Factors affecting the relationship between quantitative polymerase chain reaction (qPCR) and culture-based enumeration of Enterococcus in environmental waters.

AIMS: To determine the extent to which discrepancies between qPCR and culture-based results in beach water quality monitoring can be attributed to: (i) within-method variability, (ii) between-method difference within each method class (qPCR or culture) and (iii) between-class difference. METHODS AND RESULTS: We analysed 306 samples using two culture-based (EPA1600 and Enterolert) and two qPCR (Taqman and Scorpion) methods, each in duplicate. Both qPCR methods correlated with EPA1600, but regression analyses indicated approximately 0·8 log10 unit overestimation by qPCR compared to culture methods. Differences between methods within a class were less than half of this and were minimal for between-replicate within a method. Using the 104 Enterococcus per 100 ml management decision threshold, Taqman qPCR indicated the same decisions as EPA1600 for 87% of the samples, but indicated beach posting for unhealthful water when EPA1600 did not for 12% of the samples. After accounting for within-method and within-class variability, 8% of the samples exhibited true between-class discrepancy where both qPCR methods indicated beach posting while both culture methods did not. CONCLUSION: Measurement target difference (DNA vs growth) accounted for the majority of the qPCR-vs-culture discrepancy, but its influence on monitoring application is outweighed by frequent incorrect posting with culture methods due to incubation time delay. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to quantify the frequency with which culture-vs-qPCR discrepancies can be attributed to target difference - vs - method variability.

Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters.

AIMS: Draft criteria for the optional use of qPCR for recreational water quality monitoring have been published in the United States. One concern is that inhibition of the qPCR assay can lead to false-negative results and potentially inadequate public health protection. We evaluate the effectiveness of strategies for minimizing the impact of inhibition. METHODS AND RESULTS: Five qPCR method permutations for measuring Enterococcus were challenged with 133 potentially inhibitory fresh and marine water samples. Serial dilutions were conducted to assess Enterococcus target assay inhibition, to which inhibition identified using four internal controls (IC) was compared. The frequency and magnitude of inhibition varied considerably among qPCR methods, with the permutation using an environmental master mix performing substantially better. Fivefold dilution was also effective at reducing inhibition in most samples (>78%). ICs were variable and somewhat ineffective, with 54-85% agreement between ICs and serial dilution. CONCLUSIONS: The current IC methods appear to not accurately predict Enterococcus inhibition and should be used with caution; fivefold dilution and the use of reagents designed for environmental sample analysis (i.e. more robust qPCR chemistry) may be preferable. SIGNIFICANCE AND IMPACT OF THE STUDY: Suitable approaches for defining, detecting and reducing inhibition will improve implementation of qPCR for water monitoring.

Method repeatability for measuring Enterococcus in southern California beach sands.

AIMS: A recent study that evaluated 22 methods for enumerating faecal indicator bacteria in sand recommended standardization to a preferred method, but all researchers involved in that study had extensive experience in processing sand samples. The purpose of this study was to evaluate how well the recommended method can be transferred to laboratories without such experience. METHODS AND RESULTS: Eight southern California laboratories that rarely measure bacteria in sand processed six sand and three water samples in replicates to assess repeatability. Among-laboratory variability was found to be less than within-laboratory variability, with no significant differences in results among any of the laboratories. Moreover, within-laboratory variability was comparable between the sand and water samples, indicating that the elution procedure added little additional method error even when performed by laboratories without prior experience. CONCLUSIONS: The simple extraction method for enumerating Enterococcus in beach sands was easily transferable to and repeatable among laboratories with little or no prior experience. SIGNIFICANCE AND IMPACT OF THE STUDY: The demonstrated success of technology transfer will further demonstrate the success of method standardization and adoption, aiding in understanding of how sands affect surface water quality.

Faecal indicator bacteria enumeration in beach sand: a comparison study of extraction methods in medium to coarse sands.

AIMS: The absence of standardized methods for quantifying faecal indicator bacteria (FIB) in sand hinders comparison of results across studies. The purpose of the study was to compare methods for extraction of faecal bacteria from sands and recommend a standardized extraction technique. METHODS AND RESULTS: Twenty-two methods of extracting enterococci and Escherichia coli from sand were evaluated, including multiple permutations of hand shaking, mechanical shaking, blending, sonication, number of rinses, settling time, eluant-to-sand ratio, eluant composition, prefiltration and type of decantation. Tests were performed on sands from California, Florida and Lake Michigan. Most extraction parameters did not significantly affect bacterial enumeration. anova revealed significant effects of eluant composition and blending; with both sodium metaphosphate buffer and blending producing reduced counts. CONCLUSIONS: The simplest extraction method that produced the highest FIB recoveries consisted of 2 min of hand shaking in phosphate-buffered saline or deionized water, a 30-s settling time, one-rinse step and a 10 : 1 eluant volume to sand weight ratio. This result was consistent across the sand compositions tested in this study but could vary for other sand types. SIGNIFICANCE AND IMPACT OF THE STUDY: Method standardization will improve the understanding of how sands affect surface water quality.

