Layout 1 SPECIAL SECTION Potentially toxic cyanobacteria blooms in the Southern Alps and the Italian peninsula Introduction Over the past decades, there has been a constant increase in reports from citizens of phenomena concerning the presence of anomalous colours in surface water bodies. Often, it is not easy to identify the cause of such events, as well as to explain how these colours are not always linked to specific water pollution phenomena. However, it is important to emphasize that changes in surface water colour are not necessarily indicative of pollu- tion. Indeed, in many cases, they are associated with algal and bacterial blooms, which frequently are triggered by the natural degradation of organic matter under certain environmental con- ditions (Trüper and Genovese, 1968; Johnson et al., 2012; Belila et al., 2013; Van Vuuren and Levanets, 2020). One of the tasks of the Regional Agency for Environmental Protection and Energy of Emilia Romagna (ARPAE) is to assess the causes of these phenomena and explain them to citizens or stakeholders, informing them against common false alarms that arise in response to these anomalous colouration events. This short note will provide a brief overview of some spe- cific cases of water discolouration handled by ARPAE, with a focus on the main causes of these phenomena, which are mainly attributable to natural events. Usually, ARPAE manages these phenomena, performing both biological and chemical determinations on the collected water sample. Biological analysis involves the use of an Uther- mol sedimentation chamber and an inverted optical microscope for the characterization and enumeration of bacteria and phyto- plankton, as described in the ISTISAN 07/05 protocol. Chemical analysis generally includes the determination of pH, Biochemi- cal Oxygen Demand (BOD5), total organic carbon, and dis- solved organic carbon, following the methods APAT CNR IRSA 2060, APAT CNR IRSA 5120, and the European and Interna- tional standards UNI EN 1484:1999 and ISO 15705:2002. Moreover, given the microorganisms observed in the samples and their role in causing the anomalous colourations, the quan- tities of iron and manganese, or alternatively sulphides and sul- phates are analysed, following the European standards UNI EN Anomalous colourations in surface water bodies: causes and examples reported by the Regional Agency for Environmental Protection and Energy of Emilia Romagna (ARPAE) Michela Del Pasqua, Fabrizio Bandini, Elena Morandi, Rita Antonellini, Marcello Masi, Michele de Gioia Regional Agency for Environmental Protection and Energy of Emilia-Romagna (ARPAE), Ravenna, Italy ABSTRACT Surface water can exhibit a range of colours beyond the expected blue or green. These anomalous colourations can be triggered by different factors, both natural and anthropogenic, and not always are signs of pollution. This note explores the phenomenon of water discolouration, focusing on natural causes and analysing how factors such as algal and bacterial blooms influence water colour. The study provides examples of specific events that occurred in the Emilia-Romagna region and documented by ARPAE and highlights the importance of monitoring these colouration events in order to provide valuable insights into the health of water bodies and the sur- rounding environment. Corresponding author: Michela Del Pasqua, Regional Agency for Environmental Protection and Energy of Emilia-Romagna (ARPAE), via Marconi 14, Ravenna, Italy. E-mail: mdelpasqua@arpae.it Key words: water discolouration; natural events; surface water bodies; Euglena sanguinea; purple sulphur bacteria; iron-oxidizing bacteria. Authors’ contributions: all the authors made a substantive intellec- tual contribution. All the authors have read and approved the final version of the manuscript and agreed to be held accountable for all aspects of the work. Conflict of interest: the authors declare no potential conflict of in- terest. Funding: none. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Acknowledgements: we would like to thank Professor Aharon Oren of The Hebrew University of Jerusalem, for his help in iden- tifying the purple sulphur bacteria belonging to the genus Thio- halocapsa and Thiocapsa. Received: 29 April 2024. Accepted: 24 September 2024. Publisher’s note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affil- iated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. ©Copyright: the Author(s), 2024 Licensee PAGEPress, Italy Advances in Oceanography and Limnology, 2024; 15:12600 DOI: 10.4081/aiol.2024.12600 This work is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License (CC BY-NC 4.0). Non -co mmerc ial us e o nly M. Del Pasqua et al.6 ISO 17294-2:2016 and the methods APAT CNR IRSA 4160 and 4020, respectively. Results and Discussion Anomalous colourations due to algal blooms: the case of Euglena sanguinea As reported by Van Vuuren and Levanets (2020), freshwater ecosystems exhibit a limited diversity of microorganisms