Krka River Research Articles

Decoupling Sources of Anthropogenic Influences on Sediments of the Visovac Lake (Krka National Park, Croatia) Using Multiparametric Approach.

Historical changes of sediment characteristics and levels of inorganic and organic contaminants were studied in dated sediment cores from the Visovac Lake, situated in the Krka National Park, Croatia, to identify the main sources of anthropogenic pressures on this highly protected system. Depth distributions of lithogenic elements showed a steady decrease of terrigenous inputs due to the reduction in agricultural activities in the area, which was particularly pronounced during the 1991-1995 war in Croatia. Vertical and longitudinal distributions of Cd and Zn indicated that they are predominately of anthropogenic origin. The historical profiles of these toxic metals coincide well with the recorded production of metal industry in the upper reach of the Krka River with a sharp decrease reflecting the interruption by the war and slow recovery afterwards. By contrast, the recovery of the tourist industry in Krka NP after the war was accompanied by increasing contamination by elements characteristic of boat and car traffic (Sn, Cu, Pb) as well as oil pollution. The contamination with polycyclic aromatic hydrocarbons and polychlorinated biphenyls was only moderate. Although levels of metallic and organic contamination can be considered relatively low, the observed shift from industrial to tourism-related sources indicated that touristic activities should also be regarded as a possible threat for this vulnerable karst aquatic ecosystem.

Vanadium redox speciation in the acid-extractable phase of Krka River estuary surface sediment

This study investigated the redox speciation and mobility of V in the acid-extractable fraction of surface sediments from the Krka River estuary using an optimized IC-UV/Vis analytical method. The separation of V(IV) and V(V) redox species was done using anion-exchange based chromatographic method, while pseudo-total V concentrations were measured using HR ICP-MS analytical instrumentation. Extracted V concentrations from the sediment fraction (pH = 5, HCl) and determined pseudo-total V concentrations were used to calculate the Enrichment Factor (EF) and Risk Assessment Code (RAC), indicating potential anthropogenic influence and environmental risk. A simple PHREEQC model was developed to asses V speciation in the oxic bottom seawater layer simulating possible remobilization of the leached sediment phase. The results of the study show that minor fraction of V is present in the acid-extractable phase across the surface sediment of Krka River estuary. Higher V mobility is mostly observed at locations rich with clay minerals, terrigenous input, and carbonates. Anthropogenic influence was linked to higher enrichment but lower mobility, suggesting binding to less mobile sediment phases (reducible, organic and residual fractions). The predominance of reduced V(IV) species in the acid-extractable sediment fraction indicates a potentially low V toxicity risk in the sediments of Krka River estuary, even in cases of high potential remobilization of V. However, the model predicted complete oxidation of V(IV) to V(V) upon remobilization into the oxic bottom water layer. This highlights the complexity of V behavior in natural estuarine systems, where the toxicity risks of possible V remobilization still remain unclear. Results of this study demonstrate the need for the strengthening efforts in speciation of V in the mobile sediment phase to obtain a cohesive outlook on its potential toxicity and biogeochemical cycling.

Adsorption of trace metals onto different plastics during long-term exposure in an estuarine environment: Influence of time, stratified water column, and specific surface area

