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Within the micro and nano world, tiny membrane-enclosed bits of material are more or less free to move and act as communication tools within cells, between cells, between different tissues and between organisms in global environment. Based on the mechanism of membrane budding and vesiculation that includes all types of cells, in this review, we attempted to present a review on SARS-CoV-2 virus actions in compartments of different scales (cells and their surroundings, tissues, organisms and society). Interactions of the virus with cells on a molecular level, with neural system, endothelium, hematopoietic system, gastrointestinal system and genitourinary system. Transmission route between organisms and between mother and fetus are considered. Also, transmission of virus through contact with materials and with environment, the suggested measures to prevent contamination with the virus and to support the organism against the disease are given.

Water security and quality are a global issue of concern, which have recently become alarming due to the growth of the human population, industrialisation and expanded agricultural activities. Biologically active compounds, such as pharmaceuticals and personal care products can have major adverse effects on aquatic organisms, and are therefore one of the biggest threats in water quality. Another major concern is the spread of waterborne pathogens, including multidrug resistant (MDR) bacteria, which can cause serious illnesses in humans. In order to maintain water abundance and quality, it is necessary that adequate wastewater treatment and analytical techniques allowing for sensitive and fast-response detection of water hazards are in place. Conventional (waste)water treatment technologies often fail to adequately remove all of the water hazards detailed above. Moreover, conventional analytical techniques currently used in water quality control are, although highly selective and sensitive, time-extensive, with throughput of merely 2 to 3 samples per hour, excluding the time for sample preparation. With respect to these drawbacks, research was proposed to explore new approaches for degradation of recalcitrant compounds, inactivation of microorganisms, and fast screening methods, which are listed in the second chapter of this dissertation as research objectives. In the third chapter, an extensive theoretical background on the hazards found in aquatic environment, namely pharmaceuticals and waterborne pathogens, is given. Pharmaceuticals enter the environment through several routes (disposal of unused medication via the toilet, pharmaceuticals passing through the human body unchanged/slightly transformed, animal excretions of pharmaceutically active compounds, insufficient wastewater treatment, etc.) therefore, traces of pharmaceuticals have repeatedly been reported in surface waters, groundwater, wastewater effluents, and even drinking water. Iodinated contrast agents (ICAs), as the compounds of interest in this project are further described. ICAs are eliminated from the human body practically unchanged therefore a large proportion of them end up in municipal and hospital wastewater, where they can be present in concentrations of up to 2.4 g/L. Their ecotoxicity, degradation attempts, as well as detection monitoring in the environment are reviewed within the chapter. Additionally, waterborne pathogens, which account for 2.2 million deaths per year, are reviewed in this chapter, with emphasis on multidrug-resistant (MDR) bacteria. Although MDR infections are mostly prevalent in hospital environments, the presence of MDR bacteria in the environment is not a rarity. A high percentage of bacterial isolates in waters have been shown to be of an MDR phenotype. The theoretical background in analytical methods in water quality monitoring is also given in this chapter. Vanguard and rearguard techniques are explained, the first offering simple, cheap, and rapid sample screening, but sacrificing sensitivity and selectivity, whereas the second providing the highest quality information, excellent sensitivity and selectivity, but in expense of complicated and timely sample handling and high-cost instruments. By combining the two techniques the benefits of both can be exploited in a single system. The basic principles of thermal lens spectrometry (TLS) and its miniaturised version - the thermal lens microscopy (TLM) as fast screening methods providing high sensitivity are further explained, and their practical applications are reviewed. Furthermore, composite materials have recently been finding applications in water treatment technologies, as filter materials, adsorptives for pollutants, catalysts for degradation reactions, and disinfectants. The applications of three main types of composites: synthetic composites, biocomposites, and nanocomposites, are also reviewed within this chapter. The core of this dissertation is presented in the fourth and the fifth chapter, which examine two separate approaches for water treatment, as well as analytical methods for fast screening purposes. The fourth chapter is investigating options for degradation of iodinated X-ray agents (ICAs), namely diatrizoate, through biodegradation with extracellular enzymes of white rot fungus Dichomitus squalens, and chemical oxidation with manganese(III) acetate. Enzymatic degradation with laccase (Lac) and manganese peroxidase (MnP) at low enzymatic activities was unsuccessful, whereas at approximately 3-times higher activities the enzymes were capable of 60 % degradation in 12 days. Chemical oxidation of diatrizoate with manganese(III) acetate resulted in 85 % degradation in 12 days. Moreover, the suitability of microfluidic flow injection analysis coupled with thermal lens microscopy (μFIA-TLM) as a fast screening method for diatrizoate degradation was examined. The