re- sponsible of turning surface water from the expected blue or green colour into red colour. Among these species, the euglenoid Euglena sanguinea Ehrenberg is widely considered the most prevalent phytoplank- ton species responsible for the red colouration of freshwater ecosystems (Van Vuuren and Levanets, 2020). Documented oc- currences of blooms caused by this species have been reported extensively across the globe, resulting in the formation of red surface scums or a pronounced red discolouration of the water (Rahman and Khan, 2007; Van Vuuren and Levanets, 2020). Blooms of this species mostly occur in the spring and summer months, when waters are affected by strong solar radiation, high temperatures, and a considerable supply of nutrients (Van Vuuren and Levanets, 2020). Indeed, most of the events concerning the presence of extensive red colouration of ponds or rivers reported by citizens to ARPAE during the summer months were attributable to blooms of E. sanguinea. Among these events of algal blooms, notable cases include those reported for the Migliarina canal (MO) in August 2009 (44°49’18.58”N, 10°49’43.93”E) and the Cento canal (FE) in August 2017 (44°44’48.41”N, 11°18’45.92”E). Other events reported to ARPAE in recent years include the red coloura- tion of the Vallicella River (MO) in July 2020 (44°49’13.14”N, 11°11’5.19”E) and red scums in the river Canala dei Canali (Ra) between July and August 2020 (44°25’46.90”N, 12° 9’24.47”E). These phenomena were mainly characterized by the presence of red scum or red discolourations on the surface of the water column and by the absence of unpleasant odours (Figure 1a). The action plan typically outlined by ARPAE protocols in this scenario entails the collection of a water sample and its subsequent transfer to the laboratory units where chemical and biological analyses are per- formed. Biological analyses involve the search for certain species of algae capable of altering the water’s colour, while chemical analyses involve the measurement of pH, dissolved oxygen, and total organic carbon. Water samples collected for each of these cases were dominated by the presence of Euglena sanguinea in its characteristic red spherical shape (Figure 2a), while very low con- centrations and diversity of other algal species were present; the chemical analyses revealed high levels of dissolved oxygen and a basic pH value (8.2-8.34). Euglena sanguinea is a unicellular planktonic organism that possesses a complex of red pigments, the hematochrome, whose main function is to protect the cell from high light intensity (Van Vuuren and Levanets, 2020). In the presence of high solar radi- ation, the hematochrome pigments are distributed throughout the cell volume to protect the cell, which appears red and as- sumes a spherical shape (Figure 2a). In the evening and night hours, when low radiation is present, red pigments are concen- trated in the centre of the cell, which resumes its classic green colour (Laza-Martinez et al., 2019). Consequently, the main factors that can aid citizens in iden- tifying E. sanguinea as the main responsible for a red dis- colouration phenomenon include the formation of a red layer on the water’s surface, sometimes accompanied by the presence of red scum, while below the surface layer the water usually ap- pears clear and clean. Other factors can include the absence of unpleasant odours and the occurrence of this phenomenon dur- ing the summer months, when temperatures are extremely high and solar radiation is intense. Moreover, other conditions favourable for the development of E. sanguinea blooms are the presence of canalized waters with a high organic load and a con- siderable hydrodynamic stasis (Van Vuuren and Levanets, 2020). The alga is not harmful to humans, but it can produce a very po- tent ichthyotoxin, that is potentially harmful and lethal to fish (Zimba et al. 2004). In our cases, no detectable effects on the fish fauna were recorded. Anomalous colourations due to bacterial blooms: purple sulphur bacteria and iron-oxidizing bacteria In other cases, the phenomenon of freshwater discolouration is not due to algal blooms, but to typical ecological and geological conditions of the aquatic ecosystem that might trigger the prolif- eration of bacterial blooms, such as the presence of suspended minerals or high levels of organic load in the water column. Among the events of anomalous colouration of surface freshwater reported by citizens to ARPAE and associated with bacterial blooms, we can list some records characterized by the development of extensive pinkish-purple colouration of the water, recurring periodically in small and shallow water bodies with hydrodynamic stasis (Figure 1b). These cases occurred in the province of Ravenna, specifically in a shallow private pond in August 2017 and March 2021 (44°22’53.23”N, 12°16’11.75”E) and in a eutrophic lake within the San Vitale Pine Forest in August 2020 (44°31’23.56”N, 12°14’2.24”E). More recently, purple scums