The pervasive increase of plastics in marine systems and subsequent trace metals (TM) adsorption represents a critical environmental challenge. This long-term study is first to investigate how duration of exposure and stratified water column impact TM adsorption on various plastics in an estuary. We exposed polypropylene (PP), polyethylene (PE), poly(ethylene terephthalate) (PET), and fluorinated ethylene propylene (FEP) to estuarine conditions, categorizing them into three distinct groups to capture diverse human activities: pre-production pellets (Pellets), single-use plastics (SUP) and laboratory plastics (Lab). Plastic and water samples were analyzed before deployment and 1, 4, 7 and 16 months after deployment at three depths in the stratified Krka River estuary (Croatia). We measured TM concentrations, pH and salinity in the water column, and adsorbed TM amounts on plastics. Additionally, we examined plastic sample surfaces by Raman spectroscopy and scanning electron microscopy. TM speciation was modeled from water column data to better explain adsorption processes. Statistical analysis indicates that Zn, Cd, Pb, Ni, and Co adsorption depends on both duration of exposure and stratified water column, while Cu is influenced solely by duration of exposure to estuarine environment. Adsorbed TM amounts varied significantly among the plastic groups (Pellets, SUP and Lab), but not among different polymer types (PP, PE, PET and FEP). A noteworthy observation was the difference in the results of statistical analysis when investigating TM amounts expressed by plastic sample mass and by plastic sample surface area. Results expressed by plastic sample mass showed a difference between SUP group versus Pellets and Lab groups, while results expressed by plastic sample surface area distinguished Pellets group compared to other two groups. Therefore, we suggest presenting TM adsorption results in similar studies both by plastic sample mass and by plastic sample surface area. This approach will improve the understanding of TM adsorption on plastics, and enable more effective comparisons among the scientific literature dealing with plastics of different specific surface areas.

Mytilus galloprovincialis shell growth – Insights from shell geochemistry

Despite the wide geographic distribution of mussels and their importance in fisheries and aquaculture, understanding of temporal and spatial variations of their growth is still limited. This can be, at least partly, attributed to the lack of distinct growth lines in their shells. Our study uses geochemical properties of shells, particularly oxygen (δ18Oshell) and carbon (δ13Cshell) stable isotope values, to reconstruct mussel growth. Mytilus galloprovincialis specimens were collected from two of the most important bivalve aquaculture sites in the eastern Adriatic - Krka River estuary and Mali Ston Bay. The first site has variable environmental conditions, influenced mainly by the Krka River, resulting in a strong temperature and salinity gradient along the estuary. In contrast, Mali Ston Bay is a relatively shallow enclosed bay with more stable conditions. Samples for δ18Oshell and δ13Cshell analysis were acquired by manual microdrilling cross-sectioned shells from three similar sized shells from each site. In addition, analysis of elemental profiles (Mg/Ca, Sr/Ca, and Ba/Ca) in shells from the Krka River estuary was conducted using laser ablation ICPMS. Temperature and salinity values were obtained for each site through research and monitoring projects and used to calculate predicted δ18Oshell values. By aligning the measured δ18Oshell data along the predicted δ18Oshell curve, it was possible to reconstruct the timing and rate of seasonal shell growth. Results show that M. galloprovincialis shell deposition slows down and/or ceases during high temperature and low salinity periods. Specimens from the Krka River estuary had faster shell growth rates and were characterised by higher δ13Cshell values. Mg/Ca profiles of M. galloprovincialis specimens from the Krka River estuary, confirm growth periodicity data obtained by δ18O analysis. Ba/Ca peaks were also discussed by comparison with δ13C time-series and environmental data. Results have potential applications in fisheries studies as well as in palaeoecology and palaeoceanography research.

Interrelation between environmental conditions, acanthocephalan infection and metal(loid) accumulation in fish intestine: an in-depth study

Metal(loid) bioaccumulation in acanthocephalans (Dentitruncus truttae) and intestines of fish (Salmo trutta) from the Krka River, influenced by industrial and municipal wastewaters, was investigated in relation to exposure to metal(loid)s from fish gut content (GC), water, and sediment to estimate potentially available metal (loid)s responsible for toxic effects and cellular disturbances in biota. Sampling was performed in two seasons (spring and autumn) at the reference site (river source, KRS), downstream of the wastewater outlets (Town of Knin, KRK), and in the national park (KNP). Metal(loid) concentrations were measured by ICP-MS. The highest accumulation of As, Ba, Ca, Cu, Fe, Pb, Se and Zn was observed mainly in organisms from KRK, of Cd, Cs, Rb and Tl at KRS, and of Hg, Mn, Mo, Sr and V at KNP. Acanthocephalans showed significantly higher bioaccumulation than fish intestine, especially of toxic metals (Pb, Cd and Tl). Metal(loid) bioaccumulation in organisms partially coincided to exposure from water, sediments and food, while in GC almost all elements were elevated at KNP, reflecting the metal(loid) exposure from sediments. Seasonal differences in organisms and GC indicated higher metal (loid) accumulation in spring, which follows enhanced fish feeding rates. Higher number of acanthocephalans in the intestine influenced biodilution process and lower concentrations of metal(loid)s in fish, indicating positive effects of parasites to their host, as supported by high values of bioconcentration factors. Fish intestine and acanthocephalan D. truttae were confirmed as sensitive indicators of available metal fraction in conditions of generally low environmental exposure in karst ecosystem. Since metal(loid) accumulation depended on ecological, chemical and biological conditions, but also on the dietary habits, physiology of organisms and parasite infection, continuous monitoring is recommended to distinguish between the effects of these factors and environmental exposure when assessing dietary associated metal(loid) exposure in aquatic organisms.