degradation was monitored through the release of iodide from the diatrizoate molecule. μFIA-TLM proved to be a preferable method over UV-Vis spectrophotometry, due to its higher sensitivity, sample throughput, and simple sample handling. Limit of detection (LOD) for μFIA-TLM method was estimated to be 0.14 µM in a 100 µm channel, which is 9 times lower than LOD obtained in UV-Vis measurements. In addition to μFIA-TLM and UV-VIS, high-pressure liquid chromatography (HPLC) was used to monitor the remaining parent compound in the reaction mix. In the fifth chapter, the second water treatment approach is described. This includes synthesis of biocomposite materials from cellulose (CEL) and keratin (KER), with metal (Ag0, AgCl, Au0) nanoparticles (NPs). Materials were characterised for presence, species, and size of NPs with X-ray diffraction (XRD) and with scanning electron microscopy (SEM). Nanoparticles were confirmed to be of expected species, with sizes as follows: 6.3 ± 0.5 nm for Au NPs, 12 ± 2 nm for Ag NPs, and 22 ± 1 nm for AgCl NPs. In order to evaluate antibacterial properties of the materials, contact tests with gram-negative (Escherichia coli and Pseudomonas aeruginosa) and gram-positive bacteria (Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), and Vancomycin-resistant Enterococcus faecalis (VRE)) were conducted. Direct-contact assay over 24 hours showed a dose and species-dependent antibacterial activity of [CEL:KER + Ag NPs] materials. The highest potency against the selected bacteria (up to 6-log of reduction) was observed for the material with 500 mg of Ag NPs. AgCl NPs appeared to be less potent than Ag NPs, whereas Au NPs exhibited antibacterial activity only against MRSA and VRE. In addition, antiviral properties of materials were investigated on selected bacteriophages (MS2, phiX174, and fr). However, biocomposite materials with 500 mg of Ag NPs and AgCl NPs, as well as, 240 mg of Au NPs did not exhibit any activity against selected bacteriophages. Biocompatibility with human fibroblasts was evaluated through a direct contact assay for 3 and 7 days of exposure. High concentrations of metal NPs turned out to be cytotoxic for human fibroblasts, whereas the amount of 69 mg of Ag NPs in [CEL:KER] was low enough not to affect the viability of the fibroblasts after 3 days of exposure. Composites with Ag NPs and AgCl NPs were also tested for leachability of NPs out of the materials. Both types of NPs were leaching out in two different forms, as silver ions, and as colloidal silver. Leaching of ionic silver from both materials stabilised after 3 days, whereas colloidal silver was still leaching out on the 7th day. The overall percentage of the total silver (ionic + colloidal) leached was only 0.04 % of silver incorporated in the material. In summary, this dissertation investigates alternative approaches for water treatment technologies, which could potentially serve as unit improvements of existing technologies, or as on-point pre-treatment technologies to facilitate further conventional water treatment techniques. It also demonstrates the suitability of μFIA-TLM for fast screening measurements in aquatic samples, offering high sample throughput, simple handling of the samples and superior sensitivity over the UV-Vis spectrophotometry. Kakovost in zadostna količina vode sta v zadnjem času zaradi naraščajočega prebivalstva, industrializacije in vse bolj razširjenega kmetijstva, zaskrbljujoči na globalni ravni. Za zagotavljanje teh ciljev je potrebno zagotoviti zadovoljivo stopnjo tretiranja odpadnih voda ter razviti analitske tehnike, ki zagotavljajo občutljivo in hitro detekcijo nevarnih agensov v vodah. Eno večjih nevarnosti predstavljajo farmacevtske učinkovine in izdelki za osebno nego, saj vplivajo na kakovost vode, ker so pogosto biološko aktivni in lahko povzročijo neželene učinke na vodnih organizmih. Drug velik problem kakovosti vode so patogeni organizmi, ki se prenašajo z vodo, vključno z antibiotsko rezistentnimi bakterijami ter njihovim potencialnim širjenjem v okolje. Konvencionalne tehnologije tretiranja (odpanih) voda pogosto nezadostno odstranijo zgoraj izpostavljene nevarne snovi in organizme. Tudi analitske tehnike, ki se običajno uporabljajo pri nadzoru kakovosti vode so kljub selektivnosti in občutljivosti, zamudne, z zmožnostjo analize le 2 do 3 vzorcev na uro (izključujoč čas za pripravo vzorcev). Zaradi zgoraj navedenih pomanjkljivosti so v tej disertaciji raziskani novi pristopi k razgradnji odpornih spojin, inaktivaciji mikroorganizmov ter razvoju novih in hitrih presejalnih analitskih metod. Ti so v drugem poglavju disertacije navedeni kot raziskovalni cilji. Za boljše razumevanje problematike so v tretjem poglavju podane obsežne teoretične osnove o nevarnih agensih, ki se pojavljajo v vodnem okolju, s povdarkom na farmacevtskih učinkovinah ter patogenih organizmih, ki se prenašajo z vodo. Farmacevtske učinkovine vstopijo v okolje po različnih poteh (odstranjevanje neuporabljenih zdravil v stranišče, zdravila, ki zapustijo človeško telo nespremenjena ali le malce transformirana, živalski iztrebki, ki vsebujejo veterinarska zdravila, nezadostno tretirana odpadna voda, ipd.), zato so bile farmacevtske učinkovine v sledovih najdene v površinskih in podzemnih vodah, odpadnih vodah ter občasno tudi v pitni vodi. Nadalje so v tem poglavju opisana jodirana kontrastna sredstva, ki smo jih v tem projektu preučevali kot modelne spojine. Jodirana kontrastna sredstva se izločijo iz človeškega telesa praktično nespremenjena, zato velik delež teh konča v komunalnih in bolnišničnih odpadnih vodah, kjer so lahko prisotna v koncentracijah do 2.4 g/L. V istem poglavju je opisana tudi ekotoksičnost teh spojin, poskusi njihove razgradnje, ter njihova detekcija in monitoring v okolju. V poglavju so obravnavani tudi patogeni organizmi, ki se prenašajo z vodo ter so odgovorni za povzročitev 