Data were collected that facilitated the comparison of enterococci using 4 different methods along with comparison of these data with a series of physico-chemical and hydrometeorologic measurements. Results were evaluated for the purpose of establishing the ability of alternative approaches for predicting ENT(MF). For the IND, enterococci based on the CS and qPCR methods were significantly different from the MF method, which was used in the epidemiological studies for establishing current recreational water quality guidelines. Moreover, among the enterococci assay methods, only ENT(qPCR-a) and ENT(qPCR-b) were not significantly different for both individual sample and geometric means (DGMs). These two qPCR assays utilize identical reverse primers but use different probes that are within 5bp of each other, and different forward primers that give different sized products of 139bp for ENT(qPCR-b) and only 89bp for ENT(qPCR-a).
Although the two culture based methods for enterococci were observed to provide significantly different IND values, DGM values were observed to be not significantly different. These results indicate that the total number of beach closures would be similar regardless of whether MF or CS methods were used to evaluate the samples. However, the specific days that the beaches would be closed would be different depending upon whether MF or CS was used. Thus, an equivalency cannot be established between the two methods, on a sample to sample basis. By averaging the data over the course of the day, this variability is averaged allowing for an equivalency to be established for DGM values.
No significant correlations were observed between source tracking markers and enterococci levels, with the exception of the storm event evaluated. The lack of correlation during non-storm conditions suggests that enterococci may not be related to a single source (human, dog, or gull). During the storm, the enterococci signal coincided with the levels of Catellicoccus
gull marker. The large storm event evaluated as part of the current study occurred in March, a month characterized by large numbers of birds as documented at the study site using a digital camera (27
). In general, the greatest numbers of birds visit the site during the late winter early spring (December through March). Of interest water temperatures were inversely correlated with the concentrations of gull marker, which is consistent with the visitation of birds at the study site during the cooler months of the year. Evaluation of the levels of CATgull
observed during this study on a month to month basis indicated that the levels of this marker correlated strongly and significantly (r=0.95, p<0.01) with the reported numbers of seagulls on the beach (27
). Such associations were not observed between the numbers of bathers and enterococci or BACUCD
and between numbers of dogs and BACdog
. Wright et al. (29
) documented that the enterococci contribution from dogs for this study site is greater than that of gulls. The significance of birds during the one storm event may reflect an intermittent occurrence as the number of birds at the beach is highly seasonal and the storm event that was sampled as part of this study coincided with a time period characterized by more birds.
Environmental parameters including turbidity, tide, wind, and solar radiation were related to changes in enterococci levels. The impact of tide is likely due to the sand, in particular sand at the extreme upper reaches of the tidal line, serving as a source of enterococci to the water column (26
,). Turbidity is a measure of the resuspension of sand from the intertidal zone; the correlation of enterococci with turbidity further supports the role of sand within the intertidal zone as serving as a source of the indicator bacteria (16
). Enterococci levels based on the culture methods were inversely affected by solar radiation, whereas enterococci based upon the qPCR methods were not affected by solar radiation. This result was consistent with earlier studies that showed inactivation of FIB could be accelerated by solar radiation (26
). These earlier studies hypothesized that enterococci by the MF and CS methods reflected microbial inactivation associated with increasing solar radiation, but the qPCR methods, which could measure both viable and non-viable bacteria, were not affected by solar radiation levels (34
). Wind appears to play a secondary role in impacting enterococci levels, with wind direction serving as a significant variable for ENT(MF). Wind speed served as a negatively correlated parameter with ENT(qPCR). The association between wind direction and speed could be associated with wind-induced circulation and dilution (26
Rainfall is the main environmental variable that has been used for proactive beach water quality warnings (26
). The largest rainfalls observed during the 15-days monitoring period were 12 mm over 6 hours and 28 mm over 24 hours. The levels of enterococci in the runoff water on the beach and water were extremely high (over 103
CFU/100 mL) within a few hours after the storm events. This result was consistent with a study conducted at a southern California beach that showed that rainfall greater than 13 mm increased FIB (36
The primary disadvantage of the culture-based methods is the longer sample incubation times. Enterococci levels could be available within 4 hours by the use of qPCR method. However, the study showed that qPCR methods provided values that were significantly different from ENT(MF). Direct extrapolation of ENT(qPCR) to estimate ENT(MF) was not practical because of weak correlations between the MF and qPCR. Prediction of ENT(MF) based on ENT(qPCR) in combination with environmental parameters (e.g. solar radiation) was also not successful in this study. In addition to these challenges, limited studies at the study site have demonstrated that recreational water illnesses at the study site were only correlated with the levels of enterococci based on the MF method (11
This study showed that the regression model based on environmental variables could provide reasonable qualitative predictions of ENT(MF) for DGM levels although quantitative predictions were poor. Since regression models based upon environmental measures could potentially provide results even faster than rapid microbiological measurement techniques, there is a potential of utilizing such models to estimate average water quality conditions over a given period of time at this beach. One alternative to current practice would be to couple the use of a regression model with regular ENT(MF) measurements. Ideally the regression model should provide a value for the probability of illness which takes into the account the uncertainties associated with the model in predicting ENT(MF) and the uncertainties of the ENT(MF) in predicting health outcomes. An even more ideal scenario would be to tie the regression models to health outcomes directly (13
In addition to physico-chemical and hydrometeorologic parameters, results also suggested that bacterial indicator levels were affected by the numbers of animals on the beach which may also have seasonal patterns associated with their numbers and fecal inputs. Thus, levels of enterococci at non-point source beaches are affected by a myriad of environmental factors and input loadings which are very difficult to capture within simple regression models. For marine beach sites characterized by non-point sources, additional factors should be considered to estimate ENT(MF), including the seasonal nature and shorter term variations of different sources of enterococci (e.g. variations in humans, birds, and dogs).
Alternative methods may be capable of estimating the geometric means of enterococci by membrane filtration, but are not adequate for estimating individual sample results.