Stable isotopic composition of tissues and shells in the Mediterranean mussel, Mytilus galloprovincialis, in estuarine and aquaculture settings

The Mediterranean mussel, Mytilus galloprovincialis Lamarck, 1819 is a widely distributed commercially important bivalve species, and is one of two cultivated bivalve species in the Adriatic Sea, with the Krka River estuary being a key aquaculture location. Understating the feeding ecology of M. galloprovincialis could help aquaculture be more efficient. The aim of this study is to use stable isotopes to advance our understanding of M. galloprovincialis feeding ecology, as well as explore the potential of their shell as proxies for environmental change. We analyzed stable isotope composition (hereafter, δ13C and δ15N), of M. galloprovincialis soft tissue, as well as the δ15N signature of its shell, over one year at multiple sites in the Krka River estuary. Mussels showed differences in the isotopic niche depending on the site, indicating that feeding varies according to geographic location, likely due to specific conditions in the Krka River estuary and differences in food availability. During our entire survey period, marine organic carbon was the dominant source in the mussels cultivated in the Krka estuary. However, in the period from late summer to late autumn, the contribution of freshwater carbon sources increased for both sites situated in the middle estuary. The distinct feeding patterns observed at each sampling site could be attributed to a combination of factors such as local hydrographic conditions, food availability, and the presence of other competing species. Overall, the stable isotope results present valuable information on the feeding ecology of Mytilus galloprovincialis, highlighting the complex interactions between environmental factors and feeding habits. The results of our study contribute to the isotope data archive for M. galloprovincialis. Furthermore, this work shows the potential of M. galloprovincialis shell δ15N values as an indicator for nitrogen loading in aquaculture settings and coastal marine environments.

High diversity of macrophytes in the »green river« Krka (Slovenia)

AbstractThe presence, abundance, and distribution of aquatic macrophytes and their growth forms in the river Krka in Slovenia were studied. The studied slow‐flowing lowland River Krka is also called a »green river« since it is overgrown with macrophytes from its source to its outflow to the river Sava. The environmental parameters of the river ecosystem were also assessed using the modified version of the RCE (Riparian, Channel, and Environmental Inventory) method. Within 24 sections reaching from 100 to 250 m, 23 taxa of macrophytes were found. Sections were distributed noncontinuously from the source to the outflow more or less equally along the river course. The invasive alien species Elodea canadensis reached the highest relative abundance, followed by Ranunculus trichophyllus, Potamogeton crispus, Myriophyllum spicatum, Ceratophyllum demersum, and Potamogeton nodosus. Potamogeton crispus was present in most of the studied sections followed by Nasturtium officinale and M. spicatum. The river offers suitable conditions for the high diversity of macrophytes regarding the type of substrate, low flow velocity, heterogeneity of habitats, and relatively high concentrations of nutrients. In comparison to the survey performed in 2003, when the river Krka was surveyed continuously from the source to the outflow, we found that seven of the submerged and natant macrophytes were not detected in 2020. Macrophytes, growing in the river Krka, indicated meso‐ to eutrophic conditions. Five species recorded in the river Krka are listed on the Red list of endangered species in Slovenia, indicating the need to conserve the river ecosystem.

