Distribution, speciation and fate of trace metals in the stratified Krka river estuary

The distribution, speciation, behaviour, and fate of selected trace metals (TM) (Zn Cd, Pb, Cu, Ni, and Co) were studied in the water column of the highly stratified Krka River estuary, from 2009 to 2013, covering two contrasting seasons (summer/winter). In addition, the study involved measurements of dissolved/particulate organic carbon (DOC/POC) and major physico–chemical parameters (salinity, pH, dissolved oxygen, temperature). Water samples were taken from the three characteristic layers (brackish, the freshwater–seawater interface (FSI), and seawater). Stripping voltammetry (anodic, and adsorptive cathodic) was the primary analytical technique, while the diffusion gradient in thin films (DGT) technique was used as a complementary technique for speciation measurements. The concentrations of all TM were lower in the Krka River than in the open Adriatic which allowed the identification of pathways and processes governing the horizontal and vertical redistribution of TM across the whole estuary, whether they were added into the system by human activity or by ‘‘in-situ’’ processes (e.g. scavenging). Non-conservative behaviour for most of the studied TM observed in the surface layer of the estuary was primarily caused by their input in the Šibenik Bay area. Detailed "mapping" of the bay showed that the harbour and nautical marina are the regions with the highest concentrations of TM. A significant increase of Cu and Zn in the entire estuary transect during the summer period appeared to be directly linked to intensive nautical activity i.e. caused by the release of TM from antifouling paints. The scavenging of metals into the deeper layers, along with the longer residence time of seawater, caused an upstream increase in TM concentrations in the seawater layer. A very good agreement between the vertical profiles of DGT-labile, and dissolved TM concentrations suggests that the DGT technique can be successfully used to determine potentially bioavailable TM concentrations in estuarine conditions. The fraction of DGT-labile TM, which reflects their chemical speciation (primarily, their binding to natural organic ligands), ranged from > 90% for Cd, to < 20% for Cu, but also depends on the concentration ratios of metals and organic ligands. Voltammetric speciation of Cu showed the presence of two types of organic ligands that form strong (L1, 9.6 < logK1 < 11.9) and weak (L2, 7.8 < logK2 < 9.9) Cu complexes. The concentration of weak organic ligands (L2) is correlated with the DOC concentration, and is higher in summer, most probably due to the increased biological activity. The concentration of free Cu ions (the most bioavailable form), at ambient conditions, is regulated primarily by the complexation with strong ligands (L1). For most of the samples, the calculated free Cu slightly exceeds the toxic threshold of 10 pM. However, these values are the result of applied methodology of calculation, and does not necessarily reflect real ambient conditions.Raspodjela i specijacija odabranih metala u tragovima (TM) (Zn Cd, Pb, Cu, Ni i Co), te njihovo ponašanje i sudbina istraživani su u vodenom stupcu vertikalno uslojenog estuarija rijeke Krke tijekom dva kontrastna godišnja doba (ljeto/zima) u periodu od 2009 do 2013. Istraživanja su obuhvaćala i mjerenja otopljenog/partikulatnog organskog ugljika (DOC/POC), te osnovnih fizičko–kemijskih pokazatelja (salinitet, pH, otopljeni kisik, temperatura), a uzorkovanja su provođena u tri karakteristična sloja (boćati sloj, haloklina i morski sloj). Voltammetrija s akumulacijom (anodna i adsorptivna katodna) bila je primarna analitička tehnika, dok je za specijaciju korištena i komplementarna tehnika pasivnog uzorkovanja temeljena na principu difuzijskog gradijenta u tankom filmu (DGT). Koncentracije TM niže su u rijeci Krki nego u vodi otvorenog dijela Jadrana, što je omogućilo identifikaciju puteva i procesa koji reguliraju horizontalnu i vertikalnu preraspodjelu TM u cijelom estuariju, neovisno da li su oni u sustav uneseni ljudskom aktivnošću ili „in-situ“ procesima (npr. tonjenje -„scavenging“). Nekonzervativno ponašanje u površinskom sloju estuarija uočeno za većinu TM uzrokovano je primarno unosom metala u području Šibenskog zaljeva. Detaljno „mapiranje“ zaljeva pokazalo je da su luka i nautička marina područja s najvišom koncentracijom TM. Pokazalo se da je značajan porast Cu i Zn u cijelom estuariju tijekom ljetnih mjeseci u izravnoj vezi s pojačanom nautičkom aktivnošću, odnosno vezani su za otpuštanje metala iz protuobraštajnih boja s plovila. Vertikalni transport TM u dublje slojeve („scavenging“), te dulje vrijeme zadržavanja morske vode uzrokovali su uzvodni porast koncentracije TM u morskom sloju. Vrlo dobro slaganje vertikalnih profila DGT-labilnih i otopljenih koncentracija TM ukazuju da se DGT tehnika može uspješno koristiti za određivanje potencijalno bioraspoloživih koncentracija TM u estuarijskim uvjetima. Udio DGT-labilnih TM odraz je njihove kemijske specijacije (primarno vezanja s prirodnim organskim ligandima), te varira od > 90% za Cd, do < 20% za Cu, ali ovisan je također i o omjeru koncentracije metala i organskih liganada. Voltammetrijska specijacija Cu pokazala je prisutnost dva tipa organskih liganada koji stvaraju jake (L1, 9.6 < logK1< 11.9) odnosno slabe (L2, 7.8 < logK2< 9.9) Cu komplekse. Koncentracija slabijih organskih liganada (L2) u korelaciji je s koncentracijom DOC-a, te je veća ljeti najvjerojatnije zbog povećane biološke aktivnosti. Koncentracija slobodnih iona Cu (vrsta koja je najviše bioraspoloživa) pri okolišnim uvjetima regulirana je u većoj mjeri kompleksiranjem s jakim ligandima (L1). Za većinu uzoraka izračunate vrijednosti slobodnog Cu blago prelaze graničnu toksičnu vrijednost od 10 pM. Međutim, te vrijednosti su rezultat upotrijebljenog načina izračuna i ne odražavaju nužno stvarno stanje