2,2 milijona smrtnih primerov na leto, s povdarkom na antibiotsko rezistentnih bakterijah. Čeprav so okužbe z antibiotsko rezistentnimi bakterijami primarno problem v bolnišnicah, prisotnost teh bakterij v naravnem okolju ni izjema, saj je velik odstotek bakterijskih izolatov iz naravnih voda kazal fenotip rezistence na več antibiotikov hkrati. Še ena obravnavana tema v tem poglavju so teoretične osnove analitskih metod, uporabljenih pri monitoringu kakovosti vode. Te zajemajo “vanguard” in “rearguard” tehnike. Prve ponujajo preposto, poceni in hitro presejanje vzorcev, vendar na račun nižje selektivnosti in občutljivosti, medtem ko slednje zagotavljajo rezultate najvišje kakovosti, odlično občutljivost in selektivnost, vendar je rokovanje z njimi zapleteno, priprava vzorcev je zamudna in potrebni so dragi inštrumenti. Z združitvijo obeh vrst tehnik je v enem sistemu mogoče združiti prednosti obeh. Opisani so tudi osnovni principi spektrometrije s toplotnimi lečami (TLS) ter njene miniaturne verzije – mikroskopije s toplotnimi lečami (TLM), kot primer hitre presejalne metode z visoko občutljivostjo. Zajet je tudi pregled objav s praktičnimi aplikacijami le teh. Nazadnje pa so v poglavju obravnavani tudi kompozitni materiali, ki se v zadnjem času uporabljajo kot alternativne metode pri tretiranju (odpadnih) voda, in sicer kot materiali za filtre, adsorbenti za onesnažila, katalizatorji pri reakcijah razgradnje ter kot sredstva za dezinfekcijo. V tem poglavju je podan predgled aplikacij treh glavnih tipov kompozitnih materialov: sintetičnih kompozitov, biokompozitov in nanokompozitov. Osrednji del te disertacije je predstavljen v četrtem in petem poglavju, ki vključujeta dva ločena pristopa tretiranja voda, kot tudi analitske metode za hitro in občutljivo zaznavo spojin. V četrtem poglaviju so raziskane možnosti za razgradnjo jodiranih kontrastnih sredstev, natančneje diatrizoata, z biorazgradnjo z izvenceličnimi encimi glive bele trohnobe Dichomitus squalens, ter s kemijsko oksidacijo z manganovim(III) acetatom. Encimska razgradnja z lakazami in mangan peroksidazami je bila pri nizkih encimskih aktivnostih neuspešna, medtem ko so encimi s 3-krat višjimi aktivnostmi bili sposobni 60 % razgradnje v 12 dneh. S kemijsko oksidacijo diatrizoata z manganovim(III) acetatom pa smo dosegli 85 % razgradnjo v 12 dneh. Preverili smo tudi ustreznost mikrofluidne injekcijske analize z mikroskopijo s termičnimi lečami (μFIA-TLM) za hitro zaznavo razgradnje diatrizoata. Razgradnjo smo preverjali preko jodida, odcepljenega iz molekule diatrizoata. Ugotovili smo, da je μFIA-TLM metoda primernejša kot UV-Vis spektrofotometrija, saj je občutljivejša in omogoča analizo večih vzorcev v danem času ter zagotavlja preprostejše rokovanje z vzorci. Za μFIA-TLM metodo smo ocenili mejo detekcije (LOD) 0,14 µM v 100 µm mikrokanalu, kar je 9× nižje kot ocenjena LOD za spektrofotometrične metode. Poleg μFIA-TLM in spektrofotometrije smo razgradnjo spremljali tudi z visokotlačno tekočinsko kromatografijo (HPLC), kjer smo spremljali preostanek diatrizoata v reakcijski mešanici. V petem poglavju je predstavljen drugi pristop k tretiranju voda: sinteza biokompozitnih materialov iz celuloze (CEL), keratina (KER) in kovinskimi nanodelci (Ag0, AgCl, Au0). Material je bil okarakteriziran z rentgensko difrakcijo (XRD) ter z vrstično elektronsko mikroskopijo (SEM), s katerima smo preverjali prisotnost, zvrst in velikost nanodelcev. Potrdili smo, da so bili nanodelci pričakovanih zvrsti ter da so bile njihove velikosti sledeče: 6,3 ± 0,5 nm za Au NPs 12 ± 2 nm za Ag NPs in 22 ± 1 nm za AgCl NPs. Protibakterijske lastnosti materialov smo ocenili s kontaktnim testom na gram-negativnih (Escherichia coli in Pseudomonas aeruginosa) ter gram-pozitivnih (Staphyloccocus aureus, proti meticilinu odporni S. aureus (MRSA) in proti vankomicinu odporni Enterococcus faecalis (VRE)) bakterijah. 24-urni kontaktni test je pokazal, da so protibakterijske lastnosti [CEL:KER + Ag NPs] materialov odvisne od koncentracije in zvrsti nanodelcev. Najvišjo aktivnost (do 6-stopenj logaritemskega zmanjšanja) smo izmerili pri materialu z 500 mg Ag0 nanodelcev. Nanodelci srebrovega klorida (AgCl) so bili manj aktivni kot nanodelci elementarnega srebra (Ag0), medtem ko so bili zlati (Au) nanodelci aktivni le proti MRSA in VRE bakterijam. Na izbranih bakteriofagih (MS2, phiX174, in fr) smo raziskali tudi protivirusne lastnosti materialov, vendar pa biokompozitni materiali z 500 mg Ag in AgCl, kot tudi 240 mg Au nanodelcev, niso kazali protivirusnih lastnosti proti izbranim bakteriofagom. Biokompatibilnost s človeškimi fibroblasti smo ocenili s kontaktnim testom, kjer smo materiale izpostavili kulturi fibroblastov za 3 in 7 dni. Visoke koncentracije nanodelcev so bile citotoksične za človeške fibroblaste, medtem ko so bili fibroblasti, po 3 dneh inkubacije z materialom z 69 mg Ag NPs, viabilni in primerljivi s kontrolo. Pri kompozitih s srebrovimi (Ag0 in AgCl) nanodelci smo testirali izluževanje nanodelcev iz materialov. Iz obeh materialov so se izluževali nanodelci v dveh oblikah, kot srebrovi ioni ter kot koloidno srebro. Izluževanje ionskega srebra se je pri obeh materialih stabiliziralo po 3 dneh, medtem ko se je koloidno srebro še vedno izluževalo na 7. dan. Skupno izluženo srebro (ionsko + koloidno) je predstavljalo zgolj 0.04 % srebra vključenega v material. V celoti disertacija razišče nekatere alternativne metode tretiranja voda, ki bi lahko potencialno služile izboljšavi posameznih enot obstoječih tehnologij, ali pa tretiranju na izvoru onesnaženja, kar bi olajšajo nadaljnje konvencionalno tretiranje. Prav tako v disertaciji dokažemo ustreznost μFIA-TLM tehnike kot hitre presejalne metode za vodne vzorce. Pokazali smo, da je μFIA-TLM visoko-učinkovita metoda, ki omogoča preprosto rokovanje z vzorci in zagotavlja izredno visoko občutljivost v primerjavi z spektrofotometrijo.