were reported for the Zaniolo canal (RA) (44°29’48.22”N, 11°49’0.39”E) in July 2023, during the flood events that characterized Emilia-Romagna. During these blooms, citizens also reported a strong hydrogen sulphide smell and the presence of suspended solids. The microscopic analysis of the water samples revealed that these phenomena were asso- ciated with the presence of purple sulphur bacteria (Figure 2b, 2c), while physicochemical analysis showed a high presence of chlorides and sulphates, severe oxygen deficiency and a large amount of hydrogen sulphide responsible for the bad smell. In- deed, these organisms are classified as “anoxygenic pho- totrophic purple bacteria” as they typically grow under anoxic conditions, in presence of light and hydrogen sulphide (Pfenning and Trüper, 1992). They utilize sulphide as an electron donor for their photosynthetic processes, which is subsequently oxi- dized to sulphate. This oxidation process involves the interme- diate formation of elemental sulphur globules that are accumulated inside or outside the cell depending on the bacterial species (Pfenning and Trüper, 1992). As reported by other studies (Caumette, 1986; Belila et al., 2013), these organisms proliferate in anoxic zones and in situa- tions characterized by an overload of organic matter. These en- vironmental conditions are observed in wastewater stabilization ponds (Belila et al., 2013) or in shallow coastal lagoons that both Non -co mmerc ial us e o nly Natural causes of surface water bodies colourations 7 receive freshwater inputs with organic material from the conti- nent and marine waters with a high concentration of sulphates (Trüper and Genovese, 1968; Caumette, 1986). The purple sulphur bacteria detected in the water samples analysed by ARPAE were identified as members of the family Chromatiaceae and likely belonging to the genus Thiohalocapsa and Thiocapsa, after a careful examination from taxonomic ex- perts in this field (Figure 2b and 2c). Visual features that can help citizens understand the natural water colouring phenomenon caused by purple sulphur-oxidiz- ing bacteria include the presence of extensive pinkish-purple colouration of the water body and the occurrence of an unpleas- ant odour of hydrogen sulphide. Moreover, the proliferation of sulphur-oxidizing bacteria usually occurs in shallow water bod- ies with a small surface area, stagnant warm water, and an over- load of organic matter. In other cases, the action of other microorganisms is responsible for further events of red-rust and orange coloura- tions of surface waters reported by citizens to ARPAE. These events were characterized by the presence of orange flocculent material deposited along the water body and col- loidal material in suspension of the same colour (Figure 1c). Figure 1. Anomalous colourations in surface water bodies were detected by the Regional Agency for Environmental Protection and Energy of Emilia-Romagna (ARPAE). a) Bloom of Euglena sanguinea in the canal Canala dei Canali (RA) let the water assume a red colour with red scum. Cyanobacteria of the genus Oscillatoria were responsible for the green scum visible in the picture. b) Purple dis- colouration of a private freshwater lake in the province of Ravenna caused by purple sulphur bacteria. c) Orange flocculent material de- posited along the Samoggia stream (BO) due to the presence of iron-oxidizing bacteria. Non -co mmerc ial us e o nly M. Del Pasqua et al.8 Figure 2. Microorganisms are responsible for anomalous colourations detected in surface water. a) Euglena sanguinea in its characteristic red spherical shape (Scale bar 100 µm). b) Purple sulphur bacteria of the genus Thiohalocapsa (Scale bar 50 µm). c) Purple sulphur bacteria of the genus Thiocapsa (Scale bar 50 µm). d) Iron oxidizing bacteria of the genus cf. Leptothrix enclosed in straight sheaths (Scale bar 50 µm). e) Iron oxidizing bacteria of the genus cf. Gallionella with twisted stalks (Scale bar 50 µm). f) Iron oxidizing bacteria of the genus cf. Sphirophyllum with threads twisted in a spiral form (Scale bar 50 µm). Non -co mmerc ial us e o nly Natural causes of surface water bodies colourations 9 These cases were observed along the Samoggia stream (BO) (44°37’19.14”N, 11°12’54.78”E) in the years 2016 and 2017, along the Savio River (FC) (43°50’26.99”N, 11°58’2.12”E) in January and February 2021, and finally in Borgo Maggiore (San Marino) (43°57’12.66”N, 12°26’56.70”E) in March 2021. The microscopic analysis performed by ARPAE allowed the observation of the structure of the colloidal material and re- vealed that it was mainly composed of filamentous iron-pre- cipitating bacteria, likely belonging to the species Leptothrix ochracea Kützing and to the genera Spirophyllum and Gal- lionella (Figure 2d, 2e, and 2f). The analytical results of both water and sediment samples showed the absence of anthro- pogenic substances (hydrocarbons, heavy metals) and show a very high concentration of iron and manganese, supporting