Raspodjela, specijacija i sudbina metala u tragovima u uslojenom estuariju rijeke Krke

The distribution, speciation, behaviour, and fate of selected trace metals (TM) (Zn Cd, Pb, Cu, Ni, and Co) were studied in the water column of the highly stratified Krka River estuary, from 2009 to 2013, covering two contrasting seasons (summer/winter). In addition, the study involved measurements of dissolved/particulate organic carbon (DOC/POC) and major physicochemical parameters (salinity, pH, dissolved oxygen, temperature). Water samples were taken from the three characteristic layers (brackish, the freshwaterseawater interface (FSI), and seawater). Stripping voltammetry (anodic, and adsorptive cathodic) was the primary analytical technique, while the diffusion gradient in thin films (DGT) technique was used as a complementary technique for speciation measurements. The concentrations of all TM were lower in the Krka River than in the open Adriatic which allowed the identification of pathways and processes governing the horizontal and vertical redistribution of TM across the whole estuary, whether they were added into the system by human activity or by in-situ processes (e.g. scavenging). Non-conservative behaviour for most of the studied TM observed in the surface layer of the estuary was primarily caused by their input in the Šibenik Bay area. Detailed "mapping" of the bay showed that the harbour and nautical marina are the regions with the highest concentrations of TM. A significant increase of Cu and Zn in the entire estuary transect during the summer period appeared to be directly linked to intensive nautical activity i.e. caused by the release of TM from antifouling paints. The scavenging of metals into the deeper layers, along with the longer residence time of seawater, caused an upstream increase in TM concentrations in the seawater layer. A very good agreement between the vertical profiles of DGT-labile, and dissolved TM concentrations suggests that the DGT technique can be successfully used to determine potentially bioavailable TM concentrations in estuarine conditions. The fraction of DGT-labile TM, which reflects their chemical speciation (primarily, their binding to natural organic ligands), ranged from > 90% for Cd, to < 20% for Cu, but also depends on the concentration ratios of metals and organic ligands. Voltammetric speciation of Cu showed the presence of two types of organic ligands that form strong (L1, 9.6 < logK1 < 11.9) and weak (L2, 7.8 < logK2 < 9.9) Cu complexes. The concentration of weak organic ligands (L2) is correlated with the DOC concentration, and is higher in summer, most probably due to the increased biological activity. The concentration of free Cu ions (the most bioavailable form), at ambient conditions, is regulated primarily by the complexation with strong ligands (L1). For most of the samples, the calculated free Cu slightly exceeds the toxic threshold of 10 pM. However, these values are the result of applied methodology of calculation, and does not necessarily reflect real ambient conditionsRaspodjela i specijacija odabranih metala u tragovima (TM) (Zn Cd, Pb, Cu, Ni i Co), te njihovo ponašanje i sudbina istraživani su u vodenom stupcu vertikalno uslojenog estuarija rijeke Krke tijekom dva kontrastna godišnja doba (ljeto/zima) u periodu od 2009 do 2013. Istraživanja su obuhvaćala i mjerenja otopljenog/partikulatnog organskog ugljika (DOC/POC), te osnovnih fizičko–kemijskih pokazatelja (salinitet, pH, otopljeni kisik, temperatura), a uzorkovanja su provođena u tri karakteristična sloja (boćati sloj, haloklina i morski sloj). Voltammetrija s akumulacijom (anodna i adsorptivna katodna) bila je primarna analitička tehnika, dok je za specijaciju korištena i komplementarna tehnika pasivnog uzorkovanja temeljena na principu difuzijskog gradijenta u tankom filmu (DGT). Koncentracije TM niže su u rijeci Krki nego u vodi otvorenog dijela Jadrana, što je omogućilo identifikaciju puteva i procesa koji reguliraju horizontalnu i vertikalnu preraspodjelu TM u cijelom estuariju, neovisno da li su oni u sustav uneseni ljudskom aktivnošću ili „in-situ“ procesima (npr. tonjenje -„scavenging“). Nekonzervativno ponašanje u površinskom sloju estuarija uočeno za većinu TM uzrokovano je primarno unosom metala u području Šibenskog zaljeva. Detaljno „mapiranje“ zaljeva pokazalo je da su luka i nautička marina područja s najvišom koncentracijom TM. Pokazalo se da je značajan porast Cu i Zn u cijelom estuariju tijekom ljetnih mjeseci u izravnoj vezi s pojačanom nautičkom aktivnošću, odnosno vezani su za otpuštanje metala iz protuobraštajnih boja s plovila. Vertikalni transport TM u dublje slojeve („scavenging“), te dulje vrijeme zadržavanja morske vode uzrokovali su uzvodni porast koncentracije TM u morskom sloju. Vrlo dobro slaganje vertikalnih profila DGT-labilnih i otopljenih koncentracija TM ukazuju da se DGT tehnika može uspješno koristiti za određivanje potencijalno bioraspoloživih koncentracija TM u estuarijskim uvjetima. Udio DGT-labilnih TM odraz je njihove kemijske specijacije (primarno vezanja s prirodnim organskim ligandima), te varira od > 90% za Cd, do < 20% za Cu, ali ovisan je također i o omjeru koncentracije metala i organskih liganada. Voltammetrijska specijacija Cu pokazala je prisutnost dva tipa organskih liganada koji stvaraju jake (L1, 9.6 < logK1 < 11.9) odnosno slabe (L2, 7.8 < logK2 < 9.9) Cu komplekse. Koncentracija slabijih organskih liganada (L2) u korelaciji je s koncentracijom DOC-a, te je veća ljeti najvjerojatnije zbog povećane biološke aktivnosti. Koncentracija slobodnih iona Cu (vrsta koja je najviše bioraspoloživa) pri okolišnim uvjetima regulirana je u većoj mjeri kompleksiranjem s jakim ligandima (L1). Za većinu uzoraka izračunate vrijednosti slobodnog Cu blago prelaze graničnu toksičnu vrijednost od 10 pM. Međutim, te vrijednosti su rezultat upotrijebljenog načina izračuna i ne odražavaju nužno stvarno stanje