The usage of environmentally friendly extraction techniques for the discovery of novel synthetic compounds available in medicinal plants has a high priority with everyday advances in the field of pharmaceuticals. In this research, a supercritical CO2 extraction method was implemented in the analysis of four plants (chamomile, St. John’s wort, yarrow, and curry plant) after which the non-targeted analysis of the chemical composition, phenolic content, and antioxidant activity was evaluated. The obtained extraction yield was the highest for the chamomile (5%) sample. The chemical composition analyzed by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) demonstrated extraction of diverse compounds including terpenes and terpenoids, fatty acids, flavonoids and coumarins, functionalized phytosterols, and polyphenols. Voltammetry of microfilm immobilized on a glassy carbon electrode using square-wave voltammetry (SWV) was applied in the analysis of extracts. It was found that antioxidant activity obtained by SWV correlates well to 1, 1-diphenyl-2-picrylhidrazine (DPPH) radical assay (R2=0.818) and ferric reducing antioxidant power (FRAP) assay (R2=0.640), but not to the total phenolic content (R2=0.092). Effective results were obtained in terms of activity showing the potential usage of supercritical CO2 extraction to acquire bioactive compounds of interest.

The appearance of COVID disease and the introduction of subsequent measures and specific medical treatments throughout the world in 2020 could have been expected to cause the drug content changes in the surface waters. We have studied the lower course of the Croatian Mrežnica River at three areas (rural, small town, and large town domains) in three periods (May 2020, April and September 2021). The applied method was liquid chromatography with tandem mass spectrometry, which enabled the screening of 256 compounds with 7-9% (19-24 drugs) being detected, depending on the sampling period. In the beginning of the pandemic, total quantity of drugs was much lower in the rural and small town domain (142-187 ng L- 1) compared to the large town (954 ng L-1), whereas by the autumn 2021 marked increase of drug levels in rural and small town areas was observed (443- 481 ng L-1). Increase was especially notable for analgesics: non-opioid (aminophenazon, acetaminophen, ibuprofen) and opioid (tramadol, oxycodone). In spring of 2020, nonopioids were detectable only in large town domain (94.3 ng L- 1), and their concentrations steadily increased throughout the pandemic (up to 172 ng L-1). In the vicinity of small town, sharp increase of nonopioids occurred already in spring 2021 (235 ng L-1) and in rural area in autumn 2021 (125 ng L- 1). The opioids appeared in the river water at all studied sites in the last sampling period (46-90 ng L-1). Contrary to analgesics, decrease in quantity was recorded for the stimmulants, i.e. coffeine and cotinine (nicotine metabolite), indicating less active socializing due to the COVID epidemiological measures.

Copper (Cu) occurs in nanomolar concentrations in seawater and is an essential micronutrient to the most marine organisms, but its free ion is toxic to marine organisms at concentrations as low as 10-12 M. Copper in oxic environments such as oceans is primarily present as Cu (II), leading to speciation models assuming only Cu (II) as significant Cu state. Only few studies reported detectable amounts of Cu (I) in water, with Cu (I) ratio ranging from 5-10% of total dissolved copper in oceans and up to 80% of total dissolved copper in estuaries. Marine organisms are producing Cu binding ligands as defence mechanism against free Cu toxicity, which are likely reducing Cu (II) to Cu (I). Since it is predicted that free Cu concentration in ocean will rise by 50 % by the end of century, better understanding of copper speciation is crucial to asses its negative effects. In this work we will present adaption of solid phase extraction method for Cu (I) determination in seawater. Cu (I) is retained on column as bathocuproine (BCP) complex, while Cu (II) is complexed with ethilendiamine (EDTA) and it is not retained on column. Cu (I) is eluated with acidic methanol, irradiated under UV lamp and mixed with concentrated HNO3, HCl and H2O2 in order to destruct Cu (I)- BCP complex. Cu concentrations are measured in both Cu (II) and Cu (I) fractions, as well as in seawater samples before column separation, using anodic stripping voltammetry (ASV) with hanging drop mercury electrode (HDME).

The hyporheic zone (HZ) belongs to the local and regional interstitial groundwater ecosystems and to the most threatened aquatic environments. Bioaccumulation monitoring allows us to gain insight into the bioavailability of the pollutants in the environment and their biomagnification in the food chain. The aim of this research was to evaluate the accumulation of macro- and trace elements in stygophilous amphipod Synurella ambulans which inhabits HZ of the Sava River in Croatia. Concentrations of 12 elements (As, Ba, Cs, Rb, Se, Sr, Tl, V, Ca, K, Mg, and Na) in interstitial water and in amphipods were measured by high resolution inductively coupled plasma mass spectrometry. The study was conducted in the middle section of the Sava River at two study sites: 1) Medsave – located about 3 km upstream from the wastewater outlet of the city of Zaprešić and 2) Jarun – located about 13 km downstream from this wastewater outlet and within the Zagreb City area, during the 4 seasons: winter 2018, spring, summer, and autumn 2019. At both sampling sites the increasing order of metals in S. ambulans was as follows: Tl < Cs < V < As < Se < Rb < Ba < Sr < Mg < Na < K < Ca. In interstitial water, metals at both sampling sites had similar increasing order as in S. ambulans and the concentration ranges were the following: < 0.1 µg L-1 (Cs, Tl), < 1 µg L-1 (As, Se, V), 1 - 5 µg L-1 (Rb), > 10 µg L-1 (Ba) and > 100 µg L-1 (Sr). In general, higher metal accumulation was observed for Rb, Se, Tl, K, and Na in S. ambulans specimens from the Medsave site and for As, Ba, Cs, V, Ca, and Mg from the Jarun site in all studied seasons. Comparison between chemically similar alkali and alkaline earth macro- and trace elements in S. ambulans showed that bioaccumulation factors (BAFs) increase in the following order: Mg < Na < Ba ≈ Sr < Ca < K < Rb < Cs. In this case, the tendency to accumulate in S. ambulans increased with the relative atomic mass of the element. BAFs were generally higher at Medsave, with average values ranging from 165 to 19, 534 L kg-1, and Cs with the highest BAF. It is considered that Cs is concentrated by crustaceans probably because of its chemical similarity to K, and it may derive from the radioactive wastes in fresh water [1, 2]. Cesium has the potential to biomagnify with increasing trophic levels in aquatic food webs [3]. Also, it has been shown that Cs had an impact on the neuromuscular system by increasing the amplitude of excitatory postsynaptic potentials [4]. Biomonitoring of HZ is crucial for protection and conservation of these generally little- considered groundwater connected ecosystems. These are the first results on concentrations of presented elements in S. ambulans and as such are essential for assessing point source of water pollution and global evaluation of groundwater ecological quality.

