Ruđer Bošković Institute University of Zagreb, Faculty of Science University of Pau, France University of Bordeaux, France

Wrap-up Meeting December 2 and 3, 2019

Accumulation, Subcellular Mapping and Effects of Trace Metals in Aquatic Organisms (AQUAMAPMET) Aims of the project

 to increase our understanding and gain new knowledge on how fish, crustaceans, bivalves and fish intestinal parasites (Acantocephalans) cope with elevated metal concentrations in the freshwater ecosystems by relating data on metal concentrations in water, accumulated metals and biomarker responses in biota to data on intracellular metal mapping  to evaluate anthropogenic impact on selected freshwater ecosystems referring to metal/metalloid pollution by using integrated chemical, biochemical and biological approach Specific objectives

Evaluation of Evaluation of biological metal/metalloid effects of contamination accumulation in fish, on biota acanthocephalans, Intracellular mapping of crustaceans and bivalves trace metals in fish Evaluation of subcellular tissues, acantocephalans partitioning of and bivalve tissues, and metals/metalloids in fish, correlation between two acanthocephalans, imaging techniques – crustaceans and bivalves NanoSIMS and TEM Evaluation of biodiversity Integration of chemical, of MZB, periphyton and biochemical and cellular drift imaging approach Project team

ZRINKA DRAGUN SANJA GOTTSTEIN MARIJANA ERK JASNA LAJTNER VLATKA FILIPOVIĆ MARIJIĆ RENATA MATONIČKIN KEPČIJA DUŠICA IVANKOVIĆ MARKO MILIŠA NESRETE KRASNIĆI MIRELA SERTIĆ PERIĆ TATJANA MIJOŠEK ZUZANA REDŽOVIĆ

DIRK SCHAUMLŐFFEL ETIENNE GONTIER MARIA DE LOS ANGELES SUBIRANA MANZANARES (JULIEN MALHERBE) Timing of activities in the AQUAMAPMET project Study area – the River 1st and 2nd project year

Sampling campaigns:  autumn 2015 1  spring 2016 KNIN

CROATIA

Prokljan Lake

Border of the ŠIBENIK The Krka River – sampling locations and field work referent location – Krka River spring The Krka River – sampling locations and field work industrial area of Knin – river Orašnica

• „Hot-spot” – waste waters not adequatly treated nor managed

• the capacity of galvanized zinc plating 100,000 tons, hot dip galvanizing 30,000 tons, and heat treatment 100,000 tons per year The Krka River – sampling locations and field work downstream from Knin - wastewater discharge Basic physico-chemical water parameters

- temperature, mean O2 levels → moderate and uniform Limiting values (HR-R_12) - pH → slightly alkaline pH 7.0 – 9.0 environment -1 COD ≤ 4mg O2 L - COD and nutrient - -1 N-NO3 ≤ 0.7 mg N L concentrations → rather 3- -1 P-PO4 ≤ 0.03 mg P L low - site downstream from Knin GRC (Government of the Republic of generally showed higher Croatia), 2019. Directive on Water Quality values of majority of physico- Standard. Official Gazette No.96., Zagreb. chemical parameters

Both studied sampling sites – classified as waters of very good and good water quality Trace and macro-element concentrations • significantly higher • significantly higher values at site autumn values were downstream from Knin: recorded for Se, Rb, Sb, Li, Rb, Mo, Mn, Fe, Sr, U, Al, Ti, V, Cr, As, Na, and Ca Mg and K

 20% higher levels of Ba, Mo, Ca, Rb  2-fold higher levels of Li emerged from municipal and Sr and industrial effluents  17-fold and 38-fold higher levels of Fe and Mn from the town of Knin

 both sites in this study could be considered as uncontaminated regarding the metal concentrations  “self-purification ability” Study area – the River Sampling campaigns:  autumn 2017 3rd and 4th project year  spring 2018

1-MASLENJAČA

3-ILOVA

5-TREBEŽ 0 10 km Study area – the Ilova River Sampling campaigns:  autumn 2017 3rd and 4th project year  spring 2018

1-MASLENJAČA

3-ILOVA

5-TREBEŽ 0 10 km The Ilova River – sampling locations and field work Village Maslenjača – about 50 km upstream from Kutina The Ilova River – sampling locations and field work Village Ilova – upstream from Kutina, about 800 m from highway The Ilova River – sampling locations and field work

Village Trebež – 15 km downstream from Kutina Location of anthropogenic influence

Fertilizer factory

Lonjsko polje - Ilova

Photo: Google Earth Kutinica - before the confluence Phosphogypsum storage ponds into Ilova Phosphogypsum → a The filter-cake is slurried with waste by-product from the the water and pumped to special storage ponds where processing of phosphate the solid part is settled and rock in factories covered with highly acidic producing phosphoric transport water (pH 2–3) acid and phosphate containing high concentrations fertilizers of fluoride and elevated amounts of heavy metals.

