Copper exposure of freshwater mussels (Anodonta anatina): Some physiological effects
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COPPER EXPOSURE OF FRESHWATER MUSSELS (Anodonta anatina): SOME PHYSIOLOGICAL EFFECTS Dissertation submitted to the FACULTY OF BIOLOGY, CHEMISTRY, AND GEOSCIENCES UNIVERSITY OF BAYREUTH, GERMANY to obtain the academic degree of D R . RER . NAT . presented by A NDHIKA P USPITO N UGROHO M. Sc. born August 8, 1976 in Semarang, Indonesia Bayreuth, November 2011
COPPER EXPOSURE OF FRESHWATER MUSSELS (Anodonta anatina): SOME PHYSIOLOGICAL EFFECTS by Andhika Puspito Nugroho Environmental Chemistry and Ecotoxicology University of Bayreuth Germany Supervisor: Prof. Dr. Hartmut Frank
This doctoral thesis was prepared at the Department of Environmental Chemistry and Ecotoxicology, University of Bayreuth, from November 2010 until November 2011, supervised by Prof. Dr. Hartmut Frank. This is a full reprint of the dissertation submitted to attain the academic degree of Doctor of Natural Sciences (Dr. rer. nat.) and approved by the Faculty of Biology, Chemistry and Geosciences of the University of Bayreuth. Acting Dean: Prof. Dr. Beate Lohnert Date of submission: November 9, 2011 Date of defense (disputation): June 19, 2012 Doctoral Committee Prof. Dr. Hartmut Frank 1 st reviewer Prof. Dr. Klaus H. Hoffmann 2 nd reviewer Prof. Dr. Ch. E.W. Steinberg 3 rd reviewer Prof. Dr. Gerhard Gebauer Chairman Prof. Dr. Stefan Peiffer Prof. Dr. Britta Planer-Friedrich
I ACKNOWLEDGEMENTS Many people contributed to this dissertation in innumerable ways, and I am grateful to all of them. First and foremost I am heartily thankful to my supervisor, Prof. Dr. Hartmut Frank, for his time, advice, support, encouragement, and patience during my research at the Chair of Environmental Chemistry and Ecotoxicology, University of Bayreuth. I would like to thank Dr. Silke Gerstmann for all the guidance, instruction, and helpful discussion about my work. The support of Prof. Dr. Britta Planer-Friedrich is greatly appreciated by allowing me to use her laboratory facilities for the mussel exposure experiments and the preparation of the metal analyses. I also want to thank Prof. Dr. Klaus H. Hoffmann and Dr. Martina MeyeringVos for all their help in the laboratory and giving me access to other facilities during sample analyses and writing my dissertation. I am grateful to Dr. Gunter Ilgen and Barbara Scheitler for assisting in metal analyses. My thanks also go to all of my Indonesian friends for their nice friendship given to me during these years. I wish to express my appreciation to all of my colleagues at the Chair of Environmental Chemistry and Ecotoxicology, University of Bayreuth. I thank Dr. Huong Ngo Thi Thuy for helpful discussions; Fabian Iltzsche and Stefan Will for providing lots of technical assistance, Irmgard Lauterbach for all her help in the administrative affairs during my study; Michael Fischer, Mario Kiesewetter, Andreas Bantle, and Michael Heyers for all their support with laboratory work. I am pleased to thank to all of my colleagues at the Chair of Animal Ecology I, University of Bayreuth. I thank Ahmad Alamer, Dr. Judith Lorenz, Hassan ElDamanhouri, Marion Preiß, Dorothea Wiesner, Carmela Herrmann, Ursula Wilczek, and Intisar Taha for their kindness and help. Finally, I would like to thank my parents, wife, parents-in-law, brothers, and sisters-in-law for supporting and encouraging me always.
II TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS II LIST OF PUBLICATIONS AND AUTHOR’S CONTRIBUTIONS V LIST OF ABBREVIATIONS VI SUMMARY VII ZUSAMMENFASSUNG I X 1. General introduction 1 1.1. Copper in aquatic ecosystems and its transfer in food chains 1 1.2. Effects of copper on calcium homeostasis and cellular defense mechanisms of freshwater mussels 2 1.3. Freshwater mussels and their status .. 4 1.4. Ecotoxicological studies with Anodonta anatina ... 5 1.5. Objectives of the research 6 1.6. Methodological requirements ... 6 2. Materials and methods 7 2.1. Copper experiment with microalgae Parachlorella kessleri 7 2.2. Copper exposure experiment of mussels .. 7 2.3. Analytical methods ..................................................................... 8 2.4. Statistical analysis ...................................................................... 8 3. Results 9 3.1. Studies of copper effects on the green alga Parachlorella kessleri: Producing Cu-loaded algae for feeding experiments ... 9 3.2. Studies of different copper exposure pathways on the freshwater mussel Anodonta anatina ......................................... 9 3.2.1. Studies of copper uptake, distribution, bioaccumulation, and elimination .................................. 9 3.2.2. Studies of copper effects ............................................... 9 4. General discussion 1 0 5. Conclusions, contributions, and perspectives 12 5.1. Conclusions . 12 5.2. Contributions ... 12 5.3. Perspectives 13 6 . References 13 PUBLICATIONS Publication I : Producing Cu - loaded algae for feeding experiments: effects of copper on Parachlorella kessleri 20 Introducti on 20 Materials and methods 2 1 Glassware and reagents ... 21 Test organism, culture conditions, and toxicity testing . 22 Determination of growth and biomass 22
III Copper determination 22 Determination of chlorophyll-a, chlorophyll-b, and pheophytin-a contents ...................................................................................... 23 Protein and polysaccharide contents ......................................... 23 Data analysis .............................................................................. 24 Results 24 Discussion 27 Conclusions 28 Acknowledgements 29 References 29 Publication II : Uptake, distribution, and bioaccumul ation of copper in the freshwater mussel Anodonta anatina 32 Introduction 32 Materials and methods 33 Algal food preparation ... 33 Isotopic Cu stock solution preparation and labware . 33 Organisms ... 33 Experimental design . 34 Metal analyses 34 Statistical data analyses 35 Results 35 Discussion 39 Conclusions 42 Acknowledgements 43 References 43 Publication III : Effects of copper exposure on calcium, carbohydrate, and protein levels in the freshwater mussel Anodonta anatina 45 Introduction 45 Materials and methods 46 Chemicals 46 Animal and experimental design . 46 Calcium determination .. 47 Copper determination 47 Protein and carbohydrate determinations .. 47 Statistical data analyses 48 Results 48 Discussion 51 Con clusions 52 Acknowledgements 52 References 52 Publication IV : Effects of copper on lipid peroxidation, glutathione, metallothionein, and antioxidative enzymes in the freshwater mussel Anodonta anatina 55 Introduction 55 Material and methods 56 Chemicals 56
IV Animals and experimental design ... 56 Analytical methods . 57 Sample preparation ... 57 Total copper ... 57 Lipid peroxidation .. 57 Glutathione . 58 Metallothioneins ..... 58 Enzyme activities ... 58 Proteins ... 58 Statistical data analyses 58 Results 60 Discussion 6 2 Conclusions 6 4 Acknowledgements 64 References 64 DECLARATION AND ERKLÄRUNG 67
V LIST OF PUBLICATIONS AND AUTHOR’S CONTRIBUTIONS This dissertation is presented in cumulative form. It comprises four individual manuscripts, from which all was published. The author’s contributions to each manuscript are given below. 