Early Diagenetic Processes in the Sediments of the Krka River Estuary

To study the processes that govern the post-depositional mobility of metals in the estuarine sediment, five sediment cores were sampled in the Krka River estuary (Croatia). The obtained concentration ranges in the pore water were 0.057–49.7 μM for Fe, 0.310–100 μM for Mn, 0.068–26.8 nM for Co, 0.126–153 nM for Cu, 11.5–2793 nM for Zn, 0.222–31.3 nM for Pb, 4.09–59.4 nM for U, 38.8–2228 nM for Mo, and 0.065–2.29 nM for As. The vertical distribution of metals in the dissolved and solid fraction of the sediment, coupled with other diagenetic tracers (e.g., dissolved sulphide), demonstrate the importance of early diagenetic reactions, in particular Fe and Mn oxyhydroxide and sulphate reduction, for the cycling of metals in the sediment. The redox zonation in the sediment was compressed, and the suboxic zone occurs immediately below the sediment–water interface. The estimated benthic fluxes in the estuary were 5220 kg y−1 for Fe, 27,100 kg y−1 for Mn, 6.00 kg y−1 for Co, 20.5 kg y−1 for Cu, 5.16 kg y−1 for Pb, 111 kg y−1 for Mo, and 87.3 kg y−1 for As. The riverine input was more important than the benthic flux, except in the case of Mn and Fe.

Spatial Distribution, Ecological Risk Assessment, and Source Identification of Metals in Sediments of the Krka River Estuary (Croatia)

To evaluate the level of contamination and predict the potential toxicity risk, selected metal concentrations (Cd, Pb, Cr, Mn, Co, Ni, Cu, Zn, and As) were determined in 40 surface sediment samples from the stratified karstic Krka River estuary (Croatia). In addition, diffusive gradients in thin films (DGT) probes were deployed in situ to understand the mobilization mechanisms and bioavailability of metals in the sediment. The results show significant spatial differences between the upper and lower estuary, with the latter being more affected by anthropogenic pollution. The pollution assessment using the enrichment factor (EF), the geoaccumulation index (Igeo), and the pollution load index (PLI) showed a strong enrichment of metals in the lower part of the estuary, especially of Mn, Cu, Zn, Pb, and As. The statistical analysis (PCA) revealed the former ferromanganese factory and the port as major sources of pollution in the area. Nickel, Co, and Cr, although slightly elevated, may be attributed to the natural origin. The metal mobility in the estuarine sediment was primarily governed by early diagenetic processes (aerobic organic matter mineralization, Fe and Mn oxyhydroxide reduction), which caused the release of metals from the sediment into the pore water and subsequently into the overlying water column.

Prostorna i sezonska raspodjela partikularnog fosfora u obalnim vodama i otvorenom moru srednjeg Jadrana

The seasonal and spatial distributions of particulate phosphorus (PP) in the water column of the coastal area and open sea of the middle Adriatic Sea area were investigated for the first time. The relationship between PP and two other important forms of phosphorus (dissolved inorganic phosphorus (DIP), and organic phosphorus (P-ORG)), and the relationship between PP and other nutrients (nitrogen and silicon salts) and oceanographic parameters (temperature, salinity, oxygen saturation, chlorophyll a) from the water column were also examined. PP concentrations ranged from 0.007 μmol dm-3 in the open sea near Palagruža archipelago to 0.589 μmol dm-3 in the Krka River estuary. In the coastal area, PP concentrations were highest in autumn and lowest in winter. The vertical distribution of PP concentrations in the water column of the coastal area were highest in the surface layer during most of the year due to input of PP by freshwater inflows. In the open sea area, there were no significant seasonal or vertical differences in the distribution of PP concentrations in thewater column. Seasonal analysis of the total phosphorus pool (PP, DIP and P-ORG) in the water column indicated that PP and P-ORG concentrations were higher in the coastal area compared to the open sea, while DIP concentrations were comparable.The results further demonstrate that PP can be an important fraction of the total phosphorus pool. A significant positive correlation was found between PP and chlorophyll a, indicating that PP was mainly of biological origin, and with nutrient salts of nitrogen and silicon, while a negative correlation was found with salinity.