Polybrominated diphenyl ethers (PBDEs) and organochlorinated (OC) compounds such as polychlorinated biphenyls (PCBs) and DDT isomers are omnipresent in aquatic environments as a consequence of current anthropogenic activities or their use in the past. Due to their specific life cycle, eels are regularly used for biomonitoring contaminant concentrations reflecting environmental burden. Monitoring levels of pollutants in eels is important because it is suspected that they have contributed to the decline of the eel population over the last few decades, as well as because of the impact on human health, as eels are widely included into typical diets among people living in the area of their habitat. Persistent organic compounds (POPs) – PBDE congeners (BDE–28, −47, −99, −100, −153, −154, −183), indicator PCB congeners (PCB−28, −52, −101, −118, −138, −153, −180), and three DDT-type compounds (DDT, DDD, DDE) were analysed in 33 European eels (Anguilla anguilla) caught in the upper (S1) and middle (S2) course of the Raša River, an eastern Adriatic karstic river. Microwave-assisted extraction was used as the extraction technique followed by GC-µECD. According to the results, a low level of PBDE contamination of eels was observed, with a median total concentration (∑PBDE) of 1.76 ng g-1 lipid weight (lw) and 2.02 ng g-1 lw at locations S1 and S2, respectively. BDE-47 was a dominant congener in the samples in which it was detected, accounting for more than 50% to the ∑PBDE in 75% of all eel samples, followed by BDE-100. A relatively wide range of total PCB concentrations was detected, with a median of 29.3 ng g-1 lw and 46.2 ng g-1 lw at S1 and S2, respectively. PCB-138 and -153 were the predominant congeners in analysed eel samples, followed by PCB-180. Hexachlorobiphenyls mainly contribute the most to PCB levels in eels, but the same is also typical for different marine organisms in the Adriatic Sea. The analysed DDT- type compounds were present in all eel samples from the Raša River. The median of their total concentration was 37.3 ng g-1 lw and 53.8 same is also typical for different marine organisms in the Adriatic Sea. The analysed DDT-type compounds were present in all eel samples from the Raša River. The median of their total concentration was 37.3 ng g-1 lw and 53.8 ng g-1 lw, at locations S1 and S2, respectively. DDE was the predominant compound among all of the targeted analytes. Concentrations of targeted organic contaminants were significantly positively correlated with length, weight and the relative condition factor of eels, and even to a greater extent with the lipid content of eels. Since all of the analysed eels were in the yellow stage, such results were expected, because in this life stage, they strongly accumulate lipophilic contaminants due to their high lipid content and sedentary lifestyle. Although the OC compounds included in this study were banned decades before PBDEs were, they were still detected in higher concentrations in eels from the Raša River, confirming their persistence in biota. Generally, the levels of organic contaminants in eels measured in our study were at the lower end of levels reported for eels across Europe.

One of the main challenges of the modern world is the degradation of water quality due to human activities. The continuous interaction between sediment and water regulates the adsorption- desorption behaviour and thus the bioavailability of pollutants in rivers. The aim of this study was to compare the granulometric and geochemical characteristics of river sediments at five sites of the karst Krka River, which are under different anthropogenic impact: Krka River source (KRS, reference site), Krka River influenced by municipal wastewater (KRK-MWW), tributaries impacted by industrial effluents (Orašnica (TOR) and Kosovčica (TKO)) and by agricultural runoff (Butišnica (TBU)). After sampling the river sediments in July of 2021, carbonate content, particle size distribution, mineral oils, total organic carbon (TOC) and concentrations of Al, As, Ba, Cd, Cr, Cu, Fe, Mn, Ni, Pb, V and Zn were determined. Carbonate content ranged 22.8 (TOR) - 92.8% (KRK), while the predominant particle size fractions were sand and silt at all locations. The particle size distribution indicated multiple material sources for the studied sediments. The obtained characteristics of the sediments were typical for karst rivers, but some parameters indicated anthropogenic impact. Compared to the other sites, levels of mineral oils and common industrial metals were high (Al≥Fe>Zn>Mn>Ba) at TOR and TKO, while TOC was elevated at TKO and KRK. Geochemical data of sediment from the Krka River watercourse indicated more evidence for industrial impact than urban and agricultural. Since pollutants in the sediment from karst rivers can easily become bioavailable, strict protection plans and continuous monitoring of the Krka River are recommended.

Book of abstracts of 22nd European Meeting on Environmental Chemistry that was held in Ljubljana, Slovenia, 05 - 08 December 2022.

The process of environmental pollution is experiencing exponential and unstoppable growth and has become one of the biggest problems facing humanity today. Progress in science has also brought a new type of pollution, therapeutic drugs, or pharmaceutically active compounds that are one of the main components of inland water pollution and that have been found in the sediment and plasma of fish from the Sava River. Based on previous research, cardiovascular drugs have been chosen: amiodarone, ramipril, simvastatin and verapamil, the effect of which has been investigated, individually and in a mixture, on the development of the zebrafish embryo Danio rerio (Hamilton, 1822) in the first 96 hpf. During exposure to environmentally relevant concentrations (0.1, 1.0 and 10.0 μg/L), caused changes such as mortality and abnormality rate, larval length, hatching rate, heart rate, spontaneous movements of the pectoral fins and oxidative stress were monitored. With increasing concentration and time exposure of individuals to the tested compounds, effects on exposure to all compounds were observed, but the highest toxicity was observed on the simvastatin sample. The integration of the ultimate biological effects examined within this study confirmed the excellent cause-and-effect relationship between exposure to pharmaceutical compounds and the observed effect on the aquatic organism, thus providing a basis for biomonitoring of environmental pollution, especially freshwater ecosystems.