1. Nugroho, A.P., and H. Frank, 2011. Producing Cu-loaded algae for feeding experiments: effects of copper on Parachlorella kessleri. Toxicological and Environmental Chemistry, published (Publication I) Own contribution: idea (60%), method development (90%), data analysis and calculations (100%), writing (100%) and editing the paper (60%) 2. Nugroho, A.P., and H. Frank, 2011. Uptake, distribution, and bioaccumulation of copper in the freshwater mussel Anodonta anatina. Toxicological and Environmental Chemistry, published (Publication II) Own contribution: idea (70%), method development (90%), data analysis and calculations (100%), writing (100%) and editing the paper (60%) 3. Nugroho, A.P., and H. Frank, 2012. Effects of copper exposure on calcium, carbohydrate, and protein levels in the freshwater mussel Anodonta anatina. Toxicological and Environmental Chemistry, published (Publication III) Own contribution: idea (70%), method development (100%), data analysis and calculations (100%), writing (100%) and editing the paper (60%) 4. Nugroho, A.P., and H. Frank, 2012. Effects of copper on metallothionein, glutathione, lipid peroxidation, and antioxidative enzymes in the freshwater mussel Anodonta anatina. Toxicological and Environmental Chemistry, published (Publication IV) Own contribution: idea (80%), method development (100%), data analysis and calculations (100%), writing (100%) and editing the paper (60%)
VI LIST OF ABBREVIATIONS APW Artificial pond water ATP Adenosine Triphosphate BSA Bovine Serum Albumin CAT Catalase DNA Deoxyribonucleic Acid DTNB 5,5'-Dithio-bis-(2-nitrobenzoic acid) DTT Dithiothreitol dw Dry weight EDTA Ethylenediaminetetraacetic Acid EF Enrichment Factor EPF Extrapallial fluid GHL Gonads, Heart, Labial Palps GPX Glutathione Peroxidase GR Glutathione Reductase GSH Glutathione HML Hemolymph IC Inhibition Concentration LOEC Lowest-observed-effect concentration MDA Malondialdehyde MT Metallothionein NOEC No-observed-effect concentration OD Optical density PBS Phosphate-buffered Saline PMSF Phenylmethylsulphonyl Fluoride ROS Reactive Oxygen Species SOD Superoxide Dismutase TF Transfer Factor ww Wet weight
2 accumulate copper from water, transferring it to grazing species at the next trophic level. This contributes to biomagnification along aquatic food chains. In addition, grazing species can take copper up from the water (Connell and Sanders 1999; Edding and Tala 1996; Pinto et al. 2003). Bioconcentration occurs via uptake and retention of metals from water, across gill membranes or other external body surfaces (Kaoud and El-Dahshan 2010). The concentration of the metals in the soft tissue or particular organs can be considered as a relative measure of ambient concentrations due to the ability of mussels to accumulate copper (Kumari and Nair 1992). Since the middle of the last century, the continuously increasing technological and industrial use of copper has led to globally increased mining and translocation of copper from the earth’s crust to the surface and the corresponding trend of rising concentrations in all compartments of the anthroposphere over natural levels, especially in freshwater ecosystems of industrialized and industrializing countries. Metallurgic activities, its use in machinery such as electrically propelled locomotives, in the building sector as roofing material, for water pipes and kitchenware, for overland high-voltage power lines, and in directly dissipative ways of using it as fungicide, algicide, and molluskicide, the disposal of copper-containing waste waters, and its release and deposition of atmospheric particulate matter from coal combustion (Mohammed and Markert 2006; Momčilović 2004), all this has led to a continuously increasing Cu-burden of the biosphere. This condition entails copper transfer through food chains and its bioconcentration in mussels’ bodies. Copper accumulation in cells over the physiological requirements leads to toxic effects, depending upon its bioaccumulation beyond the optimum level within the respective organism (Nott 1998). 1.2. Effects of copper on calcium homeostasis and cellular defense mechanisms of freshwater mussels Calcium (Ca) is an essential macronutrient for mussels. It has an almost universal importance for nerve conduction, mussel contraction, as second messenger for regulation of carbohydrate metabolism such as controlling the activation of glycogenesis, regulation of mitochondrial electron transport, the metabolism of carbohydrate intermediates of the tricarboxylic acid cycle, and of
