Application of 2D fluorescence spectroscopy on faecal pigments in water – Characterization of wastewater fluorescence and potential indication of faecal pollution Bärbel Daub Master’s Thesis 45 credits Second cycle, A2E Uppsala 2017 Institutionen för Energi och Teknik Examensarbete 2017:01 Department of Energy and Technology ISSN 1654-9392 Uppsala 2017 Title: Application of 2D fluorescence spectroscopy on faecal pigments in water Supervisor: Annika Nordin, Swedish University of Agricultural Sciences, Department of Energy and Technology Assistant Supervisor: Martin Wagner, DVGW Technologiezentrum Wasser (Water Technology Center), Ger- many, Außenstelle Dresden Examiner: Björn Vinnerås, Swedish University of Agricultural Sciences, Department of Energy and Technology Credits: 45 ECTS Level: Advanced level, A2E Course title: Independent project in Environmental Science – Master’s thesis 45.0 credits Course code: EX0828 Programme/education: Soil and Water Management – Master’s programme Series title: Examensarbete (Institutionen för energi och teknik, SLU), 2017:01 ISSN: 1654-9392 Place of publication: Uppsala Year of publication: 2017 Online publication: http://stud.epsilon.slu.se Keywords: wastewater, faecal pollution, faecal pigment, fluorescence, urobilin, stercobilin Sveriges lantbruksuniversitet Swedish University of Agricultural Sciences Faculty of Natural Resources and Agricultural Sciences Department of Energy and Technology Abstract Drinking water pollution by faeces and associated enteric pathogens can cause serious health issues and outbreaks of diseases. A fast and reliable indication of faecal pollution is necessary to prevent the con- sumption of polluted water. This work aims at identifying faecal pigments in wastewater and discusses the possibility of using on-line fluorescence monitoring of faecal pigments in water as a tool for the detection of faecal pollution. Three faecal pigment standards, urobilinogen, urobilin, and stercobilin, as well as wastewater in- and outflows from five German wastewater treatment plants (WWTPs) were characterized by 2D fluorescence spectroscopy (using Excitation Emission Matrices), and by high per- formance liquid chromatography (HPLC) coupled with absorption (DAD) and fluorescence detection (FLD), as well as mass spectrometry (MS). Furthermore, tests on faecal pigment stability, reaction to zinc addition, kinetics, and pH influence on faecal pigment fluorescence were performed. With the ob- tained fluorescence data, a parallel factor analysis (PARAFAC) model for the detection and quantifica- tion of urobilin and stercobilin in real water samples was developed. An addition of zinc to the pigments in real water lead to a time-dependent fluorescence intensification of a factor >30 and red shift of the pigments’ fluorescence spectra, which can be used as a tool to detect low concentrations of faecal pig- ments in water. Urobilin and stercobilin were identified in all examined WWTP inflows. The results and literature study indicated that a degradation of faecal pigments during wastewater treatment may have taken place. In the wastewater of one treatment plant, fluorescein was detected. Fluorescence detection and quantification of faecal pigments in wastewater was possible with the help of zinc addition or prior enrichment, but more studies are needed to enhance the sensitivity of the method to be sensitive enough to detect faecal pollution in concentrations relevant for drinking or surface water monitoring. It was concluded that fluorescence detection of faecal pigments in water is promising as an early warning sys- tem, but in this study it did not prove sensitive enough to be used as a stand-alone method. Keywords: wastewater, faecal pollution, faecal pigment, fluorescence, urobilin, stercobilin Popular Science Summary It is not new that one can get sick by the consumption of polluted water. Many of the diseases spread with polluted water are caused by organisms living in faeces, which are present in the water because there has been some faecal contamination. Understandably enough, ingesting faeces and pathogens with water is not a nice thought. For this reason it is very important to detect any faecal pollution of drinking or recreational water as fast as possible. For the detection, the yellow-brownish pigments that determine the colour of urine and faeces can potentially be used as indicators. Some of these pigments are fluores- cent, which means that they emit light with a characteristic colour when they are illuminated and this can be used to identify them. There are already other methods to detect faecal pollution of water, but the established methods take a lot of time and labour is necessary to handle water samples in the laboratory. To detect faecal pollution with the help of fluorescence techniques would have the advantage of being very fast and requiring little handling. Especially a fast result is important to warn people before they drink polluted water or swim in it. In this study, it was investigated how well the fluorescence detection of faecal pigments works under different conditions. One aim was to find the pigments in wastewater with the help of fluorescence techniques. Also, an unidentified fluorescence signal that had been found in an earlier study in wastewater was examined. For this purpose, faecal pigments (urobilin, urobilinogen and stercobilin standards) were characterized in different water media with fluorescence spectroscopy as well as with some reference methods. Samples from 5 different wastewater treatment plants were taken and also characterized with the same methods as the pigment standards. Then the results were compared. To make the identification of faecal pigments easier, a model was developed that was supposed to recognize faecal pigments in water by interpreting fluorescence data. It was found out that the unidentified fluorescence signal in wastewater was caused by several sub- stances, both faecal pigments and also another substance, which is called fluorescein. As the name says, this is a chemical which has a strong green fluorescence and can be used to mark the flow of water, for example when leaching from a pipe is suspected (it is also said to have been used earlier to dye the Chicago river green on St. Patrick’s Day, but that is another story). The faecal pigments could be found in the inflow to all wastewater treatment plants, but they could not be found in the cleaned outflow of the plants. It was not possible to recognize the pigments by fluorescence only, because wastewater con- tains many other substances. An enrichment of the wastewater, meaning that it was concentrated, could help. Adding zinc to the wastewater also improved the fluorescence signal and might make an enrich- ment unnecessary. However, the fluorescence intensity after zinc was added decreased again, so more tests need to be done to get a reliable method. Fluorescein was mainly found in water from one of the treatment plants. It looks very similar to faecal pigments in its fluorescence, causing a risk to confuse the substances. The model was able to solve this problem and could distinguish between faecal pigments, their zinc complexes and fluorescein. However, the model needs to be tested on more data to ensure its reliability. In conclusion, there are still some uncertainties and the method is not yet sensitive enough, but after some more development, fluorescence detection of faecal pigments in water has a great poten- tial. Table of contents Glossary ...................................................................................................................................... 6 1 Introduction ......................................................................................................................... 7 2 Aim ...................................................................................................................................... 8 3 Background .......................................................................................................................... 9 3.1 Faecal pigments ........................................................................................................... 9 3.1.1 The heme metabolism ............................................................................................ 9 3.1.2 Indicator function ................................................................................................. 11 3.1.3 Metal complex formation ..................................................................................... 12 3.2 Fluorescence .............................................................................................................. 12 3.2.1 Fluorescence spectroscopy .................................................................................. 13 3.2.2 Fluorescence spectrometer design ...................................................................... 14 3.2.3 Fluorescence influencing factors and quenching ................................................. 15 4 Materials and methods ..................................................................................................... 16 4.1 Materials .................................................................................................................... 16 4.1.1 Chemicals .............................................................................................................. 16 4.1.2 Real water samples .............................................................................................. 16 4.2 Analytical