Disinfectants are one of the key tools in the process of controlling the spread of hospital infections. Given that there is a growing interest in the development of more environmentally and human-friendly, but also economically affordable agents, the reduction of active substances in prepared working solutions is desirable. At the same time, the prepared solutions must keep or even shorten needed contact times, which are necessary to achieve the required effects according to standardized efficacy testing procedures. Quaternary ammonium compounds and amines represent one of the most common active groups of substances used in preparations to disinfect surfaces, instruments, equipment, or remaining medical devices. The reason for their widespread use is that these substances enable different ways of application, good compatibility with materials, and generally proven effectiveness. The problem of monitoring such a large group of compounds in so many different media is needed in an extremely large number of analytical methods, which include chromatography (liquid or gas coupled with various detectors), mass spectrometry, electrochemical, and many other methods. A reliable analytical method is also vital in process of proving compliance with legislation in the field of biocides, especially in the field of medical devices. Antimicrobial substance N-(3-aminopropyl)-N-dodecylpropane-1, 3-diamine (BAPDA) is a novel compound used for disinfection purposes. Methodologies for BAPDA detection such as HPLC-MS have already been developed, but our scope of research was oriented to the development of a simple method, suitable for the determination of residues on various hygienic and medical surfaces. We propose HPLC-DAD method with the prior derivatization of the amine, present in the sample. The derivatization procedure was carried out as described earlier [4]. Briefly, amine was dissolved in a 0.1 M HCl aqueous solution or ethyl acetate. Sodium hydroxide (2 M) was added followed by the addition of benzoyl chloride. The reaction took place at different temperatures for a specific time interval. The acylation was stopped by adding saturated NaCl solution and performing further extraction with diethyl ether. The upper organic layer was dried and redissolved in methanol for HPLC analysis. Method optimization included: the selection of solvent for a working solution, temperature, and derivatization time optimization. Obtained results showed that ethyl acetate was a better initial solvent, with 50 min as an optimal time at 30°C with a linear response curve for amine concentrations from 0.2 to 1 mg/ml. The method was evaluated on several biocidal products.

Brown macroalgae have recently been the focus of many research due to their composition and the accumulation of specific metabolites with several health benefits and biological activities. Additionally, the extracts of Halopteris scoparia (HASC) have shown to have protective and preventive activity against human cancer cells. In this study, the evaluation of HASC against harmful ultra-violet (UV) light was done in regard to different activities, namely pigment determination by HPLC analysis, the transmission of erythema (Te) and pigmentation (Tp), along with the calculation of sun protective factor (SPF), antityrosinase and antioxidant activities. Antioxidant activity was also analysed before and after irradiation using UV-B light (8 W, 2000 μW cm−2) for different time periods (30, 60, and 120 minutes). Analysis was performed on two different fractions obtained with the solid-phase extraction (SPE) method, methanolic F3 and dichloromethane F4 fractions, from three different harvesting seasons (February, April, and June 2021). HPLC analysis revealed several pigments in HASC samples. The most abundant were fucoxanthin, lutein, and β-carotene. The highest concentrations were found in the sample from February. Interestingly, the less lipophilic pigments such as fucoxanthin, lutein, astaxanthin, and canthaxanthin, were observed in the F3 fraction, while the F4 fraction contained more lipophilic pigments chlorophyll a and β-carotene. The highest antioxidant activity using DPPH and ABTS assays was also observed for the February sample which could be explained by the presence of pigments such as β-carotene with known activity against free radicals. SPF was high for all HASC samples when compared to a known UV filter Benzophenone-3 (BP-3). % Te and % Tp showed a high protective effect for all samples, with the lowest transmission for HASC from February, especially the F4 fraction which can be attributed to the presence of β- carotene. No significant difference was observed in antioxidant activity after UV-B irradiation for all tested time periods and samples. Only the HASC June samples have shown a slight decrease in antioxidant activity after 120 minutes. Additional chemical characterization of obtained extracts is in progress to further evaluate additional bioactive compounds present in samples that could influence the photostability of these samples and could have applications as natural sun-protective agents.

The lower part of the Mrežnica River has long been exposed to industrial pollution from the cotton industry near the Town of Duga Resa. To assess the level of metal pollution of the Mrežnica River affected by anthropogenic activities, total metal concentrations were measured in liver and muscle of northern pike (Esox lucius), as a representative fish species. The liver was selected as the main metabolic site in organism, while the muscle, as the edible part, could pose a health risk to humans in case of elevated metal levels. Fish were caught in autumn 2021 at two sites: upstream of the known pollution source as the reference site (REF) and downstream of the Town of Duga Resa (DRT) as the contaminated site. Tissue samples were digested with mixture of HNO3 and H2O2, and metal concentrations (As, Bi, Cd, Co, Cs, Cu, Fe, K, Mg, Mn, Na, Pb, Rb, Se, Tl and Zn) were measured using HR ICP-MS. The concentrations of Pb were below detection limits in both organs, and of Cd only in the muscle. Obtained results confirmed liver as metabolically more active organ than muscle, as accumulation of most elements was 2-250 times higher in liver. The most prominent difference was observed for Fe, Cu, Co and Zn at both investigated sites, with concentrations in the liver higher 10-250 times compared to the muscle. However, Cs, K, Mg and Rb showed higher bioaccumulation in the muscle. Despite different roles and structures of the two organs, they reflected environmental conditions in a similar manner. Significant differences between REF and DRT sites were evident for Bi, Rb and Se in both organs, and for Cd, Mn and Zn only in liver. Bismuth and Zn showed higher levels at DRT, whereas enhanced accumulation of Cd, Mn, Se and Rb was observed at REF. These results can be partially explained by differences of metal exposure from water and sediments. Namely, patterns of Bi, Cd, Mn, and Zn are consistent with their exposure levels in sediments or water [1], while the causes of increased Se and Rb accumulation at REF should be further investigated. Some spatial variability in both organs were also observed for As, Co, Fe, K, Mg and Na, but were not shown significant. Comparison to literature revealed that metal(loid) concentrations in analyzed organs of northern pike indicate certain increase of bioaccumulation due to anthropogenic pressure. The concentrations in the fish muscle, however, were acceptable at both sites based on the national and European legislations [2, 3], which provide permissible levels for As, Cd and Pb. Therefore, moderate impact of the historical contamination was observed in the Mrežnica River, and further monitoring of the environment and biota is still recommended

Coal combustion produces nearly 1 billion tonnes of coal combustion residuals (CCR) annually, of which 40-50% is disposed. Regulated landfills and ash ponds are usually properly lined and have leachate collection systems, but old landfills usually lack these pollution prevention measures. Two such landfills are located in Croatia and were characterized by various physical and chemical analysis techniques to evaluate soil formation, trace element distribution and release of contaminants. The ashes have predominantly silt and sand fractions and a relatively large specific surface area. However, they differ in chemical and mineralogical composition, as indicated by different concentrations of trace elements, pH, and calcite content. The release of contaminants differs between landfills due to various processes such as cementation, depletion of elements and pedogenesis. This study highlights the neglect of old abandoned landfills and their changes as the waste ages.

