Key abiotic and biotic determinants of occurrence and toxicological imapct of cyanobacterial blooms in a lowland dam reservoir of Sulejów, Poland

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1 N FKBR E ARE AH RE GR E Białko reporterowe (lucyferaza) Designed by M. Łapińska Substancje stanowiące wzorce obcości Substancje o charakterze trwałych zanieczyszczeń środowiska Substancje wywołujące stres oksydacyjny Substancje zaburzające szlaki endokrynne Key abiotic and biotic determinants of occurrence and toxicological imapct of cyanobacterial blooms in a lowland dam reservoir of Sulejów, Poland Joanna Mankiewicz-Boczek

2 Study site Sulejów Reservoir Established in 1973 Max surface [m2] mln Average depth [m] 3.30 Volume [m3] 78.9 mln Average flow for multi-year period [m3/s] Average water retention time [day] N 42 Used for retention & recreation Serves as alternative source of drinking water for Łódź aglomeration (till 2004 as main source drinking water) cyanobacterial bloom near the dam, 2012 Fot. A. Skowron

3 Cyanobacterial studies from 1997 IDENTIFICATION of key abiotic (physico-chemical, hydrological) parametersaffecting the development of toxic cyanobacterial blooms METHODS ELABORATIONfor monitoring of toxic cyanobacteria (application of molecular methods for risk assessment and early warning system) OPTYMISATION of biological structureof Pilica river floodplain for selfpurification enhancement and REDUCTION of diffusive and point sources pollutionin the Pilica basin IDENTIFICATION of the impact of biotic parameters and interactions on the trail: cyanobacteria / cyanophages / bacteria / cyanoabcterial toxins / other organisms ESTIMATION OF BIOLOGICAL POTENTIAL of cyanobacteria and cyanotoxins (cellular biosensors for detection and evaluation of novel mechanisms of noxious bioactivity of cyanobacteria

4 Cyanobacterial monitoring Proposed integral procedure of microcystin-producing cyanobacteria monitoring for bathing water quality Spring/Summer Determination of physico-chemical parameters including nutrients concentration: P-PO 4, TP (>0.1 mg/l*), N-NO 3, N-NH 4, TN (>1.5 mg/l*) Chlorophyll a (> 10 µg/l**) Phytoplankton analysis Occurrence of Microcystis, Planktothrix, Anabaena Detection of toxigenic (potentially toxic) strains of cyanobacteria PCR amplification of mcy genes (polymerase chain reaction) Occurrence of microcystins Application of screening tests: determination of microcystins concentration ELISA (enzyme-linked immunosorbent assay) determination of microcystins toxicity PPIA (protein phosphatase inhibition assay) Confirmation of microcystins if ELISA showed > 2.5 µg/l Quantitative and qualitative analysis of microcystins HPLC (high performance liquid chromatography) Transdisciplinary interpretation of results Following the first and second principle of Ecohydrology, the identification of cause-effect relationship with comparative studies of the lake/reservoir typology, hydrochemistry, phytoplankton diversity and water toxicity are fundamental for developing a strategy to reverse eutrophication. (Zalewski 2000; Wagner et al. 2009) Mankiewicz-Boczek et al. in Chorus[ed.], 2012, Current approaches to Cyanotoxin risk assessment, risk management and regulations in different countries. Mankiewicz-Boczek in Zalewski M., Urbaniak M. [eds.] Adaptation of ecohydrological system solutions and biotechnologies for Africa. Note: * critical values for eutrophication recommended by OECD (1983); ** relatively low probability of adverse health effect recommended by WHO (2003)

5 Influence of environmental factors on toxigenic activity and cyanobacterial toxicity Designed by M. Łapińska Interaction between cyanobacteria toxic genotypes toxin production- reservoir hydrology conditions Average water retention time: 55 days 18 days 1 µg/l average microcystins concentration 1 µg/l average microcystins concentration 1% toxic Microcystis genotypes 70% toxic Microcystis genotypes Gągała et al., 2014, Microbial Ecology Project NSC 0964/B/P01/2010/39

