrete-lined, effluent-dominated environments (35) but 2 orders of magnitude greater than stream in southern California. Because the wastewater values reported for host-specific viral predation (∼0.1 µL effluent discharged to this stream is disinfected, FIB in the -1 h-1)(36). Protistan grazing has been suggested as the stream are likely from nonpoint sources, such as bird and primary mechanism by which allochthonous bacteria, such dog feces, homeless persons, illegal dumping, illicit con- as EC and ENT, are removed from stream ecosystems (37, 38) nections to the storm sewer system, and/or environmental and coastal waters (39), and it is generally regarded as the growth. Mass and volume balance calculations indicate that primary mechanism that regulates prokaryotic biomass in treated wastewater is not a significant source of FIB to the aquatic environment (40). Cucamonga Creek. Further, FIB were detected in only one microcosm constructed from treated wastewater and only The results presented in this paper suggest that the then after the wastewater was mixed with nutrient brothsa environmental growth of FIB in urban streams, such as state of nutrient abundance unlikely to be replicated in the Cucamonga Creek, requires ambient DOC concentrations field. In the microcosm containing treated wastewater and in excess of 7 mg/L. Once DOC concentrations exceed this nutrient broth, both culture-dependent and culture-inde- threshold, the final FIB concentration will likely be deter- pendent methods demonstrated substantial increases in the mined by resource competition among the heterotrophic concentration of ENT with time, implying that new ENT cells bacteria present in the stream (“bottom-up control”) and/or were being created. Thus, the increase in culturable ENT predation by (presumably lytic bacteriophage) and measured in this microcosm cannot be attributed solely to protistan grazers (“top-down control”). Recent studies of resuscitation of chlorine-injured ENT released with the natural microbial populations suggest that bottom-up control wastewater effluent. may prevail in nutrient rich (eutrophic) environments, while Runoff from the urban landscape appears to be the top-down control may prevail in nutrient poor (oligotrophic) primary source of FIB loading to Cucamonga Creek during environments (40). This observation implies that DOC and both dry weather and wet weather periods. FIB survival in FIB concentrations in runoff should covary, which is indeed microcosms containing unfiltered runoff appears to be the case both at Cucamonga Creek and in many agricultural modulated by nutrient concentrations, in particular DOC and urban streams along the California coast (41). These and phosphorus. Below threshold concentrations of 7 and results are not consistent with the hypothesis that FIB are 0.07 mg/L, respectively, FIB decay with time. Above these static contaminants (like sediments or nutrients) with well- thresholds, EC and ENT exhibit either exponential growth or defined and land-use-specific export coefficients, as has been a periodic-steady state pattern in which bacterial concentra- suggested for catchments in the United Kingdom (42). Rather, tions fluctuate around some mean value. These nutrient our data suggest that nonpoint source FIB impairments in thresholds are not an artifact of weather conditions at the southern California are best viewed as an ecological phe- time of sampling, because EC grew in microcosms prepared nomenon, in which a dynamic balance between FIB sources, from runoff collected during both wet weather (10/17/05) nutrient availability, competition with other heterotrophic and dry weather (10/12/06) (Table 1). Intriguingly, these bacteria, and predator prevalence determines the magnitude microcosm results mirror, at least for DOC, what we observed and extent of FIB pollution and its human health implications. during the field sampling campaign at Cucamonga Creek: of all the nutrients measured, only DOC was significantly and Acknowledgments strongly correlated with FIB concentrations in runoff from sites A and E. This study was funded by a joint grant from the National The lowest phosphorus concentration measured in our Water Research Institute (03-WQ-001) and the US Geological runoff samples (ca., 25 µg/L) is about 5 times growth-limiting Survey National Institute for Water Research (UCOP-33808), values reported for heterotrophic bacteria growing in the with matching funds from the County of San Bernardino in laboratory (160 nM or 5 µg/L) (33). Because FIB in the California. C.Q.S. was funded by the United States Envi- environment must compete with other, perhaps better ronmental Protection Agency (EPA) under the Science to adapted, heterotrophic bacteria for scarce resources (33), it Achieve Results (STAR) Graduate Fellowship. EPA has not is possible that FIB die-off observed for phosphorus con- officially endorsed this publication, and the views expressed centrations <0.07 mg/L is caused by phosphorus starvation. herein may not reflect the views of the EPA. The authors It should be noted that the analytical method used to measure thank the San Bernardino Flood Control District for providing soluble phosphorus detects primarily orthophosphate, and the motivation for this study and the staff to support sampling consequently, total phosphorus in the runoffswhich includes operations. Numerous UCI students participated in the organic phosphorus and polyphosphatesscould be higher sampling and laboratory activities; without them, this study than reported here. would not have been possible. Three anonymous reviewers The periodic steady-state pattern observed in our mi- provided constructive comments for the improvement of crocosm experiments closely resembles the predictions of a this paper.

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