Assessing ecological integrity in marine waters, using multiple indices and ecosystem components: challenges for the future.

During the last decade, there have been substantial scientific advances in the development of indices that measure the condition of biological ecosystem elements in coastal and estuarine waters. Though successful, these advances were only the initial steps and a special session on use of indices in ecological integrity assessments was held at the Coastal and Estuarine Research Federation meeting to focus the field on the most appropriate directions for the next decade. The session identified four primary scientific challenges: (i) reduce the array of indices by identifying the index approaches that are most widely successful; (ii) establish minimum criteria for index validation; (iii) intercalibrate methods to achieve uniform assessment scales across geographies and habitats; and (iv) integrate indices across ecosystem elements. Where an explosion of indices characterized the last decade, the next decade needs to be characterized by consolidation. With increased knowledge and understanding about the strengths and weaknesses of competing index approaches, the field needs to unify approaches that provide managers with the simple answers they need to use ecological condition information effectively and efficiently.

Comparison of total coliform, fecal coliform, and enterococcus bacterial indicator response for ocean recreational water quality testing.

In July 1999, California's ocean recreational bacterial water quality standards were changed from a total coliform (TC) test to a standard requiring testing for all three bacterial indicators: TC, fecal coliforms (FC), and enterococci (EC). To compare the relationship between the bacterial indicators, and the effect that changing the standards would have on recreational water regulatory actions, three regional studies were conducted along the southern California shoreline from Santa Barbara to San Diego, California. Two studies were conducted during dry weather and one following a large storm event. In each study, samples were collected at over 200 sites which were selected using a stratified random design, with strata consisting of open beach areas and rocky shoreline, and areas near freshwater outlets that drain land-based runoff. During the dry weather studies, samples were collected once per week for 5 weeks. For the storm event study, sampling occurred on a single day about 24 h following the storm. The three indicator bacteria were measured at each site and the results were compared to the single sample standards (TC > 10,000; FC > 400 and EC > 104 MPN or cfu/100 ml). EC was the indicator that failed the single sample standards most often. During the wet weather study, 99% of all standard failures were detected using EC, compared with only 56% for FC, and 40% for TC. During the Summer Study, EC was again the indicator that failed the single sample standards most often, with 60% of the failures for EC alone. The increased failure of the EC standard occurred consistently regardless of whether the sample was collected at a beach or rocky shoreline site, or at a site near a freshwater outlet. Agreement among indicators was better during wet weather than during dry weather. During dry weather, agreement among indicators was better near freshwater outlets than along open shoreline. Cumulatively, our results suggest that replacement of a TC standard with an EC standard will lead to a five-fold increase in failures during dry weather and a doubling of failures during wet weather. Replacing a TC standard with one based on all three indicators will lead to an eight-fold increase in failures. Changes in the requirements for water quality testing have strong implications for increases in beach closures and restrictions.

Comparison of bacterial indicator analysis methods in stormwater-affected coastal waters.

Membrane filtration (MF) and multiple tube fermentation (MTF) have been used for decades to measure indicator bacteria levels in beach water samples, but new methods based on chromogenic substrate (CS) technology are becoming increasingly popular. Only a few studies have compared results among these methods and they have generally been based on samples collected from a limited number of sites during dry weather. In this study, samples were collected from 108 sites the day after a major rainstorm, and three indicator bacteria (total coliforms (TCs), fecal coliforms (FCs) or E. coli, and enterococci (EC)) were each measured using MF, MTF, and CS. Sampling sites were selected using a stratified random design, stratified by open sandy beach, rocky shoreline, and beach areas near urban runoff outlets. The CS results were found to be highly correlated with both MF and MTF for all three indicators regardless of whether the samples were taken along open shoreline or near a runoff outlet. While correlated, TC values were higher using the CS method, consistent with other studies that have demonstrated false positives with this method. FC values were 12% lower with CS, reflecting the specificity of the CS method for E. coli rather than for the entire FC group. No significant differences were observed for EC, although some differences were observed within specific laboratories. Differences for all of these indicators were small enough that, when assessed categorically, there was more than 90% agreement between CS methods and either MF or MTF methods as to whether State of California Beach Water Quality Standards were met or exceeded.