6 Interaction CYANOBACTERIA / CYANOPHAGES Designed by M. Łapińska Detection of cyanophages (g91gene) capable of degrading cyanobacterial cells SpearmanRankOrder Correlation(p<0.05) Cyanophages (g91) 2010 (n=9) 2013 (n=10) Total Microcystis(16S rrna) Toxigenic Microcystis(mcyA) Phylogenetical analysis Cyanophage g91 BLAST homology analysis indicated 90% similarity to g91 gene described for cyanophage from the genera Myoviridae strain Ma-LMM01 14 ACCTAACCAGATTG 1 70 GCTGGAGTATTAGAGTTAMCAAG-AST-T--TCCTCTGTGCCCATCTCTAGCGGCGACCT ACATCAGCGTTCGTTTCGGCACTGTAGCCGGTGCAGCCCTCAWTATAGTAGAGGGTAATA 71 Study supported by the National Science Centre, project number UMO-2013/11/N/NZ8/00607

7 Interaction CYANOBACTERIA / OTHER BACTERIA Designed by M. Łapińska Detection of bacteria capable of degrading microcystins cyanobacterial hepatotoxins Microcystistoxigenic strains (mcya)/potential MCs degraders (mlra) [gene copy number per µl] mcya Microcystis (395 pz) [gene copy number/µl] mlra (120 bp) [gene copy number/µl] MCs [µg/l] MCs concentration [µg/l] Tresta Phylogenetical analysis Bacteria mlra BLAST homology analysis indicated 95% similarity to gene mlra described for bacteria from the genera Sphingopyxis sp. C1 andstenotrophomonas sp. 1 CTCCTCCCACAAATCAGGACGAGCCCAATGGCCACGGCGAATTCSACGAAATCCCAAGGG CGCCACCCGAGCCCTGCAACCGTTGGGGCCCACTCGGCAGTGACGTTTACGCCCAGTTCG TTATGGATCGCGTGAATGAGCACGCCACCCCACATCGATCCACCGAGCTTGTTGCATACG AAGACAGCGATGTTGGTGCCGGCAATGAACCCCGGAGCGATAACGAATTGCTTGACAATA ACGCCCCAGGCCGCGCCAGGATCGCCGGAGAACAGTGTCGGCAGGTCGCGCGGCAAATGC CAAGCCCACCACATTATGCCGAGGATCGCCGCTGCGGTCAGGGGGTCAAACTTCTTCAGG AGCTGCGGCAGCGCAGAGCCGCGCCAGCCCAGTTCTTCGAGCAGCGGGCCAGGGCTGAGT AGCAGCGATGCTGCCAGCCATCACATAAATGGCGA 455 Mankiewicz-Boczek et al. 2015; Open Life Sciences formerly Central European Journal of Biology Study supported by the National Science Centre, projects number 0964/B/P01/2010/39 and UMO 2012/07/N/NZ8/00599

