The monitoring of hygienic quality of recreational water is generally performed by culture based methods for detection of fecal indicator bacteria, like fecal coliforms, Escherichia coli
and intestinal enterococci, which take 18–48 h before the result is available. Several water samples are analyzed on preset dates during the bathing season, and at the end of the season the bathing beaches are classified based on a set of criteria [1
]. Such monitoring and classification of beaches into quality classes (excellent, good, sufficient or poor) give a good impression of the general hygienic water quality at the beach. However, for urban recreational areas with sporadic poor water quality due to short-term pollution, for example if the area is located near discharges from combined sewer overflows (CSOs), results from routine monitoring are of limited value for real-time decision-making.
In such areas, rapid, simple methods that provide an estimate of the level of fecal contamination in the water are needed for supporting decisions about whether to give advice against recreational activities and if so, for how many days after a short-term pollution event. Such rapid methods are also useful for detection of wastewater contamination and location of its points of ingress into storm sewer systems, e.g., due to cross-connection in separate sewer systems, illicit connections or overflows and leakages through broken sewers [2
An alternative indicator can be defined as an organism or non-biological constituent of fecal pollution or sewage that is used to indicate the presence of fecal pollution. Constituents can range from commensal organisms found only in one type of host species to viruses, caffeine, or optical brighteners [3
]. Epidemiological studies and quantitative microbial risk assessment indicate a stronger association between fecal indicators and waterborne diseases when sewage contamination is present compared to the presence of fecal pollution from non-human (animal) sources [4
]. One reason for this may be that viruses, which are human-specific, cause over half of gastroenteritis cases associated with recreational water use worldwide [6
]. A rapid method that mainly reflects the level of sewage/human fecal pollution, and to a lower extent the sporadic fecal contribution from birds/animals, may therefore potentially better reflect the risk of gastroenteritis associated with recreational activities. Methods for detection of human specific markers (based on PCR) have been developed and are useful for microbial source tracking [3
], but the methods are still too complicated to be implemented in fully automated on-line instruments and require special laboratory facilities and trained personnel.
Measurement of enzyme activities in water samples, like the β-d
-galactosidase (GAL)- or β-d
-glucuronidase (GLU) activity, is less complicated, and easy-to-use field kits and automated on-line instruments have been developed [7
]. GAL activity is suggested as a rapid “early warning” indicator of gross sewage contamination [10
]. The enzyme GAL hydrolyzes β-d
-galactosides, including lactose, to monosaccharides by cleaving the β-glycosidic bond formed between a galactose and a second moiety. Several substrates, mainly chromogenic and fluorogenic, have been developed to measure the activity of this enzyme [13
]. Common culture-based methods for detection of coliform bacteria or fecal coliform bacteria are based on growth of the coliform bacteria on solid- or in liquid media, which contain inhibitors to suppress the growth of non-target bacteria, inducers to induce the formation of GAL and chromogenic/fluorogenic substrates, with subsequent detection of a fluorescent or colored end product. The sum of selective growth and GAL activity ensure the specificity of such methods [14
]. By using sensitive instruments for detection of the end product, GAL activity can also often be measured directly in contaminated environmental water samples without pre-cultivation [15
]. Such direct measurement of GAL activity in a water sample is not a measurement of culturable coliform bacteria, but of all enzymes (compounds that may hydrolyze the actual substrate) initially present in the water samples [12
]. The enzyme GAL is ubiquitous in nature and has been isolated from bacteria, yeasts, molds, plants and animals [17
]. The activity per cell may, however, vary among the different species/strains, and high temperature (44 °C) during the measurement may to some extent select for the detection of activity from heat-stabile enzymes [18
]. GAL synthesis is, in some strains, induced by lactose or galactose and repressed in the presence of glucose [19
], e.g., E. coli
grown in the presence of the inducer isopropyl-β-d
-thiogalactopyranoside (IPTG) showed >3 LOG10
higher GAL activity than E. coli
grown in the absence of an inducer [18
]. Since the incubation time in rapid methods (<2 h) is too short to induce the synthesis of new enzymes [20
], the measured GAL activity of a water sample will depend on the “historical” growth conditions of the organisms that have synthesized the enzymes.
Some beaches are exposed to birds and bird feces, or feces from other animals, which periodically may influence the measured number of fecal indicator bacteria. It may be hypothesized that the GAL activity in human feces (and sewage), due to the human diet, is relatively higher than in some other potential sources of GAL activity, e.g., birds and adult animals feces. This hypothesis was further tested in the presented paper.