Due to the increasing human population and the negative impact of fossil fuel combustion on the environment, there is an ever-increasing need for sustainable energy development [1]. Since microalgae offer several advantages (high growth rate, high oil content, etc), they have been considered a promising candidate for alternative renewable fuel sources (biodiesel, bioethanol, etc.). The two most important factors affecting biodiesel production, oil content, and microalgal fatty acids profile, are highly dependent upon the used microalgae species and cultivation conditions. Furthermore, studies have confirmed the beneficial effect of trace elements (Co, Cr, Mn, Mo, Cu, Fe, Zn) on microalgal growth and lipid content [2]. This research aimed to assess the dependence of total fatty acids content and profile in marine microalgae Dunaliella tertiolecta upon exposure to different concentrations of sodium selenite (Se) during two-stage cultivation. Total lipids were determined as total fatty acid methyl esters (FAME) by in-situ transesterification, while the FAME content was determined using gas chromatography [3]. According to the obtained results, the total FAME content of the control treatment group (no Se addition) was approximately 2-fold higher compared to the 20.00, 40.00 and 80.00 mg L-1 selenite treatment groups, which can be explained by the excessive exposure time of microalgae cells to Se which leads to a decrease in lipid synthesis. Furthermore, it was reported that in the control medium, γ-linoleic acid (C18:3 (c6, c9, c12) and palmitic acid (C16:0) were present in a higher proportion. Increasing the Se concentration in the culture medium increases the proportion of C16:0, while the proportion of C18:3 (c6, c9, c12) decreases, which indicates that the addition of Se in a culture medium, as a stress factor, contributes to the synthesis of monounsaturated fatty acids (MUFA). Since highly saturated fatty acids (e.g. C16:0) contribute to the satisfactory value of cetane number and oxidative stability parameters, which represent important biodiesel standards, it can be concluded that the present study reports valuable data regarding the influence of Se on fatty acids content and profile in microalgae D. tertiolecta and therefore potentially sustainable biodiesel production.

Chlorella vulgaris is a green unicellular that has been subjected to extensive studies within the last decade and has experienced imense applications in food and cosmetic industries [1]. C. vulgaris biomass represents a valuable and sustainable source of numerous bioactive compounds that can positively affect human health, such as proteins, vitamins, polysaccharides, microelements and polyunsaturated fatty acids (PUFA) [2]. Proteins and PUFAs recently gained a special interest of the nutraceutical sector due to their ability to accelerate wound healing, decrease blood pressure and cholesterol levels, but also improve bone health and neurological function [3]. For those reasons, particular focus is placed on the microalgal potential to enhance the nutritional value of conventional foods. Drying harvested microalgae while at the same time preserving all beneficial activities has shown to be a challenging step for the industry. To deepen the knowledge gap, C. vulgaris biomass was dried at different temperatures (lyophilized, 25, 40, 60 and 100), followed by determination of protein, carbohydrate and lipid contents. The highest protein value was observed for C. vulgaris dried at 25°C (58.5377±0.0007% (µg/µg), followed by samples dried at 40°C and 100°C. According to the findings, lower-temperature dried samples had higher levels of chlorophyll a and b, as well as their derivatives pheophytin a and b. The biomass dried at 40°C had the highest amount of carotenoids. The lipid profile determined by gas chromatography analysis showed a predominance of monounsaturated and polyunsaturated fatty acids, in specific essential omega-6 (linoleic acid) and omega-3 (docosahexaenoic acid) fatty acids. The highest total lipid content of C. vulgaris was around 5% for samples dried at 60 and 100°C. The results obtained confirmed C. vulgaris as a microalga with a great potential source of proteins, pigments and essential fatty acids, but also emphasized the importance of optimization of drying techniques.

The hyporheic zone (HZ) belongs to the groundwater connected ecosystems and as a key component of fluvial ecosystems, it plays a crucial role in ecohydrology, biogeochemical cycling, nutrient turnover, retention and biotransformation of trace organic compounds, remineralisation of organic matter, retention of fine particulate matter and pollutant attenuation [1, 2]. The aim of this study was to assess anthropogenic impact on the environmental quality of the HZ of the Sava River by measuring basic physico-chemical parameters, nutrients, organic carbon and metal contaminants in the interstitial water and sediment at two gravel bar locations (Medsave and Jarun) during four seasons (winter 2018, spring, summer and autumn 2019). Medsave is located 3 km upstream from the wastewater discharge of the town of Zaprešić as a point source of contamination, and Jarun 13 km downstream from it. Physico-chemical parameters, including concentrations of nutrients and organic carbon were affected by wastewater discharge, but also by hydrometeorological conditions, such as water level and river discharge. In summer and autumn, at both sampling sites lower average water level (-249 cm) and discharge (122 m3/s) were recorded, compared to spring and winter (-214 cm ; 179 m3/s). Nitrates were higher in spring and autumn at both sampling sites, which can be linked to higher atmospheric precipitation and leaching from agricultural lands. At the Jarun site, phosphate levels in interstitial water in the spring, summer, and autumn indicated less than good water quality status according to the water quality standards for the respective river type HR-R_5b [3]. The observed increased concentrations of dissolved organic carbon in water and total organic carbon in sediment at Medsave may be related to the intensive use of agricultural fertilizers. The concentrations of macro elements (Na, K, Mg and Ca) in interstitial water from the HZ were higher at the Jarun sampling site than at Medsave. Their enrichment in the HZ compared to the surface water was observed at Jarun. In the interstitial water from the HZ, the concentrations of common environmental contaminants Ni and Pb were higher at the Jarun sampling site, while Cd and Zn were higher at Medsave. Higher concentrations of Ni and Pb were observed in interstitial water than in river water. Sediment concentrations of Mo, Ni and Pb normalized to Al showed slightly higher values at Jarun. However, the concentrations of ecotoxic elements were below the environmental quality standards set for surface freshwaters [3]. In conclusion, the environmental quality of the HZ of the Sava River was slightly affected by anthropogenic influences, although it was not yet severely disturbed. Therefore, monitoring of basic parameters and contaminant concentrations in the HZ is recommended.

