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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.

Introduction: Dentitruncus truttae is an acanthocephalan species endemic to limited areas in Croatia, Italy, and Bosnia and Herzegovina. Since these parasites are characterised as effective metal accumulators, we estimated for the first time application of D. truttae as bioindicator of metal exposure. Therefore, metal distribution, seasonal variation and accumulation were compared in D. truttae and its host Salmo trutta from the Krka River in order to evaluate their sensitivity and indications of metal exposure in the moderately contaminated watercourse. Methods: Sampling was conducted in autumn 2015, spring 2016 and autumn and spring 2021 at Krka River source (KRS) as reference site, and location near the Town of Knin (KRK) impacted by industrial and municipal wastewaters. Molecular analysis confirmed species D. truttae and its taxonomic classification in the family Illiosentidae. Metals were measured by HR ICP-MS in aciddigested acanthocephalans and fish intestine, and their ratio was used to calculate bioconcentration factors (BCFs = C[parasite]/C[host intestine]), as possible indication of exposure duration. Results: Parasite prevalence ranged 92-100% at KRS and 80-97% at KRK. Fish and acanthocephalans showed higher levels of most elements at KRK compared to KRS, while metal accumulation was more efficient in acanthocephalans than fish. That is especially valid for Cd, Cu, Mn, Pb or Sr, resulting in higher BCFs and therefore, reflecting their higher input into Krka River watercourse in recent times. Conclusions: D. truttae showed more effective metal accumulation than fish and even significant differences in metal accumulation between two sites, confirming this parasite as a promising bioindicator species sensitive even to low environmental metal concentrations

Knowledge of population structure and dynamic in crustaceans is fundamental for the understanding of population ecology and protection of the aquatic ecosystems. The hyporheic zone (HZ) is a unique dynamic ecotone that is located at the interface of surface water and groundwater [1]. This habitat belongs to the groundwater connected ecosystems (GWCEs) which are threatened by human activities due to accumulation of pollutants in aquifers. Species Synurella ambulans is a small freshwater stygophilous amphipod inhabiting a relatively wide range of water bodies, including the HZ. The objective of the study was to determine population dynamic and reproductive biology in S. ambulans from HZ of the Sava River (sampling sites: Medsave and Jarun) sampled in December 2018, April, July and October 2019. The following life history traits were analysed: body length of individuals and sex ratio (female:male). The reproductive season of S. ambulans was determined to be in early spring (from March to April) because juveniles appeared during the spring (April 2019). The parenting generation died out after the spring season (May to June) and was subsequently replaced by juveniles which started to reproduce at the beginning of the following spring (March). In the Jarun study site the total body length (TBL) of adult females ranged between 3.38 and 6.00 mm (mean = 5.08 mm ; SD = 0.67), whereas in Medsave ranged between 4.13 and 6.38 mm (mean = 5.22 mm ; SD = 0.49), in all seasons. Adult male TBL ranged between 3.00 and 4.50 mm (mean = 3.57 mm ; SD = 0.32) in Jarun, and between 3.00 and 5.00 mm (mean = 3.68 mm ; SD = 0.40) in Medsave. Results revealed that mean body length of females was greater than that of males. These values were comparable to the literature data, where the maximum body length of females and males was found to be 7.0 and 4.7 mm, respectively [2]. Data on the sex ratio of S. ambulans indicate that males were more abundant than females in all seasons, while the opposite was found in populations from Poland [2]. Distinct seasonal trend in sex ratio throughout the sampling period was observed, ranging from 0.44:1 in October to 0.08:1 in July. Current results concerning the life cycle and reproduction of S. ambulans shed light on this poorly investigated species and its temporal dynamics in the vulnerable groundwater-connected habitat. ACKNOWLEDGMENTS The financial support of the European Regional Development Fund for the Qua/Qua Protein project (KK.01.1.1.07.0023) and of Croatian Science Foundation for the AQUAMAPMET project (IP-2014-09-4255) are acknowledged. We are grateful to Mr. Ivica Barač for his great help in the field work. REFERENCES [1] T. Orghidan, Bul. Stiint. Sect. Biol. Stiinte Agric. Sect. Geol. Geog. 7 (1955) 657–676. [2] A. Konopacka, M. Błazewicz-Paszkowicz, Pol. Arch.Hydrobiol. 47 (2000) 597–605.