3 almost all aspects of cellular metabolisms and growth (Albert et al. 1994; Sick et al. 1979). Moreover, calcium is required by young and adult mussels for shell formation (calcification) (Sick et al. 1979). Mussel cells require calcium (Ca) in specific limits of cytosolic concentrations (≤ 10 -7 M). Calcium homeostasis is maintained by extrusion and compartmentalization systems (Viarengo et al. 1993). In addition, mussels require carbohydrates as main energy source for their metabolic processes (Honkoop et al. 1999) and for shell formation (Marie et al. 2007; Marin and Luquet 2004). Proteins are also required by mussels for catalyzing biochemical reactions, transport and storage of molecules in and out or within cells, and have structural and mechanical functions (Albert et al. 1994). Exposure of mussels to copper at 0.35 µmol L -1 leads to an interference with the systems responsible for maintaining Ca homeostasis in gills, digestive gland, and kidney (Santini et al. 2011). This is followed by uncontrolled, increased cytosolic Ca concentrations activating various Ca-dependent catabolic processes such as phospholipid hydrolysis, protein degradation, and DNA fragmentation, ultimately leading to cell death (Viarengo et al. 2002; Viarengo 1994). In addition, high copper levels can entail decreased carbohydrate levels in gills and mantle (Satyaparameshwar et al. 2006). Mussels have developed detoxification mechanisms to cope with copper challenge. In the cytosol, glutathione (GSH), a tripeptide which contains sulfhydryl (SH) groups with strong affinity for copper cations and found in high concentrations (0.2 – 10 mM) (Monostori et al. 2009), can provide a first line of defense against increased cytosolic levels of free copper by binding the metal to its SH-groups. Increased copper also induces synthesis of metallothioneins (MT), specific SH-rich proteins having the capacity to bind copper (Conners and Ringwood 2000; Viarengo et al. 2002).Increased cytosolic copper can induce oxidative stress because copper may be involved in the formation of reactive oxygen species (ROS) by catalyzing the generation of • OH from H 2 O 2 and O 2•- through a Haber-Weiss cycle (Lackner 1998; Pinto et al. 2003). During aerobic respiration, oxygen is reduced to water through four steps of electron transfer resulting in oxygen intermediates which are highly reactive and toxic ROS, i.e. the superoxide anion (O 2•- ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical
4 ( • OH) (Lackner 1998). In healthy aerobic cells, there is a balance between ROS production, molecular oxidation, and antioxidant consumption. Mussel cells have a wide range of antioxidative enzymes, neutralizing ROS and keeping their concentrations at very low levels. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) represent a group of enzymes having antioxidative roles (Isani et al. 2003; Pinto et al. 2003; Viarengo et al. 2002). SOD catalyzes the disproportionation of O 2•- to O 2 and H 2 O 2 , whereas CAT and GPX catalyze the production of H 2 O from the degradation of H 2 O 2 and ROOH, respectively. SOD is the cell’s first line of defense against ROS because it controls O 2•- which can be a precursor to several other highly reactive species (Pinto et al. 2003). If these mechanisms are challenged beyond their protective capacities and ROS production rates are higher than the rates of its inactivation by antioxidant defense systems, oxidative stress conditions arise. In such case, free ROS can react quickly and indiscriminately with biomolecules such as lipids, proteins, and nucleic acids, resulting in lipid peroxidation, formation of protein carbonyl groups, and DNA strand breaks. Determination of lipid peroxidation allows to assess oxidative stress levels in cells (Company et al. 2008; Lackner 1998). 1.3. Freshwater mussels and their status Freshwater mussels are invertebrate animals which have two shells (bivalve shell) as mirror images of each other, connected by a hinge-like ligament. Adult mussels have a variety of sizes, colours, and shapes, depending on the species. They are sedentary and inhabit the bottom of freshwater ecosystems such as creeks, rivers, streams, ponds, and lakes. They have a muscular foot which helps them anchor against strong currents and allows limited movements. Mussels are ecologically important in aquatic ecosystems comprising a significant proportion of the total standing crop in freshwater benthic communities, cycling calcium in lakes, removing suspended detritus and cleaning the water, mixing surficial sediments through bioturbation, and serving as food for aquatic mammals (Box et al. 2006; Naimo 1995; Nedeau and Victoria 2003). The family of Unionidae is the most endangered of all aquatic animal species. Alterations of mussel habitats potentially influence the survival of the