The Evaluation of γ-Zein Reduction Using Mass Spectrometry—The Influence of Proteolysis Type in Relation to Starch Degradability in Silages

The starch availability and nutritional value of corn (Zea mays L.) are affected by zein proteins. The aim of the study was to see whether the proposed reduction of γ-zeins during the fermentation of silages is a result of either the enzymatic proteolytic activity or of the acidic environment, and how this reduction affects starch availability and degradability in high-moisture corn. A mass spectrometry (MS) technique was used to quantify the 16- and 27-kDa γ-zeins. Briefly, two-dimensional gel electrophoresis (2-DE) was used for γ-zein separation, followed by densitometry for protein quantification and matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF/TOF) for protein identification. The results show that the reduction in γ-zeins induced by the ensiling led to a more pronounced starch availability and in vitro degradation, and this reduction was dependent on the type of proteolysis. More specifically, the results indicate that the reduction of γ-zeins in the ensiled corn was primarily driven by the enzymatic proteolysis. Furthermore, we demonstrated that 2-DE followed by densitometric quantification and the mass spectrometry analysis for protein identification can be used as a state-of-the-art method for γ-zein evaluation both in fresh and fermented/ensiled corn samples

Evaluation of Discrete and Passive Sampling (Diffusive Gradients in Thin-films – DGT) Approach for the Assessment of Trace Metal Dynamics in Marine Waters – a Case Study in a Small Harbor

Two complementary approaches, based on discrete and passive samplings (diffusive gradients in thin-films - DGT), supported by the speciation modeling, were evaluated for the assessment of distribution and operational speciation of trace metals (Zn, Cd, Pb, Cu, Ni and Co) within a small marine harbor (Rijeka, Croatia). Concentrations of dissolved metals were relatively low and comparable to, or slightly above those found in coastal Adriatic region. Compared to higher variability of dissolved metal concentrations due to the discrete sampling, smoother temporal distribution was recorded for DGT-labile metals. The percentages of DGT-labile metal concentrations, which reflect their affinity to organic matter, varied among metals with the following order: Cu<Zn≈Co<Ni<Cd≈Pb. DGT-labile metal concentrations predicted by speciation modeling were in a good agreement with the measured ones for Zn, Cd and Ni, while they are underestimated for Pb and Cu, and overestimated for Co. In-situ DGT technique is recommended for the assessment of the water quality status in marine environment. This work is licensed under a Creative Commons Attribution 4.0 International License