the hypothesis that these phenomena of reddish-orange colouration of water bodies were attributed to the presence of iron-oxidiz- ing bacteria. The presence of ferrous and manganese ions in the water samples can be attributed to the environmental char- acteristics of the groundwater and likely linked to the minerals present in the soils and rocks of the area under investigation, such as the provinces of Bologna and Forlì-Cesena. Thanks to their copious metallic depositions, these organ- isms can be identified by their morphological characters visible under an optical microscope. Members of the genus Leptothrix are single thread form composed of cylindrical cells placed end to end and generally enclosed in sheaths, usually straight or curved (Figure 2d), while species belonging to the Gallionella genus present long branching twisted stalks coated with ferric oxides (Figure 2e) (Harder, 1919; Johnson et al., 2012). Spiro- phyllum spp. are the most abundant of the iron thread bacteria, and they have the form of flat ribbon-like threads twisted in a spiral form (Figure 2f) (Harder, 1919). These bacteria can thrive thanks to the energy obtained from the oxidation of ferrous and manganese ions present in the water (Harder, 1919; Emerson and Revsbech, 1994; Wang et al. 2011). This process leads to the deposition of ferric hydroxide that can be stored inside the cell or accumulate as extracellular matrices in the form of reddish-orange deposits (Harder, 1919). Factors that can help citizens recognize the natural phenom- enon of water colouration caused by iron-oxidizing bacteria in- clude the presence of orange colouration in the water body with reddish-orange deposits and the absence of unpleasant odours. Moreover, unlike other algal and bacterial blooms that are more prevalent during warmer months, iron bacteria can persist and cause discolouration events throughout the years, including au- tumn and winter months. Iron-oxidizing bacteria thrive even in well-oxygenated water bodies with flowing, non-stagnant waters. These microorganisms have been found in a range of iron- rich environments, such as freshwater wetlands, marine and fresh- water hydrothermal vents, water distribution pipes, and salt and freshwater marshes (Karl et al., 1988; Johnson et al., 2012). All these cases suggest the ubiquitous presence of iron-oxidizing bac- teria within wetland soils and sediments, supporting their pivotal role in metal cycling in aquatic and wetland environments. This study aimed to highlight natural water discolouration phenomena that are poorly understood and documented in Italy, with the goal of sharing important information within the scien- tific community. To begin addressing these little-known phenom- ena, ARPAE’s technical approach was grounded in basic research, employing fundamental techniques such as optical microscopy for the taxonomic characterization of these microorganisms. As the Agency’s knowledge of these phenomena has expanded, future perspectives will involve the development of more advanced tech- nical methodologies to refine analyses and obtain more accurate results. In this regard, the use of molecular techniques could be integrated with the taxonomic approach to better characterize the microbial communities responsible for water discolouration and to provide more accurate identification. Conclusions Reports of water discolourations recorded by ARPAE and associated with algal and bacterial blooms are constantly in- creasing, probably due to a growing public awareness of envi- ronmental issues as well as a rising frequency of changes in environmental conditions. Often it is not easy to immediately identify the cause of such events, as well as to explain to citizens that these phenomena are not always linked to anthropogenic causes or specific water pollution. Therefore, it is crucial to em- phasize the importance of correctly contextualising these phe- nomena, providing comprehensive answers to citizens in order to avoid and limit their concerns. The increased experience gained by ARPAE regarding these phenomena and collaboration among various professional fig- ures within the Agency has allowed a clearer understanding of these events over the years. There are various results of this col- laboration, including timely communication to the police en- forcement engaged in environmental protection, as well as the effective response to citizen reports. Moreover, proper commu- nication regarding the nature of these events through publication on local websites and institutional platforms promotes a greater reassurance among the population but can also contribute to cre- ating in citizens the feeling of effective and professional envi- ronmental surveillance by the Agency. This, in turn, contributes to the development of a reciprocal relationship of collaborative trust between citizens and the public administration. References Belila A, Abbas B, Fazaa I, et al, 2013. 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