5 mussels because several stages of the mussels’ life histories such as sperm release by adult males into the water column, uptake of sperms by siphoning females, fertilization of ovae, release of viable larvae (glochidia) from females, and attachment of glochidia to suitable host fish by encystations for transformation to free-living juvenile mussels are critical stages which ultimately can all contribute to a decreased mussel reproduction and population development. Laboratory experiments have shown that the early life stages of freshwater mussels are sensitive to many chemicals including copper and ammonia (Cope et al. 2008). Due to the importance of mussels in aquatic ecosystems, protective and conservative actions are required for maintaining healthy mussel populations and for recovering endangered ones by investigation of freshwater mussel biology, the preservation of water and riparian resources, and the control and/or elimination of threats to these animals (Watters et al. 2009). 1.4. Ecotoxicological studies with Anodonta anatina Ecotoxicological investigations concerning the effects of contaminants are devoted to reveal at which dose or concentrations they become toxic. Investigations on contaminant uptake and elimination rates, distribution among mussel tissues and organs, and the relationship between contaminant accumulation and biological responses at each level of organization are required. Anodonta anatina is a freshwater mussel species of the family Unionidae which is widely distributed in Europe in a variety of freshwater ecosystems and is used for biological monitoring surveys (Mäkelä et al. 1995; Mäkelä and Oikari 1990). The species has been used for uptake and body distribution studies of contaminants such as chlorinated phenolics (Mäkela and Oikari 1990), for accumulation and monitoring studies of 2,4,6-trichlorophenol (Englund and Heino 1996), pentachorophenol (Mäkela and Oikari 1995), other chlorinated phenolics (Mäkelä et al. 1991), 45 Ca accumulation (Pynnönen 1991), and uptake and cadmium accumulation and depuration (Holwerda et al. 1988). Other ecotoxicological studies on contaminant impacts at biochemical and physiological levels of A. anatina have been reported, such as the effects of copper on Ca-ATPase and carbonic anhydrase (Santini et al. 2011), effects of
6 cadmium on calcium metabolism (Ngo et al. 2011), and effects of crude oil on cytogenetic damage (Baršienė et al. 2006). 1.5. Objectives of the research This present research project aims to study the importance of two different copper exposure pathways, i.e. via water or food, on uptake, distribution, accumulation, and elimination in the freshwater mussel Anodonta anatina, and their potential physiological impacts. In order to reveal the effects of copper via food, the mussel must be fed by copper-containing algae which have normal nutritional value to avoid secondary effects. Therefore, before the actual mussel experiments, microalgae Parachlorella kessleri are raised while being exposed to various copper concentrations, to find the limit at which the physiological state and nutritional value is comparable to non-exposed algae and to be used as 63 Cu-loaded food for the mussels (Publication I). In the following experiments, A. anatina are exposed to Cu via water or food. Distribution, bioaccumulation, and elimination of the trace metal among the organs of the exposed mussels are investigated (Publication II). The effects of elevated copper levels on the levels of calcium, soluble carbohydrates and proteins (Publication III), on metallothionein induction and glutathione levels, on the activities of antioxidative enzymes, and on lipid peroxidation (Publication IV) in various organs and tissues are examined. 1.6. Methodological requirements Since mussels contain endogenous copper, the stable isotope 63 Cu is used as tracer to differentiate between endogenous and exogenous copper. According to Croteau et al. (2004), stable isotopes of metals can be used as markers to help investigating directional uptake pathways and their bioaccumulation and elimination from aqueous and dietary sources. Use of inductively-coupled plasma mass spectrometry (ICP-MS) as analytical tool allows to determine the isotopes at low concentrations as individual masses.
