The EEMs from samples collected from the domestic raw sewage and urban river water have been evaluated and show 3 peaks (T1, T2, B1) with the strongest fluorescence intensities (Fig. 2). More information concerning the EEM spectra of these samples collected is listed in Additional file 1: Figs. S1 and S2 for domestic sewage and river water, respectively.
EEM characteristics of domestic sewage
Figure 2a shows that basically there are two main fluorophores in the spectrum of domestic sewage. The first fluorescence intensity peak is at Ex/Em of 275/350 nm, which corresponds to tryptophan-like peak T1 components. The second fluorescence intensity peak is at Ex/Em of 225/350 nm, which is associated with tryptophan T2 fluorescent components. The detected tryptophan-like fluorescent components are mainly protein-like substances, which may be derived from the human activities such as human excreta, food residues and cooking oils [44]. Based on Fig. 2b, the averaged fluorescent peak intensity of tryptophan-like T1 and tryptophan-like T2 material are 802 a.u. and 261 a.u., respectively, with an average T1/T2 ratio of 3.1. Tryptophan-like T1 materials are related to soluble microbial products, and tryptophan-like T2 materials are associated with proteins of aromatic structures [45].
In addition, a distinct narrow band occurs at \(\lambda_{\text{ex}} \, = \, 3 30{-} 3 7 5\) nm for \(\lambda_{\text{em}} \, = \, 4 10{-} 4 50\) nm. This is can be explained by the fluorescent whitening agents from the grey water such as dishes and clothes washing, which also indicates the contribution of the domestic sewage to some extent. Considering domestic sewage is transported through pipelines and has no direct contact with soil, the possibility of humus from the soils is small. Therefore, the humic-like fluorescent components in domestic sewage are mainly derived from the humic acids originated in tap water supply [46]. From this perspective, the intensity of humic-like substances is much less pronounced than the protein-like ones in the domestic raw sewage samples.
EEM characteristics of river water
As indicated in Fig. 2c, there are two distinct fluorescence peaks in the EEMs of the river water samples, with the Ex/Em at 230/340 nm and 275/305 nm, respectively. The peak at EEM of 230/340 nm corresponds to tryptophan-like T2 materials, and the peak at EEM of 275/305 nm corresponds to tyrosine-like B1 materials. Both the tryptophan-like T2 and tyrosine-like B1 belong to protein-like materials.
As for the humic or fulvic substance, a fluorophore was also generated at with the Ex/Em at 250/435–455 nm. However, its fluorescence intensity was not as high as that of tryptophan-like T2 and tyrosine-like B1 substances in this case. Previous studies by Hudson et al. [38] showed that in clean rivers, peaks C1, C2 and A predominated. This is because DOM originating from clean river water is dominated by natural organic matter from plant material, whereas sewage-derived DOM is dominated by organic matter originating from microbial activity. By contrast, our study showed that tryptophan-like or tyrosine-like substances, instead of humic-like or fulvic-like substances, were the indication of polluted urban river water. Therefore, with increasing urbanization and anthropogenic activities, the fluorescent signature of urban waters changed with increasing human impact from being humic-rich to protein-rich with peaks T and B.
Figure 2d further shows the measured fluorescent peak intensities of urban river water at six sampling stations. Among the six sites, the S1 monitoring site is located in Shang’aotang River, where the sluice gates are installed (see Fig. 1). As a result, Shang’aotang River is separated from other rivers including Puhuitang River and Caohejing River, which are polluted by the dry-weather as well as wet-weather outflow from the storm drains of this catchment. By contrast, no storm drain outfalls are directly connected to the Shang’aotang River. Therefore, water quality at S1 station was better than that of the other five sampling stations. However, the fluorescence intensities of tryptophan-like T2 and tyrosine-like B1 at the S1 station were not considerably different from those at other five stations. This showed that fluorescence property of urban rivers was determined by materials more than sewage-derived DOMs in this catchment.
Differences of EEM characteristics between domestic sewage and urban river water
Our study showed that domestic sewage featured most strongly tryptophan-like peaks T1 and T2, whereas urban river water featured tryptophan-like peak T2 and tyrosine-like peak B1. Both domestic sewage and urban river water had the characteristics of tryptophan-like T2 fluorescence; however, it was interesting that tryptophan-like peak T2 in the urban river water was more intense than that in the untreated domestic sewage.