The nanoscale secondary ion mass spectrometry (NanoSIMS), as a type of imaging mass spectrometry, represents a technique for elemental imaging that has not been widely applied in parasitology. Acanthocephalans are intestinal parasites of Mandibulata and Gnathostomata, and are recognised as bioindicators of metal exposure due to their effective metal accumulation. It was reported that acanthocephalans accumulate metals, especially toxic ones, at much higher concentrations than other indicator organisms, like fish, bivalves or crustaceans. Till now, there are no data on metal localisation in different body parts of acanthocephalans, even though these data may help us in understanding their successful metal uptake and storage. In the present study, the NanoSIMS was applied for localization of 11 elements, N, Fe, S, P, O, C, Na, Ca, Cu, Tl and Pb in three body parts of an acanthocephalan: trunk spines, inner part of the proboscis receptacle and the outer layer of the tegument. The chemical composition of the acanthocephalan body indicated complex structures that differed in the localization of the studied elements. In trunk spines, the highest element accumulation was found in the middle part, especially of S and Na, which were highly concentrated in the central ring, while Ca and C were mostly found in the whole spine area. The lower content of S, Ca and P in the proximal spine region might provide spine flexibility. In the proboscis receptacle the most dominant element was Ca, mostly located in the cytoplasmic inner lining and proboscis retractor muscles. The same pattern was found for Na, Fe, Cu, S, C and P, but in much lower concentrations. Considering outer layers of the acanthocephalan body, S and Na were mostly localised in contractile filaments of the muscle and intracellular matrix, while Ca was mostly found in the central cytoplasmic core of longitudinal muscles and neck retractor. Toxic metals were distributed along investigated structures without clear patterns and in very small quantities. The highest density in all studied structures was confirmed for Na, Ca, S, C, N, O, as important and constitutive elements in living cells, and among metals Fe was predominant in all analysed structures.

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.

Due to the growing impact of human activities on aquatic ecosystems, it is important to carry out biomonitoring programmes and assess the ecological status of waters in order to adequately protect aquatic ecosystems. The hyporheic zone (HZ) is a linking ecotone between surface and groundwater bodies. It is exposed to constant anthropogenic influences, which can affect the quality of surface running waters as well as aquifers. The aim of this study was to investigate the accumulation of metals in the stygophilous crustacean species Synurella ambulans (F. Müller, 1846) in order to assess its potential as a bioindicator of water quality in HZ. Amphipods were sampled by Bou-Rouch pump in the HZ of the Sava River gravel bar on the average depth of cca 50 cm once per season in winter 2018, spring, summer and autumn 2019 at two localities. First was located about 3 km upstream from the wastewater outlet of the town of Zaprešić, and second about 13 km downstream from the outlet situated in the city of Zagreb. Through the sewerage system of the town of Zaprešić treated wastewaters from the pharmaceutical industry are discharged together with treated municipal wastewaters. Measured concentrations of Cd, Cr, Cu, Co, Fe and Mn in the interstitial water of HZ, were not significantly different between two locations in most seasons, while Pb and Zn were below the detection limit of the method. Ni was significantly elevated at downstream location, and Al at upstream location in most seasons. According to the environmental quality standards, the metal concentrations in the interstitial water of the Sava River do not exceed the maximum permitted concentrations for groundwater. Concerning the bioaccumulated concentrations, it was found that Pb, Ni, Cr and Al were significantly elevated in S. ambulans at downstream location, while for Cd, Cu, Co, Zn, Mn and Fe no difference was observed between locations in most seasons. Elevated concentrations of metals in S. ambulans compared to the interstitial water of HZ indicate their high bioaccumulation in organisms. Because of their capacity to bioaccumulate metals, stygophilous amphipods may be useful in research on the long-term risk of metal discharges into subterranean habitats. In conclusion, biomonitoring of HZ is crucial for protection of this transitional ecosystem and S. ambulans can be used as potential bioindicator of metal pollution.

Marine ecosystems are threatened by variety of anthropogenic activities and pollutants, of which metals present serious threat due to their persistence, toxicity and potential to bioaccumulate. Metal concentrations in water may not reflect the chronic pollution load so bioindicator organisms such as native fish species are used to get an overall measure of contaminant exposure in the aquatic environments (Kraemer et al., 2006). Our goal was to estimate metal exposure in the area impacted by harbor activities, agriculture and tourism near the Port of Ploče and Neretva River mouth in the Southern Adriatic Sea in Croatia, using 30 individuals of three fish species (Mullus barbatus Linnaeus, 1758, Merluccius merluccius Linnaeus, 1758, Pagellus erythrinus Linnaus, 1758) as bioindicators. Biometric characteristics revealed the highest total length and masses of M. merluccius compared to other two species. Fulton condition index, as a measure of fish feeding and health, pointed to better condition of M. merluccius and M. barbatus compared to P. erythrinus. Metal exposure was assessed by determination of metal concentrations in water and fish muscle, as well as biomarker responses of metal exposure (metallothioneins), oxidative (malondialdehyde) and general stress (total proteins) in fish muscle. Accumulation of the most elements showed considerable species-specific differences except for Al, Fe, K and Mg, which concentrations were comparable in all species. The most elements showed significantly higher metal accumulation in M. barbatus (As, Co, Cu, Mn, Pb, Rb) and the lowest in M. merluccius (Co, Cu, Pb, Sr and Zn) when compared with other two species. Species-specific differences were also observed in biomarker responses, possibly as an adaptive response to the present ecological disturbances. Due to the complexity of anthropogenic releases of contaminants and their potential toxic effects, it is crucial to analyze larger set of relevant chemical and toxicological variables including metals and biomarkers, which can be preferably assessed in combination to provide information on marine ecosystem health. Such comprehensive research can provide adequate monitoring strategies in order to preserve the nature and living organisms, but also to provide marine ecosystems goods and services to human population