Changing anthropogenic influence on the Santa Monica Bay watershed.

Santa Monica Bay is an open coastal embayment located directly seaward of Los Angeles, California. The Bay provides vital economic value through its water-dependent activities, such as swimming, diving, boating, and fishing. An increase from 100,000 residents in 1900 to 10 million in 2000 has imposed numerous environmental stressors on the Bay, including urbanization of the watershed. Pollutant discharges into the Bay increased throughout the early part of the century, but declined following passage of the Clean Water Act in 1972. Since that time, the predominant source of pollutant inputs has changed from point sources to non-point urban runoff. To assess how present-day and historical pollution interact to affect the environmental quality of Santa Monica Bay, three organizations collaborated on a multi-disciplinary study in 1997, towards which this volume is focused. This paper details the temporal patterns of anthropogenic influence on Santa Monica Bay to provide context for the papers that follow.

Effect of sampling frequency on shoreline microbiology assessments.

More than 80,000 shoreline bacteriological samples are collected annually in southern California to protect beachgoer health, but sampling frequency varies from daily to monthly among sampling sites. To assess the effectiveness of various sampling frequencies, we used five years of data from 24 Los Angeles area sites that have been monitored daily to simulate five alternative sampling strategies: five weekdays, five days per week including a weekend day, three days per week, weekly, and monthly. For each of these sampling strategies, we included in the simulation the local custom of adaptive sampling, in which a site is resampled the following day if bacterial concentrations exceed the State of California's beach water quality standards. We found that sampling five times per week resulted in observing about 80% of the events in which State standards were exceeded. This frequency dropped to 55%, 25%, and 5% for three times per week, weekly, and monthly sampling, respectively. Adaptive sampling was ineffective because nearly 70% of the water quality exceedences were single-day events, even at the most frequently contaminated sites. This high frequency of single-day events is of concern because the public is typically notified about water quality conditions 24-48 h after samples are collected, meaning that most warnings are out-of-date when they are issued.

A comparison of neustonic plastic and zooplankton abundance in southern California's coastal waters.

The density of neustonic plastic particles was compared to that of zooplankton in the coastal ocean near Long Beach, California. Two trawl surveys were conducted, one after an extended dry period when there was little land-based runoff, the second shortly after a storm when runoff was extensive. On each survey, neuston samples were collected at five sites along a transect parallel to shore using a manta trawl lined with 333 micro mesh. Average plastic density during the study was 8 pieces per cubic meter, though density after the storm was seven times that prior to the storm. The mass of plastics was also higher after the storm, though the storm effect on mass was less than it was for density, reflecting a smaller average size of plastic particles after the storm. The average mass of plastic was two and a half times greater than that of plankton, and even greater after the storm. The spatial pattern of the ratio also differed before and after a storm. Before the storm, greatest plastic to plankton ratios were observed at two stations closest to shore, whereas after the storm these had the lowest ratios.

A comparison of plastic and plankton in the north Pacific central gyre.

The potential for ingestion of plastic particles by open ocean filter feeders was assessed by measuring the relative abundance and mass of neustonic plastic and zooplankton in surface waters under the central atmospheric high-pressure cells of the North Pacific Ocean. Neuston samples were collected at 11 random sites, using a manta trawl lined with 333 u mesh. The abundance and mass of neustonic plastic was the largest recorded anywhere in the Pacific Ocean at 334271 pieces km2 and 5114 g km2, respectively. Plankton abundance was approximately five times higher than that of plastic, but the mass of plastic was approximately six times that of plankton. The most frequently sampled types of identifiable plastic were thin films, polypropylene/monofilament line and unidentified plastic, most of which were miscellaneous fragments. Cumulatively, these three types accounted for 99% of the total number of plastic pieces.

Comparability of bioaccumulation within the sanddab guild in coastal Southern California.