Figure 3 shows that organic matter in the investigated urban rivers exhibits seasonally different fluorescence intensities over the sampling period. Tryptophan-like peak T2 as well as tyrosine-like peak B1 was observed to be more intensive in winter as compared to that in summer. This was related to elevated river water levels due to stormwater runoff discharge into rivers and the resulting dilution effect in summer to some extent. However, despite being diluted by stormwater runoff, fluorescence intensities in peaks T and B of river water samples were still higher than those of sewage samples. The explanation is that peaks T and B are related to microbial activity [47] and may be transported into a system (allochthonous) or be created by microbial and biological activity within a system (autochthonous). Intensive tryptophan-like T2 and tyrosine-like B1 are related to biological activity particularly in areas of high primary productivity, that is, surface waters with phytoplankton or algal activity. Under this circumstance, tryptophan-like T2 and tyrosine-like B1 fluorescence may be present as ‘free’ molecules or else bound in proteins or humic structures of algae cells and their remnants in river waters. In Shanghai area, majority of aquatic plants grow in the spring and summer season and decay in the winter season [48]. Decomposition of aquatic plants contributed to the intensified tryptophan-like T2 and tyrosine-like B1 substances. Such explanation could be strengthened by recently reported protein-like fluorescence in Taihu Lake, China, where the tryptophan-like components of DOM were significantly higher in winter than in summer and autumn, due to the degradation of phytoplankton [32]. Moreover, at lower water temperature, dinoflagellates or diatoms are the predominant algal species. Correspondingly, there is a good correlation between peaks T and B materials produced by such algal species [49], as proved by this study. In this case, a strong linear relationship between the fluorescence peak intensities of tryptophan-like T1 and tyrosine-like B1 was demonstrated, with average correlation coefficient of 0.96 (p < 0.01).
Another explanation is associated with hydrologic characteristics of Shanghai that features tidal river network area. Due to the tidal force, the river water in this study site comes from Huangpu River, the largest river in Shanghai, which receives water from upstream Taihu Lake watershed. The cities in the upstream Taihu Lake watershed have developed light industries including numerous textile enterprises. In view of this, protein-like peaks in local river waters could be related to upstream inflow with chemical tracer of dispersant MF, a kind of sulfonated naphthalene formaldehyde condensates from dyeing process [37]. It was reported that many river waters in this region, even for Taihu Lake itself, featured fluorescent fingerprints same with that of textile wastewater [50,51,52,53]. Therefore, upstream inflow from a distant hydrological regime could also exert important influence on features of fluorescent peaks in local surface waters.
Based on above discussions, higher T2 fluorescence intensity observed in the urban river water was related to algal activity in the surrounding river and distant upstream inflow from neighboring cities with developed textile industries. Average T2 peak intensity in urban river water samples was 732 ± 304 a.u. If excluding the river fluorescent data disturbed by stormwater runoff, average T2 peak intensity in urban river water was up to 998 a.u., but average T2 peak intensity in untreated domestic sewage was only 261 a.u. The intensities in peak T2 between urban river water and domestic sewage differed by 3.8 times. This revealed that tryptophan-like peak T2 would be a distinct tracer in indicating urban river water and associated river water intrusion into urban drainage system in the urbanized catchment, e.g., the cities of Taihu Lake watershed, China.