Growth Modulation of Human Cells in Vitro by Mild Oxidative Stress and 1,4-Dihydropyridine Derivative Antioxidants

Reactive oxygen species and lipid peroxidation products are not only cytotoxic but may also modulate signal transduction in cells. Accordingly, antioxidants may be considered as modifiers of cellular redox signaling. Therefore, the effects of two novel synthetic antioxidants, analogues of 1,4-dihydropyridine derivatives, cerebrocrast and Z41-74 were analysed in vitro on human osteosarcoma cell line HOS, the growth of which can be modulated by lipid peroxidation. The cells were pretreated with either cerebrocrast or Z41-74 and afterwards exposed to mild, copper induced lipid peroxidation or to 4-hydroxynonenal (HNE), the end product of lipid peroxidation. The results obtained have shown that both antioxidants exert growth modulating effects interfering with the lipid peroxidation. Namely, cells treated with antioxidants showed increased metabolic rate and cell growth, thereby attenuating the effects of lipid peroxidation. Such biomodulating effects of cerebrocrast and Z41-74 resembled growth modulating effects of HNE, suggesting that the antioxidants could eventually promote cellular adaptation to oxidative stress interacting with redox signaling and hydroxynonenal HNE-signal transduction pathways. This may be of particular relevance for better understanding the beneficial role of hydroxynonenal HNE in cell growth control. Therefore, cerebrocrast and Z41-74 could be convenient to study further oxidative homeostasis involving lipid peroxidation

Growth Modulation of Human Cells in Vitro by Mild Oxidative Stress and 1,4-Dihydropyridine Derivative Antioxidants

Reactive oxygen species and lipid peroxidation products are not only cytotoxic but may also modulate signal transduction in cells. Accordingly, antioxidants may be considered as modifiers of cellular redox signaling. Therefore, the effects of two novel synthetic antioxidants, analogues of 1,4-dihydropyridine derivatives, cerebrocrast and Z41-74 were analysed in vitro on human osteosarcoma cell line HOS, the growth of which can be modulated by lipid peroxidation. The cells were pretreated with either cerebrocrast or Z41-74 and afterwards exposed to mild, copper induced lipid peroxidation or to 4-hydroxynonenal (HNE), the end product of lipid peroxidation. The results obtained have shown that both antioxidants exert growth modulating effects interfering with the lipid peroxidation. Namely, cells treated with antioxidants showed increased metabolic rate and cell growth, thereby attenuating the effects of lipid peroxidation. Such biomodulating effects of cerebrocrast and Z41-74 resembled growth modulating effects of HNE, suggesting that the antioxidants could eventually promote cellular adaptation to oxidative stress interacting with redox signaling and hydroxynonenal HNE-signal transduction pathways. This may be of particular relevance for better understanding the beneficial role of hydroxynonenal HNE in cell growth control. Therefore, cerebrocrast and Z41-74 could be convenient to study further oxidative homeostasis involving lipid peroxidation

Dissolved organic matter dynamics in the pristine Krka River estuary (Croatia)

The karstic Krka River is characterized by having lower dissolved organic carbon (DOC) concentrations (~30 μM) than coastal seawater (~60 μM). This peculiarity, together with the pristine nature of this area, makes the Krka River estuary a natural laboratory where it is possible to discriminate among the different dissolved organic matter (DOM) sources (riverine, marine and produced in-situ) and to study the main processes of DOM production and removal. The hypothesis behind this work is that in winter, due to the high discharge of the river, most of the DOM has a terrestrial signature, whereas in summer autochthonous DOM compose the main fraction of the DOM pool because of the reduced discharge, the high temperature and primary production. Our data shows that DOM in the river mainly consists of terrestrial molecules, as suggested by the high chromophoric content and low spectral slope (S275–295) values, as well by the predominance of humic-like substances. DOM in the seawater features the concentration and optical properties of the “typical” marine DOM from open sea waters. In summer, low riverine discharge and high temperature promote the intense biological activity, with an increase in DOC concentrations of up to 148 μM, resulting in a non-conservative behavior of DOM in the estuary. The high stratification combined with a decoupling between production and removal processes can explain the observed DOM accumulation. In the bottom layer DOM was released and quickly removed when oxygen was available, whereas in hypoxic waters the production of DOC, chromophoric DOM (CDOM) and fluorescent DOM (FDOM) was linearly related to oxygen consumption. Our work highlights the need of further studies combining chemical and biological information in order to gain new insights into the main processes responsible for DOM dynamics in this system