8 Interaction CYANOBACTERIA / OTHER BACTERIA Designed by M. Łapińska Detection of bacteria capable of degrading microcystins cyanobacterial hepatotoxins Loss of microcystin-lr after one week [%] Kontrola Control- - woda destylowana withoutbacteria Mieszanina Mix 011 Mieszanina Mix 022 Mieszanina Mix 03 3 Mieszanina Mix 044 Mieszanina Mix 055 Mieszanina Mix 066 Mieszanina Mix 077 Mieszanina Mix 088 Mieszanina Mix 099 Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina Mix Mieszanina 30 Mix 30 Mieszanina 31 Mix 31 Mieszanina 32 Mix 32 Degradation efficiency 0.6 µg/ml/day BLAST homology analysis indicated 94% similarity to gene 16S rrna described for bacteria Aeromonas veroniiw-s AGCGGCGGACGGGTGAGTAATGCCTGGGGATCTGCCCAGTCGAGGGGGATAACTACTGGA AACGGTAGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCG CGATTGGATGAACCCAGGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGAC GATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACT CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGC CGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAAGGTTGGT AGCTAATAACTGCCAGCTGTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCA GCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCAC GCAGGCGGTTGGATAAGTTAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTAA AACTGTCCAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAG GTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT GTCGATTTGGAGGCTGTGTCCTTGAGACGTGGCTTCCGGAGCTAACGCGTTAA-TCGACC GCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGC GGTGGAGCATGTGGTTTAATTCGATGCAACGCGAARAACCTTACCTGGCCTTGACATGTC TGGAATCCTGTAGAGATRCGGGAGTGCCTTCGGGAATCAGAACACAGGTGCTGCATGGCT 1022 phylogenetically identified colonies of bacteria, from which tested mixtures were created Mankiewicz-Boczek et al. 2015; Open Life Sciences formerly Central European Journal of Biology Study supported by the National Science Centre, projects number 0964/B/P01/2010/39 and UMO 2012/07/N/NZ8/00599

9 Designed by M. Łapińska ESTIMATION OF BIOLOGICAL POTENTIAL of cyanobacteria and cyanotoxins Biosensor activation (fold of control value) Biosensor activation (fold of control value) NFKBRE Hep3B, crude cyanobacterial extracts CME1 467 µg MC/ml CME2191 µg MC/ml CME321 µg MC/ml Concentration [ppm] NFKBRE Hep3B, purified toxin preparations PME1 268 µg MC/ml PME2 94 µg MC/ml PME3 18 µg MC/ml Concentration [ppm] Pilotestudy Comparisonof influence of purifiedand crude cyanobacterial extracts on pattern recognition receptor Conclusions: 1. The activity of NFKBRE Hep3B cell line was different for crude and purified cyanobacterial extracts. 2. Response of NFKBRE Hep3B cell line was similar despite different microcystins concentration in extracts (both in case of crude and purified extracts). 3. Crude cyanobacterial bloom extracts contained other than microcystins metabolites, which activated cellular biosensor NFKBREbased on Hep3B cell line. Study supported by the National Science Centre, project number UMO 2012/07/B/NZ8/03991

10 Designed by M. Łapińska Elaboration of system solutions Demonstration zone in Zarzęcin: reduction ofgroundwater POLLUTION WITH PHOSPHORUS COMPOUNDS, by strengthening the plant ecotone zone with geochemical barrier based on limestone. ( Phosphates concentration [mg/l] Z1 Z2 Z3 Z4 Z Critical level for occurence of cyanoabcterial blooms (fot. EKOROB) GEOCHEMICAL BARRIER

11 Increasing the efficiency of the buffer zone by incorporation of geochemical barrier Designed by M. Łapińska (fot. EKOROB) 14 reservoir groundwater level river Phosphates concentration in groundwater [mgpo 4 /l] przed befor barrier za after barrier low concentration of phosphates in groundwater (fot. EKOROB) high concentration of phosphates in groundwater Izydorczyk et al. 2013, Ecohydrology & Hydrobiology

12 European Regional Centre for Ecohydrology PAS Prof. dr hab. Joanna Mankiewicz-Boczek Dr Katarzyna Izydorczyk Dr Ilona Gągała Mgr Aleksandra Jaskulska Dep. of Applied Ecology, University of Lodz Prof. dr hab. Joanna Mankiewicz-Boczek Dr Tomasz Jurczak Institute of Medical Biology PAS, Łódź Prof. dr hab. Jarosław Dziadek Dr hab. Łukasz Pułaski Dr Dorota Jaros Dr Jakub Pawełczyk Mgr inż. Iwona Karwaciak Department of Hydrobiology, Faculty of Biology, Adam Mickiewicz University, Poznań Dr hab. Mikołaj Kokociński Dziękuję za uwagę! Thank you for your attention! Gracias por su atención!