Conservative behavior of fluorescence peaks in sewage and surface water
Changes in observed fluorescence peak intensities of domestic sewage and river water samples during the experiments are shown in Fig. 4 and Additional file 1: Fig. S3. As seen in Fig. 4a and b, for the domestic sewage samples, lab incubation experiments conducted in May showed fluorescence peak intensities of tryptophan-like T1 and tyrosine-like B1 decreased over time rapidly within the experimental duration. Fluorescence intensity of peak T1 and B1 decreased by 70.8% and 66.1%, respectively, on average after 24 h. In this process, fluorescence intensities of other fluorescent components did not increase correspondingly (see Additional file 1: Fig. S3). This result indicated that the fluorescence intensities of the tryptophan-like T1 and tyrosine-like B1 substances converted into non-fluorescent substances with time. As discussed above, the tryptophan-like T1 and tyrosine-like B1 component in domestic sewage are considered to be microbial by-products, and therefore changes in their fluorescence intensities are related to microbial metabolism. For this scenario under the lab temperature of 25 °C, active microbial activity dramatically degraded the fluorescent substances of T1 and B1. Reynolds concluded that microbial activity, measured by oxygen depletion in the BOD5 test, correlated well with the T1 fluorescence intensity of raw sewage [54]. Therefore, T1 is presented as a bioavailable substrate. However, for the incubation experiments conducted in January, the fluorescence signature of tryptophan-like T1 and tyrosine-like B1 component in domestic sewage almost remained unchanged with time. This can be explained by inhibited microbial activity under lower lab temperature (e.g., the temperature of 5 °C), leading to relatively stable fluorescence peak intensities.
Changes of fluorescence signature of tryptophan-like T2 with experimental duration was also probed for domestic sewage and surface water samples. Figure 4c shows that tryptophan-like T2 in domestic sewage samples exhibits conservative behavior, independent of environment temperature. This can be explained by that protein-like peak T2 is associated with proteins of aromatic structures, which are less likely to be degraded as compared to protein-like peak T1 of soluble biodegradable microbial products [45]. A similar finding was demonstrated in experiments of river water samples, in which fluorescence intensities of tryptophan-like T2 substances did not change within the experimental duration of 72 h as well (see Fig. 4d). As discussed above, intensified peak T2 in the river water is influenced by upstream inflow featuring chemical tracer of dispersant MF, a kind of sulfonated naphthalene formaldehyde condensates from dyeing process. This tracer would contribute to the non-biodegradability of fluorescent substances in the river water. Additionally, it was reported that protein-like peak T2 in river water was given by compounds of aromatic rings like polycyclic aromatic hydrocarbons (PAHs), exhibiting bio-refractory behavior as well [19].
Based on above discussion, tryptophan-like T2 can be used as an ideal marker to quantify percent wastewater and urban river water with inappropriate entry into storm drains on dry-weather days, which features conservative characteristics in both of the misconnected source types. Thus, the uncertainty of fluorescence mass balance model arising from chemical or biological reaction within the storm pipes could be minimized as much as possible.
Determination of percent surface water and wastewater into the storm drains
To determine the percent surface water into storm drains, the fluorescence mass balance model was set up with fluorescence intensity measurements of the samples from the river water, domestic sewage and catchment outlet. Information concerning detected EEMs for all of the catchment outflow samples is provided in Additional file 1: Fig. S4. It was found the center positions of the two identified peaks T1 and B1 of the three flow types were significantly different, whereas the center positions of peak T2 among these types were very concentrated. The identified center positions of peak T2 were at Ex/Em 225/350 nm, 230/340 nm and 225/350 nm for wastewater, river water and catchment outflow, respectively. The slight shift of the peak T2 reflected the inevitable difference between the DOM compositions in different types of sources. Comparison of fluorescence peak T2 among domestic sewage, river water and catchment outflow is presented in Fig. 5.
As seen in Fig. 5, for both summer and winter season periods, the fluorescence intensities of peak T2 in the catchment outflows were higher than those in the domestic sewage, due to occurrence of river water intrusion. For the catchment outflow, generally fluorescence intensities of tryptophan-like T2 in winter were higher than that in summer. There are two essential criteria for being selected as a tracer for misconnection source apportionment. First, the concentration or level of the tracer should be significantly different between end members. Second, the tracer behaves conservatively during the mixing and transport processes. The significant differences in fluorescence intensities of peak T2 of two sources (Fig. 5) and its conservative behavior (Fig. 4c, d) made it fit the essential criteria for being selected as a tracer. Therefore, fluorescence spectrometry has the promise for indicating the presence of river water intrusion into storm drains as a rapid, reagentless technique that requires little sample preparation.