IMPURITIES: radionuclides, and trace metals like As, Pb, Cd, Cr, Mn, Fe, Ni, Cu, Zn, Photo: Google Earth Sb, V Phosphogypsum storage ponds Basic physico-chemical water parameters

- 3- Site Location O2 O2 pH Conductivity CODKMnO4 N-NO3 P-PO4 -1 -1 -1 -1 -1 no. (mg L ) % (µS cm ) (mg O2 L ) (mg L ) (mg L ) 1 Maslenjača 8.5 86 8.03 332 3.9 1.13 0.10 3 Ilova 8.2 82 7.55 328 9.0 2.89 0.7 5 Trebež 5.0 52 7.39 473 6.8 4.54 0.23 (937) Kutinica 2.8 36 7.63 753 6.7 2.14- 0.56 11,688

Limiting values (HR-R_4): pH 7.0 – 9.0 Maslenjača → only one of -1 COD ≤ 5.5 mg O2 L all studied sampling sites – - -1 N-NO3 ≤ 1.3 mg N L classified as good water P-PO 3- ≤ 0.1 mg P L-1 quality 4 Directive on Water Quality Standard. Official Gazette No.96(2019), Zagreb. Dissolved trace elements in water of Ilova

Cadmium (µg L-1) Antimony (µg L-1) Copper (µg L-1) 0.250 1.0 2.5

0.200 0.8 2.0

0.150 0.6 Ilova 1.5 Ilova Ilova Pakra 0.100 0.4 Pakra 1.0 Kutinica Kutinica Kutinica 0.050 0.2 0.5

0.000 0.0 0.0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Arsenic (µg L-1) Vanadium (µg L-1) 6.0 20.0 5.0 15.0 4.0 Ilova Ilova 3.0 10.0 Pakra Pakra 2.0 5.0Kutinica Kutinica 1.0 0.0 0.0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Nickel (µg L-1) Iron (µg L-1) Manganese (µg L-1) 6.0 70.0 400 5.0 60.0 300 4.0 50.0 Ilova 40.0 Ilova Ilova 3.0 200 30.0Pakra Pakra Pakra 2.0 20.0Kutinica 100 Kutinica Kutinica 1.0 10.0 0.0 0.0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Trace elements (normalized to Al) in surface sediments of Ilova

5.0E-05 6.E-04 4.5E-05 Cd 3.78 Pb 44.61 4.0E-05 5.E-04 3.5E-05 4.E-04 3.0E-05 2.5E-05 3.E-04 16.20 17.86 2.0E-05 1.5E-05 2.E-04 1.0E-05 1.E-04 5.0E-06 0.15 0.19 0.0E+00 0.E+00 • Increasing Maslenjača Ilova Trebež Maslenjača Ilova Trebež concentration gradient in 4.0E-04 31.55 2.5E-03 Cu Zn downstream 3.5E-04 162.39 2.0E-03 3.0E-04 direction 2.5E-04 15.20 1.5E-03 2.0E-04 63.96 1.5E-04 6.88 1.0E-03 1.0E-04 35.50 5.0E-04 ANTROPOGENIC 5.0E-05 0.0E+00 0.0E+00 IMPACT Maslenjača Ilova Trebež Maslenjača Ilova Trebež

Concentration in mg/kg (dry sediment) Published results

http://bib.irb.hr/lista-radova?&lang=EN Browsing Project number: HRZZ-IP-2014-09-4255

Journal articles and review articles in CC journals: 8 Scientific papers in other journals: 1 Conference reports (abstracts) in CC journals: 1 Conference reports (abstracts) in other journals: 2 Papers in the publishing process: 1 Other refereed conference papers: 1 Abstracts in Book of abstracts: 23 Graduation thesis: 2+1 Rector’s award paper: 1

Young researchers – short-term research visits, workshops, courses, trainings...