Molecular Analysis of Two Endemic Squalius Species: Evidence for Intergeneric Introgression among Cyprinids and Conservation Issues

Conservation of indigenous species, especially endemic ones, is of the utmost importance. Morphological determination of species is usually not sufficient; therefore, molecular phylogenetic analyses of the Illyrian chub, Squalius illyricus, and the Zrmanja chub, Squalius zrmanjae, from the Krka River were performed. For the genetic characterization of the mitochondrial gene cytochrome b and the non-coding nuclear region Cyfun P, 15 specimens from each species were subjected to analysis. The obtained sequences were aligned with similar ones from GenBank to determine the taxonomic and phylogenetic position of these species. The obtained molecular results imply that S. zrmanjae from the Krka River has a nuclear region that resembles Dalmatian rudd, Scardinius dergle. This result implies an introgression event and the transfer of genetic information between the two genera. The investigated species are on the IUCN Red List of Threatened Species, their biological data are scarce, and further investigation and protection are needed.

Spatial and temporal variability of dissolved metal(loid)s in water of the karst ecosystem: consequences of long-term exposure to wastewaters

The metal(loid) variability in the sensitive karst ecosystem was addressed as possible indicator of short- and long-term exposure to the wastewaters, recognized as the main metal contamination sources. Industrial and municipal wastewaters are released without proper purification only 2 km upstream from the Krka National Park (KNP) in Croatia. The variability of dissolved metal(loid)s was studied in four seasons and eight locations: Krka River source (reference site, KRS), municipal wastewaters from the Town of Knin (KRK), industrial wastewaters from the screw factory (IWW), Brljan Lake in the KNP (KBL), and the tributaries Krčić (TKR), Kosovčica (TKO), Orašnica (TOR) and Butišnica (TBU). Water taken directly from IWW had several times higher concentrations of all elements than other locations and indicated industrial wastewater as the primary Mn, Zn, Co, Cs, and Fe source. Tributary Orašnica, flowing by IWW, was the most affected site, although higher metal concentrations were also found at other locations compared to KRS. Overall, spatial metal contamination followed the order: TOR>KRK>TKO>TBU>KBL>KRS. Seasonality was not pronounced, although the highest levels for most metals were observed in summer, dry season when the self-purification processes are reduced. Almost all elements had low tendency to bind with particles, therefore showing high presence in dissolved fraction and confirming their bioavailability and potential toxicity. Although metal concentrations increased over time, they were still low compared to metal-contaminated rivers. However, observed metal exposure and inter-site differences present a warning and indicate the need for the targeted continuous monitoring of potential hotspots to protect this karst ecosystem.

Integrated Reconstruction of Late Quaternary Geomorphology and Sediment Dynamics of Prokljan Lake and Krka River Estuary, Croatia

The upper part of the Krka River estuary and Prokljan Lake are a specific example of a well-stratified estuarine environment in a submerged river canyon. Here, we reconstructed the geomorphological evolution of the area and classified the data gathered in the study, integrating multibeam echosounder data, backscatter echosounder data, side-scan sonar morpho-bathymetric surveys, and acoustic sub-bottom profiling, with the addition of ground-truthing and sediment analyses. This led to the successful classification of the bottom sediments using the object-based image analysis method. Additional inputs to the multibeam echosounder data improved the segmentation of the seafloor classification, geology, and morphology of the surveyed area. This study uncovered and precisely defined distinct geomorphological features, specifically submerged tufa barriers and carbonate mounds active during the Holocene warm periods, analogous to recent tufa barriers that still exist and grow in the upstream part of the Krka River. Fine-grained sediments, classified as estuarine sediments, hold more organic carbon than coarse-grained sediments sampled on barriers. A good correlation of organic carbon with silt sediments allowed the construction of a prediction map for marine sedimentary carbon in this estuarine/lake environment using multibeam echosounder data. Our findings highlight the importance of additional inputs to multibeam echosounder data to achieve the most accurate results.