Using the maximum fluorescence intensities at each peak T2 center of domestic sewage, river water and catchment outflow to get Friver, Fsewage and Foutflow in Eq. 3, the Bayesian inference results of the percent river water into the storm drains for the two season periods are shown in Fig. 6. It shows that the maximum a posteriori probability (MAP) estimate of the percent is 12.7% and 20.8% in summer season and winter season, respectively. Considering the center shifts of the peak T2, the Bayesian estimation was also done using the fluorescence intensities of peak T2 at Ex/Em of 230/340 nm for all the three types of samples. The percentage share of river water backflow into the storm drains in summer season and winter season was 9.5% and 22.5%, respectively (Additional file 1: Fig. S8), a difference of − 3.2% and 1.7% for summer and winter as compared to the results using maximum fluorescence intensities (Fig. 6). This is within the acceptable range of the source appointment estimate. The small differences can be explained by two reasons. First, the shifts of the chosen peak T2 among the three types were small enough, i.e., one interval of excitation step and two intervals of emission step. Second, the differences in the fluorescence intensities of the chosen peak T2 were large enough among the three types. This further strengthens that peak T2 has great potential for tracking and quantifying dry-weather misconnections into storm pipes.
Theoretically, the amount of river water inflow is determined by the pressure head between surface water and storm drains; the higher the river water level, the larger the amount of river water inflow. Therefore, higher MAP in winter was associated with lower water level of storm drains of non-flood season period. However, uncertainty of Bayesian result in summer was higher than that in winter. This is possibly due to frequent rainfall events and intense precipitation during summer season. Storm water discharges led to significant variations of fluorescent substances in the three surrounding rivers (see Fig. 5), resulting in the increased uncertainty of percent river water intrusion.
The Bayesian inference results were validated with the estimated time-series river water inflow in winter season in this case. Figure 7 shows the measured historical real-time water levels between surrounding rivers and terminal outfall within 1 month of winter season period (i.e., Dec., 2008). Using the mathematical function between river water inflow and pressure head established in this catchment (i.e., Qriver = 0.104*Δh1/2, Qriver is river water inflow and Δh is the sum of real-time water pressure head between river water level and terminal wet-well level within 1 day) [6], the daily river water inflow was accordingly determined, as seen in Fig. 7. It was known from this figure that when storm pumps operated to lower the terminal wet-well level to the lowest alarm level, the significantly increased pressure head drives large amounts of river water inflow in a short period of time. For the non-pumping discharge periods, river water inflow is much smaller as compared to that under the pumping discharge periods. In this figure, the daily river water inflow ranged from 145 to 11,705 m3/day, with an av data of 4261 m3/day.
Our previous study showed that catchment outflow on dry-weather days was approximately 19,350–21,600 m3/day [6]; therefore, maximum likelihood value of river water inflow in winter season was about 4025–4493 m3/day based on MAP estimate for percent surface water intrusion. The daily averaged river water inflow based on Fig. 7 coincided well with the MAP estimate, with a relative error less than 10%. This demonstrates that FMBM is robust in quantifying surface water intrusion into storm drains.
Environmental implications
The use of fluorescence to predict the presence and quantity the dry-weather misconnection into a storm drainage system has significant environment implications. Recent advancements in sensor technology and the development of reliable and specific fluorescence probes have increased our ability to monitor organic matter characteristics in near real-time. The application of real-time data and fluorescence mass balance model would allow water managers to track and quantify the dry-weather misconnection without high costs associated with labor-intensive investigations or complex analytical approaches. For example, establishing a fitted analytical function to determine the river water inflow, have to depend on long-term real-time synchronous flow discharge and pressure head data (river water level versus catchment water level) for each catchment. By contrast, if aided with tryptophan-like T2 sensor and FMBM introduced in this study, administrative managers would be provided with a useful, fast and cheap alternative way for the investigation of dry-weather misconnections into urban storm drainage system.
The study was conducted on polluted urban rivers, whereas there may be dissimilarities in the organic matter of other surface water bodies or misconnected source types. However, we can develop same approaches to identify and quantify illicit discharges, considering custom sensors have already been constructed to capture the key peaks such as the strong microbial fluorescence signal. Although peak-picking method was used in this study for selecting the tracer peak, other EEM analysis methods such as parallel factor analysis (PARAFAC) would also be useful for the determination of the marker peak and designation of sensors for the same purpose. Additionally, the presented fluorescence approach could be combined with other chemical tracers to present an in-depth insight into illicit discharge investigation of various source types from a wider perspective, and also determine the best times to perform other chemical analyses if necessary.