The aim of the present study was (1) to measure the GAL activity relative to other fecal indicators in different fecal sources, like sewage, human and animal feces, as well as in recreational water and river water exposed to combined sewer overflows during heavy rainfall, for evaluating its usefulness as a rapid indicator of sewage contamination in urban areas and (2) to test and demonstrate measurement of the GAL activity in urban river water in a fully automated set-up.
Several studies have shown quite a good correlation between GAL activity or GLU activity and fecal indicator bacteria in water samples, but some studies have shown no or low correlation, in particular at low levels of fecal contamination [11
]. The present study also indicated a low correlation between GAL activity and E. coli
in coastal water at levels <1000 E. coli
per 100 mL, but too few samples were included to determine a potential relationship. Lack of correlation has been explained by enzyme activity from sources other than the culturable fecal indicator bacteria (E. coli
, FC or total coliforms) that were used for comparison, for example, activity from non-fecal sources like algae and marine vibrios, non-specific cell-free enzymes or other cell-free substances capable of hydrolyzing the actual substrate, GAL-positive fecal microorganisms others than coliforms or by active, but non-culturable coliforms, and variations in the contribution from such sources [11
]. Disinfection or environmental stresses are shown to reduce the number of culturable fecal indicator bacteria more than the GAL activity, which may often explain why the enzyme activity is high even if the numbers of culturable coliforms are low [27
In the present study, the GAL-activity per gram of feces from birds was found to be 3 LOG10
lower than the activity per gram of human feces, even though the E. coli
numbers per gram of bird- and human feces (logarithmic mean) were not significantly different. This may explain why the GAL-activity at some bird-affected urban beaches was low, even though the E. coli
numbers were high. A minor reduction (less than 50%) of the GAL activity was also observed if the salinity of the water sample was increased, which may further affect the correlation between GAL activity and E. coli
in coastal water. High levels of suspended solids or chemicals are also reported to potentially reduce the enzyme activity of water samples [31
The GAL-activity per gram of feces from sheep and cattle was also lower than from human feces (about 2 LOG10
). Only a few samples of animal feces were analyzed, from animals from the same farm with a quite similar diet, so further studies are required to obtain a more comprehensive picture of the variations in GAL activity in feces from different animals, at different age groups and diets. In a study by Rada et al.
(2010), the GAL activity in calf feces was found to be similar to the activity in feces from infants [33
]. Our initial work, however, indicate that high levels of GAL-activity in urban water samples may reflect the impact of human fecal pollution to a greater extent than the total fecal pollution.
This study also indicates that the main GAL activity in human feces is caused by sources other than culturable coliforms, since the GAL activity was high and relatively constant in all of the tested human feces samples, but the E. coli
/coliform numbers varied by >6.5 LOG10
. The human gut contains a significant number of different bacteria, which are potential contributors to the total fecal GAL-activity [34
]. Measurement of GAL activity therefore represents an alternative indicator of fecal contamination than the traditional culturable coliforms, FC or E. coli
. Although the E. coli
and intestinal enterococci numbers showed high variation within the individual human fecal samples, the average of all of the individual samples was as expected, based on levels in sewage and black water (which represent a natural average from several persons).
Davies and Apte (2000) reported that at FC concentrations above 2.3 × 103
cfu/100 mL in environmental water samples, the GAL activity was related to FC concentration, but below this concentration, a large background signal was observed, which was independent of the FC concentration [11
]. Despite this apparent limitation, the GAL measurement was concluded to have potential as an early warning indicator of treatment process failure and gross sewage contamination and leakage in situations where FC concentrations exceed 2.3 × 103
cfu/100 mL. This conclusion was supported by the results from this study, where a good correlation was observed between the GAL activity and E. coli
in samples taken from urban rivers before, during and after heavy rainfall. The ratio between GAL activity and E. coli
was similar in sewage and in the urban river water (slope = 1.00 in LOG-LOG-plot), indicating that fresh sewage was the main source of fecal contamination in the rivers during the rainfall episodes.
Bird feces were shown to contain low GAL activity, but high levels of E. coli, which may explain why rapid methods based on GAL measurement do not always detect high levels of E. coli at some bird-affected beaches. The GAL activity of all of the tested human fecal samples was, however, high and stable, and the GAL activity of sewage-polluted river water correlated well with the presence of fecal indicator bacteria. A rapid method based on GAL measurement can only be used to quantify high levels of human fecal pollution, corresponding to about 0.1 mg human feces/liter (or 103 E. coli/100 mL) since below this limit, GAL-activity from non-fecal environmental sources may interfere. However, since the GAL activity can easily be measured by field instruments, or as shown in the present study, by a fully automated instrument, it may be a useful surrogate parameter for detecting high levels of sewage contamination in urban waters. The simple method, based on direct mixing of the water sample and medium, is best suited for fresh water, since high salt concentrations were shown to reduce the GAL activity.