Most assessments of fish contamination in Southern California use ecologically different species from different sites. Use of ecologically similar species (a guild) might provide better assessments of fish contamination across different sites and depths. In July-August 1997, we collected samples of four sanddab guild species at 22 sites where species pairs co-occurred and determined total DDT concentrations in homogenized whole fish composites. Log-transformed DDT concentrations were highly correlated among all species pairs within this guild. All relationships were linear over the range observed, with slopes not statistically different from unity. The variability in response among species was about four times the variability encountered among replicates within species, but 15 times smaller than the variability among sites. Together, these results suggest that the sanddab guild, widespread on soft bottoms of the Southern California coastal shelf, can be used as a "superspecies" in bathymetrically diverse regional assessments of fish tissue contamination.

Composition and distribution of beach debris in Orange County, California.

Many studies have quantified debris collected on beaches around the world. Only a few of those studies have been conducted in the United States, and they are largely limited to semi-quantitative efforts performed as part of volunteer clean-up activities. This study quantifies the distribution and composition of beach debris by sampling 43 stratified random sites on the Orange County, California coast, from August to September 1998. We estimated that approximately 106 million items, weighing 12 metric tons, occur on Orange County beaches. The most abundant items were pre-production plastic pellets, foamed plastics, and hard plastics. Debris density on the remote rocky shoreline was greater than that on high-use sandy beaches for most debris items. This finding partially reflects the periodic clean-up of high-use beaches by local municipalities, and also indicates that a high percentage of the observed debris was transported to the site from waterborne sources.

A comparison of neustonic plastic and zooplankton at different depths near the southern California shore.

Previous studies of neustonic debris have been limited to surface sampling. Here we conducted two trawl surveys, one before and one shortly after a rain event, in which debris and zooplankton density were measured at three depths in Santa Monica Bay, California. Surface samples were collected with a manta trawl, mid-depth samples with a bongo net and bottom samples with an epibenthic sled, all having 333 micron nets. Density of debris was greatest near the bottom, least in midwater. Debris density increased after the storm, particularly at the sampling site closest to shore, reflecting inputs from land-based runoff and resuspended matter. The mass of plastic collected exceeded that of zooplankton, though when the comparison was limited to plastic debris similar to the size of most zooplankton, zooplankton mass was three times that of debris.

Microbiological monitoring of marine recreational waters in southern California.

An inventory was conducted to assess the number, type, spatial distribution, and costs of microbiological monitoring programs in southern California marine waters from Point Conception to the US/Mexico International Border. The location of each sampling site was determined using global positioning system (GPS), and estimates of geographic coverage were determined using geographic information system (GIS) techniques. Twenty-one programs conducted 87,007 tests annually at 576 sites in the study area. The largest number of sites was sampled in Orange County, whereas the largest number of analyses was performed in Los Angeles County because monitoring programs in this area focused on daily monitoring. Fifteen of the 21 programs were managed by National Pollutant Discharge Elimination System (NPDES) permitted sewage effluent dischargers who sampled both offshore and shoreline waters and typically tested for three indicator bacteria (total coliform, fecal coliform, and enterococcus). Their combined efforts comprised 82% of all of the microbiological indicator analyses conducted on an annual basis. Five of the remaining monitoring organizations were public health agencies, which typically focus their efforts on testing only total coliforms. Laboratory methodology also varied considerably, with NPDES permittees predominantly utilizing membrane filtration while public health agencies generally used multiple tube fermentation or premanufactured test kits. Nearly three quarters of all the effort expended in southern California occurred along the shoreline as opposed to offshore locations. Two thirds of this shoreline effort was focused on high-use sandy beaches and in proximity to perennial fresh-water outlets (storm drains and creeks). Most sampling occurred at a set of fixed sites that were revisited frequently, but only represented about 7% of the total shoreline. We estimated that roughly $3 million is spent annually on monitoring bathing water quality in southern California, exceeding that spent in any other part of the country.

Early life stage survival of striped bass in the Delaware River, USA.

Four 96-h in situ bioassays were conducted on or near the striped bass spawning grounds in the Delaware River within the States of Delaware, New Jersey and Pennsylvania, USA during 1989 to determine if water quality was sufficient to support larval survival. Tests were accomplished by holding 500 striped bass yolk-sac larvae in each of two 75 L chambers at four locations in the river ranging from north of Philadelphia, PA to Salem, NJ and at one location in the Chesapeake and Delaware Canal. Survival varied significantly among stations; highest survival was in the Chesapeake and Delaware Canal, where it averaged more than 50%. Lowest survival occurred at the station near Salem, NJ, approximately 30 km downriver of the primary spawning grounds, where less than 1% of the larvae survived in any of the tests. Survival at stations closest to the primary spawning grounds was weather-dependent; survival was over 47% during periods of little rainfall, but only 11% following periods of higher than average rainfall.