First insight in element localisation in different body parts of the acanthocephalan Dentitruncus truttae using TEM and NanoSIMS

Acanthocephalans, intestinal parasites of vertebrates, are characterised by orders of magnitude higher metal accumulation than free-living organisms, but the mechanism of such effective metal accumulation is still unknown. The aim of our study was to gain new insights into the high-resolution localization of elements in the bodies of acanthocephalans, thus taking an initial step towards elucidating metal uptake and accumulation in organisms under real environmental conditions. For the first time, nanoscale secondary ion mass spectrometry (NanoSIMS) was used for high-resolution mapping of 12 elements (C, Ca, Cu, Fe, N, Na, O, P, Pb, S, Se, and Tl) in three selected body parts (trunk spines, inner part of the proboscis receptacle and inner surface of the tegument) of Dentitruncus truttae, a parasite of brown trout (Salmo trutta) from the Krka River in Croatia. In addition, the same body parts were examined using transmission electron microscopy (TEM) and correlated with NanoSIMS images. Metal concentrations determined using HR ICP-MS confirmed higher accumulation in D. truttae than in the fish intestine. The chemical composition of the acanthocephalan body showed the highest density of C, Ca, N, Na, O, S, as important and constitutive elements in living cells in all studied structures, while Fe was predominant among trace elements. In general, higher element density was found in trunk spines and tegument, as body structures responsible for substance absorption in parasites. The results obtained with NanoSIMS and TEM-NanoSIMS correlative imaging represent pilot data for mapping of elements at nanoscale resolution in the ultrastructure of various body parts of acanthocephalans and generally provide a contribution for further application of this technique in all parasite species.

A new tool for the determination of humic substances in natural waters: Pulsed voltammetry approach

Humic substances (HS) in natural waters can be determined with a new, simple and sensitive method based on their influence on the background current in a differential pulse - adsorptive cathodic stripping voltammetry. The proposed method, termed PB-HS (pulsed background - humic substances) is discussed in detail, including its application in natural samples from the Krka River estuary. The method was additionally compared with absorbance measurements as well as with the typical electrochemical HS quantification in natural waters based on HS complexation with molybdenum (Mo). A good correlation between methods was observed, with PB-HS showing slightly better sensitivity to humic compounds than classical spectrophotometry. Higher HS concentrations measured with the Mo-method may be due to the enhanced hydrophobicity reached at pH 2 that is required by the method. Advantages of the proposed PB-HS method, compared to existing voltammetric methods for HS quantification, are that it does not require any reagent addition (except buffer) and that it can be used at the natural pH of water as well as in a wide salinity range, which is crucial for its application in estuarine waters.

Diversity of protist genera in periphyton of tufa-depositing karstic river

PurposeIn aquatic ecosystems, protists play a crucial role and cover numerous ecological functions. The karstic Krka River (Croatia) is a unique hotspot for high diversity of aquatic organisms, especially protists. The main objective of the present study was to obtain a detailed overview of the protist community structure in the periphyton of the Krka River and to determine the differences in protist diversity along the river.MethodsProtist diversity was detected by amplicon sequencing of the hypervariable region V9 of the 18S rRNA gene, using the universal eukaryotic primer pair.ResultsThe three main groups of protists were as follows: Ciliophora, Cercozoa, and Bacillariophyta. In terms of abundance of protist OTUs, the shade plot revealed an evident difference from the upstream to downstream river section, which increased between locations from Krka spring to Skradinski buk. Diversity was explored using measures of alpha and beta diversity. Alpha diversity showed an increasing trend in the downstream direction of the river. The location effect, or clustering/grouping of samples by location, was confirmed by the PERMANOVA permutation test of beta diversity.ConclusionThe combination of alpha and beta diversity can help provide deeper insight into the study of diversity patterns, but also point out to decline in species diversity and allow for effective ways to protect aquatic karst habitats in future management.