Groundwater organisms are exposed to progressive pollution and multi-xenobiotic stressors that influence the organism’s physiology, metabolism and finally ecosystem functioning and biodiversity. A useful index of stress response at the cellular level is the adenylate energy charge (AEC). AEC is determined as the ratio of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) as follows AEC= ([ATP]+1/2[ADP])/([ATP]+[ADP]+[AMP]). The aim of the study was to quantify for the first time adenine nucleotides in stygophilous crustacean Synurella ambulans and compare the AEC at two sampling sites with various levels of pollution in four seasons. Separation and quantification of nucleotides was performed using ion-pair reversed phase HPLC, with UV detection at 260 nm. Amphipods were collected from the hyporheic zone (HZ) of the Sava River at Jarun-Zagreb and Medsave-Zaprešić (upstream of pharmaceutical wastewater outlet) sampling sites in December 2018 and April, July, October 2019. The Medsave population had significantly higher AEC values (0.41 ; 0.45) than the Jarun population (0.35 ; 0.31) in winter and spring sampling campaigns, indicating higher energy supply and thus lower metabolic stress. The possible impact of concentrations of metals/metalloids in interstitial water and in amphipods on seasonal and spatial variation of the AEC are discussed. AEC was shown as a useful index of environmental stress in S. ambulans since it can directly measure the change in available energy and thus the metabolic stress experienced by a given organism. In conclusion, it appears that the seasonal and spatial variations of the AEC values reflect the ecological state in the HZ. The financial support of the European Regional Development Fund for the Qua/Qua Protein project (KK.01.1.1.07.0023) and of Croatian Science Foundation for the AQUAMAPMET (IP-2014-09-4255) are highly acknowledged.

Modern life results in continuous impact of different pollutants to aquatic ecosystems, of which marine plastic is considered as global environmental problem. According to United Nations, 30% of marine fish contained microplastics in their tissues, which consequently can lead to gut blockages, reduced energy budgets or starvation and toxic effects (Rochman et al., 2019). Our research was conducted in Croatia, in the Southern Adriatic Sea near the Port of Ploče and Neretva River mouth, known by the pollution impact of harbor activities, agricultural runoff and tourism. Three fish species, Mullus barbatus Linnaeus, 1758, Merluccius merluccius Linnaeus, 1758, Pagellus erythrinus Linnaeus, 1758, were used as bioindicators of contaminant exposure by measurement of oxidative stress biomarkers in muscle tissue, comparing fish from nearshore and offshore area. Oxidative stress is a commonly observed response to microplastic impact, but also to metal and organic exposure (Qiao et al., 2019). Our goal was to compare different biomarkers in native fish as indication of oxidative stress (malondialdehyde, MDA and glutathione, GSH), organic and metal exposure (acetylcholinesterase, AChE) and general stress (total proteins, TP). Results showed that 10% of the total trawl catch contained waste, mostly plastic. Isolation of microplastics from the fish gut content confirmed its presence in almost all selected individuals. Although Adriatic Sea was reported as the third location in Europe by plastic waste abundance (Zeri et al., 2018), our results pointed that fish species still don’t show significant differences in biological responses to oxidative stress or metal and organic exposure between nearshore and offshore area. But presence of microplastics in the gut content of sampled fish highlights the importance of continuous biomonitoring and assessment of plastic accumulation in marine environment and organisms.