Brown macroalgae have recently been in the focus of many researches due to their composition and the accumulation of specific metabolites with great antioxidant potential. Food, cosmetics, and pharmaceutical industries are constantly working to find natural, non-toxic compounds that can be used in their products. In this study, we evaluated the antioxidant activity of two (methanolic and dichloromethane) different fractions from three brown macroalgae, Dichtyota dichotoma, Dichtyota fasciola, and Halopteris scoparia. Several methods were used for the evaluation of antioxidant activity including the reduction of the radical cation (ABTS), the 2, 2-diphenyl-1-picryl-hydrazyl (DPPH), the Folin–Ciocalteu, and the oxygen radical absorbance capacity (ORAC) assays. Also, total protein content was determined by the Lowry method with slight modifications, while chlorophyll and carotenoid content were determined spectrophotometrically by the Lichtenthaler method. The toxicity of samples was determined using the zebrafish embryotoxicity test up to 96 h of embryonal development. All methanolic fractions showed higher antioxidant activity than the dichloromethane fractions, while H. scoparia methanolic fraction showed the highest activity (IC50 concentration of 1.947 mg/mL). The highest protein value was observed for Halopteris scoparia (20.7%) followed by Dichtyota dichotoma (7.4%) and Dichtyota fasciola (3.3%). Because of their good antioxidant potential, all three researched brown macroalgae have the potential to be used as a source of natural antioxidants in the cosmetic industry.

Arthrospira platensis is a promising and sustainable microalga characterized by easy growth without the need for herbicide or pesticide usage, and the possibility to utilize the whole microalgae without waste and harmful greenhouse gas emissions. It represents a valuable source of bioactive compounds with different health-promoting properties which make A. platensis an attractive ingredient for the formulation of eco-friendly cosmetics. As dehydration is one of the crucial production steps in the cosmetic, pharmaceutic, and food industries, the aim of this work was to investigate the influence of different drying temperatures on the chemical composition of A. platensis. Antioxidant activity of methanol: dichloromethane (1:1) extracts from differently dried (40, 50, 60, 80, and 100°C) samples were analyzed using 2, 2-diphenyl-1-picryl-hydrazyl (DPPH), reduction of the radical cation (ABTS), ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. The influence of drying temperature on total protein, carbohydrate, pigment, and lipid content was evaluated, while gas chromatography was carried out to determine fatty acid composition. By implementing ABTS and DPPH methods, the highest antioxidant activity was observed for the sample dried at 40°C followed by 50 and 80°C dried samples with 1.5- and 2.5-fold lower activity (p

Industrial activities are nowadays one of the most important sources of metal contamination in the aquatic environments. Such example in Croatia is the Krka River, which lower part is protected as the national park, but upstream part is impacted by improperly purified industrial effluents from the screw factory. Industrial impact was assessed in July 2021 by comparison of element concentrations (As, Ba, Co, Cu, Fe, Hg, Mo, Mn, Na, Ni, Sr, U, Zn) in water and sediments from three sites: industrial wastewaters (IWW), the Orašnica tributary (TOR) directly affected by IWW and Krka River source (KRS) as the reference site. In addition, physicochemical parameters in water were considered. Chemical oxygen demand, ammonia and total nitrogen indicated degraded water quality at IWW and TOR, which was related to significant increases of element concentrations in sediments and water compared to KRS. The highest concentrations of all elements were evident at IWW, except higher Zn in water and Hg, Mn in sediments at TOR. The highest increase was observed for Fe, Mo, Co, Ni and Cu, elements commonly used in industry. Compared to KRS, IWW had up to three orders of magnitude higher element levels in water (Mn>Co>Fe) and even more in sediments (Zn>Co>Mo). TOR, which pose the direct threat to the Krka River, as IWW basins sometimes overflow into TOR, also had much higher concentrations of all elements than KRS. Thus, presented research indicates a deterioration of the Krka River by industrial effluents and highlights the need of adequate purification and monitoring strategies to protect the living world and tufa barriers of this sensitive karst ecosystem.

Green macroalgae Ulva lactuca, also known as marine green salad has high amount of different biologically active compounds such as polysaccharides, pigments, terpenes, etc. [1]. In this study, the aim was to evaluate conventional (successive solvent extraction – hexane, chloroform, methanol) and modern (solid-phase extraction, SPE, using methanol and dichloromethane) extraction procedures, as well as an environmentally-friendly option (ethanol, further fractionated using non-toxic solvents – hexane, chloroform, ethyl acetate, and butanol) for purpose of extracting pigments such as lutein, chlorophylls, and others that could posses’ activity against tyrosinase, an enzyme that has an an important role in melanogenesis and enzymatic browning [2]. High-pressure liquid chromatography (HPLC) was used for the successful identification of lutein, chlorophyll b, and canthaxanthin. The highest content of lutein was found in SPE methanolic fraction (3.422 ± 0.007 mg/g), followed by chloroform extract (1.638 ± 0.002 mg/g). Chlorophyll b was most abundant in the ethyl acetate fraction (0.443 ± 0.001 mg/g), while the canthaxanthin content was also the highest in SPE methanol fraction (0.091 ± 0.000 mg/g). In SPE dichloromethane fraction and butanol fraction no pigments were identified. Antioxidant activity using both DPPH and ABTS methods has shown to be the highest for SPE methanol fraction. The highest monophenolase activity was observed for ethanol extract (20.39 ± 8.01 %) followed by chloroform (14.36 ± 4.31 %) and hexane extracts (7.51 ± 1.02 %), while no activity was observed for all other extracts and fractions. Samples showed better diphenolase activity. The highest activity was observed for methanol extract (57.33 ± 2.53 %), followed by chloroform extract (54.09 ± 1.05 %) and methanol fraction (51.78 ± 0.95 %). All the results suggest that not only pigments exert antityrosinase activity, but also other isolated molecules. SPE has shown to be the best-used method for lutein isolation from U. lactuca and has been shown to significantly contribute to antioxidant activity.