One new species of Aneumastus D.G. Mann et Stickle (Bacillariophyceae) from Krka River, Croatia

Karst rivers in Croatia are common phenomena and known biodiversity hotspots for many different groups of organisms. Due their importance from a natural, cultural and socio-economic point of view, most of them are protected as National Parks. Diatoms in these habitats are not well known, and recently detailed study has been started. During observations of samples from Lake Visovac on Krka River, one <i>Aneumastus</i> species with specific morphological features, has been recorded. The species is characterized by linear valves with almost parallel margins in the mid-valve, rostrate apices and striae that are partly biseriate, coarsely punctate and distantly spaced. In this study, it is described as a new species <i>Aneumastus </i><i>visovacensis</i> sp. nov. Its morphology is documented with light and scanning electron microscopy, described in detail, and compared with the most similar species from <i>A. tusculus </i>species complex.

Microplastics assessment in the Krka river estuary surface water

Microplastics (MPs), commonly defined as particles less than 5 mm, are a persistent ubiquitous anthropogenic contaminant that can be found in every environment, making it a global environmental, health, and socioeconomic problem. Due to their high surface area, MPs adsorb toxic pollutants that become bioavailable to organisms upon ingestion as they are often mistaken for food leading to biomagnification (Bule et al., 2020). The sampling area represents the lower part of the Krka River Estuary and is under direct anthropogenic influence from the city of Šibenik runoff waters, nautical and communal ports, city harbor, tourism, mariculture, and fishing. Estuaries and harbors have been recognized as hotspots and transfer pathways for MPs primarily because of the vicinity of the urban environment that emits contaminants from various sources (Miller et al., 2021). The main focus of this research was to determine MPs size, shape, color, surface area, and abundance in surface water using volume-reduced samples collected by a net. Laboratory protocol included sieving, wet peroxidation (H2O2), density separation (saturated NaCl solution), sonication, and filtration. Filter papers were then visually inspected for MPs. Image processing and measurements were carried out with ImageJ/Fiji open-source software.

Changes in Phylogenetic and Functional Diversity of Ciliates along the Course of a Mediterranean Karstic River.

Ciliates are a group of phagotrophic protists found in a wide variety of ecosystems. This study builds on recent studies of ciliates in the Krka river and investigates changes in the phylogenetic and functional diversity of ciliates in biofilm to predict the phylogenetic and functional structure of ciliates in other karstic rivers. Biofilm samples were collected from four representative locations: upstream (Krka spring), midstream (Marasovine), and downstream (Roški slap, Skradinski buk) of the Krka river to test for differences in phylogenetic and functional diversity of ciliates in relation to location and positioning on tufa stones (light/dark-exposed side of tufa stone). Our results showed that Krka spring had higher phylogenetic species variability, lower phylogenetic diversity, and lower functional richness than Skradinski buk, suggesting phylogenetic overdispersal at Krka spring. This could be due to environmental filtering, competitive exclusion, or a combination of these factors. As the first study of its kind in the Mediterranean, our results shed light on the phylogenetic and functional diversity of ciliates in karst ecosystems and provide a basis for future ecological and conservation efforts.

Microplastic Distribution through the Salinity Gradient in a Stratified Estuary

Despite the extensive and rapidly growing literature on microplastics in oceans and coastal seas, little information exists on microplastic distribution through the salinity gradient. This study is the first one to evaluate microplastic distribution through the salinity gradient of a highly stratified estuary. A total of 910 microplastic particles were collected from 12 different sampling events in the Krka River estuary, Croatia. The number of detected particles ranged from 389 in the surface layer to 63 in the deepest marine layer. The highest plastic abundance was found in the surface layer (3.68 particles/m3) and the lowest in the marine layer (0.13 particles/m3). The measured values of the cross-sectional area indicated an ellipsoidal cross-sectional shape of the particles. It was also found that the majority of microplastic particles belonged to the small microplastic class (&lt;1 mm). The Nile Red (NR) staining method was used to visualize fluorescent microplastic particles, while quantification was performed using ImageJ/Fiji software. The strong salinity stratification in the studied estuary did not alter the usual distribution of microplastic particles in the water column, and there was no significant accumulation on the halocline.