The aim of the study was to determine and describe morphometric characteristics, length– weight relationship, condition factor and heavy metal concentrations in the muscle tissue of Atlantic chub mackerel (Scomber colias) from the North Adriatic Sea. Sampling (n=100) was performed during November 2018 by a purse-seine net. Morphomeristics were obtained with graph paper and calliper for all sampled fish, while heavy metal concentrations were measured in digested fish muscles (n=10) by high resolution inductively coupled plasma mass spectrometry (HR ICP-MS, Element 2, Thermo Finnigan). The maximum ratio range of body proportion was noted in standard and fork length and the minimum was noted in proportion of body depth and fork length. Length–weight relationships of Scomber colias indicate positive allometry growth W=0.003 x L3.339 with condition factor values as noted CF = 0.88±0.08. Measured metals in fish muscle tissue showed the following concentration ranges: a) macro elements: K>Na>Mg>Ca (< 7 mg kg-1) ; b) essential trace metals/metalloids: Fe>Zn>Se>Cu>Mn (< 5 mg kg- 1), Ni (< 0.03 mg kg-1) ; c) nonessential metals/metalloids: As>Rb>Sr>Cs (< 5 mg kg-1), Cd>Tl>Ag>Sb (< 0.03 mg kg-1). Correlation among various metals and fish morphometry was not significant, so potential metal accumulation with fish growth was not confirmed. Average Cd levels in fish muscle (0.8 mg kg-1) did not exceed maximum permitted levels for Cd in foodstuffs (50 mg kg-1), pointing to safe human consumption of Atlantic chub mackerel from the North Adriatic Sea.

The hyporheic zone (HZ), a subterranean/surface water transitional interface, is a complex shallow interstitial groundwater flow system situated lateral and beneath a stream. HZs belong to the most threatened aquatic environments. The extreme characteristics of the HZs, including dynamic and highly heterogeneous hydrologic and biochemical processes, small physical size and exchange flow, and full darkness, cause higher concentration of major ions and metals and unpredictable changes in the rate of dissolved oxygen. To cope with oxygen deprivation, subterranean organisms have evolved different strategies such as reduced metabolism [1], high amounts of stored glycogen and phosphagen arginine phosphate [2] and utilization of anaerobic routes. [3] In this study, the activities of three enzymes involved in energy metabolism were measured in small stygophilous amphipod Synurella ambulans to assess its anaerobic potential. Specimens were sampled from two localities: the HZ of the Sava River (Zagreb, Jarun) and acid springbrook Stalnjak (Zaprešić). The data on metabolic enzyme activity of this species are lacking since this amphipod species hasn’t been intensively investigated. The ratio of pyruvate kinase (PK) over phosphoenolpyruvate carboxykinase (PEPCK) activities was used as an index of aerobic relative to anaerobic capacity of an animal, and lactate dehydrogenase (LDH) activity as an indicator of anaerobic glycolytic potential. Stalnjak population had lower PK/PEPCK ratio and lower LDH activity than population at the Sava River indicative of higher dependence on anaerobic pathways compared with the population from the Sava River HZ. PK/PEPCK ratio in S. ambulans was ~2- 3-fold higher compared with the published values for two other stygobiont species (Niphargus virei and Niphargus rhenorhodanensis), but ~6-fold lower than in an epigean species Gammarus fossarum (Hervant, 1997). LDH activity in S. ambulans was similar to that of N. rhenorhodanensis and N. virei. Overall, relatively low PK/PEPCK ratio and high LDH activity indicate increased anaerobic potential of S. ambulans and its ability to tolerate low oxygen concentrations underground.

In addition to chemical water analysis, assessment of biologically available pollutants involves application of bioindicators, mostly presented by fish, crustaceans and bivalves. Regarding metal exposure, fish intestinal parasites acanthocephalans were suggested as promising bioindicators, since it was reported that metal accumulation was higher in acanthocephalans compared to the other bioindicator organisms. Thus, the aim of the present study was to compare spatial and seasonal metal accumulation efficiency in acanthocephalans (Dentitruncus truttae Sinzar, 1955) and commonly used bioindicators, fish brown trout (Salmo trutta Linnaeus, 1758) and gammarids (Gammarus balcanicus Koch, 1836). Metal exposure was evaluated in the wastewater impacted Krka River (polluted site) and in the river source (reference site) in autumn (2015) and spring (2016). Metals were measured by high resolution inductively coupled plasma-mass spectrometry in fish intestine, whole gammarids and acanthocephalans, which were wet digested in a drying oven at 85°C for 3.5 hours using concentrated HNO3 and H2O2. The highest levels of Zn>Fe>Rb were found in fish, Sr>Fe>Zn in gammarids and Zn>Fe>Cu in parasites. In moderately polluted Krka River, acanthocephalans showed higher metal accumulation efficiency than fish and gammarids for Cd, Mn, Pb, Tl, while lower for Cs, Fe, Rb. Bioconcentration factors indicated 4-55 times higher metal levels (Cd>Tl>Pb>Sr>Cu>Ca>Mn) in parasites compared to fish and 3-22 times (Tl>Cd>Pb>Mn) compared to gammarids. Spatial differences were evident in both seasons and in all organisms as higher Co, Fe and Mn at polluted and Cd and Tl at reference site. Consequently, further investigation on sediment and dietary metal sources are needed.