7 2. Materials and methods 2.1. Copper experiment with microalgae Parachlorella kessleri P. kessleri (SAG Culture Collection, University of Goettingen, Germany) is exposed to Cu at various concentrations for 96 hours to find the most suitable Cu concentration which does not affect its nutritional values, being used to produce 63 Cu-loaded algae as food for mussel experiment. Details of copper experiments with the algae are described in Publication I. 2.2. Copper exposure experiment of mussels The duck mussel A. anatina can take copper up from water or food, so three groups of 21 mussels each are used, one as control and the two other groups to be exposed to the stable isotope 63 Cu via water or food for 24 days followed by 12 days of depuration. Seven samplings of three mussels each every sixth days are taken to study the time-dependent Cu accumulation and elimination. Copper exposure for 24 days represents a long-term copper exposure (Company et al. 2008), while the depuration period of 12 days allows to investigate how fast the levels return to control values, reflecting also the halflife of copper. Details of copper exposure experiments with the mussel are described in Publication II. During the experiments, total copper and isotopic copper ( 63 Cu and 65 Cu) are determined in the hemolymph (HML), the extrapallial fluid (EPF), gills, mantle, kidney, digestive gland, foot, adductors, intestines, and the collective remaining organs, i.e. gonads, heart, and labial palps (GHL) (Publication II). Effects of copper on calcium homeostasis, proteins, and carbohydrates are studied in all these body compartments. The relationships between copper and Ca, carbohydrate, and protein levels respectively, and between calcium and proteins are examined in the compartments (Publication III). Effects of copper on metallothionein, glutathione, lipid peroxidation, and antioxidative enzymes are also examined (Publication IV).
8 2.3. Analytical methods 2.3.1. Determinations of total Cu, isotopic Cu, and total calcium Total Cu and isotopic Cu in lyophilized tissue fractions are determined by inductively-coupled plasma mass spectrometry (ICP-MS), total Ca by inductively-coupled plasma atomic emission spectroscopy (ICP-AES). Determinations and calculations of the concentrations of the elements are described in detail in Publications II and III. 2.3.2. Determinations of carbohydrates and proteins Carbohydrates are determined by the phenol-sulfuric acid assay (Masuko et al. 2005), proteins by the dye-binding assay (Kruger 1994). Details of the determinations are described in Publication III. 2.3.3. Determination of metallothionein Metallothionein (MT) concentrations in the gills, mantle, digestive gland, and kidney are determined using the spectrophotometric method described by Viarengo et al. (1997) and modified by Verlecar et al. (2008). Details of metallothionein determination are described in Publication IV. 2.3.4. Determinations of glutathione, antioxidative enzyme activities, and lipid peroxidation Glutathione levels are determined according to Anderson (1985). The activities of the antioxidative enzymes catalase, glutathione peroxidase, and superoxide dismutase are assayed according to the methods of Rao et al. (1996), Paglia and Valentine (1967), and Beauchamp and Fridovich (1971), glutathione reductase activities are assayed following the method of Massey and William (1965). For lipid peroxidation assay, the method of Buege and Aust (1978) is employed. Details of the determinations are described in Publication IV. 2.4. Statistical analysis Data of mussel experiments are transformed to log (X+1) units before statistical analysis for homogeneity of variance and normality. The variability of all parameters with exposure time and copper exposure pathways are tested in
9 each organ by two-way analysis of variance (ANOVA). Details of statistical analyses were described in Publications II and III, and Manuscript IV. 3. Results 3.1. Studies of copper effects on the green alga Parachlorella kessleri: Producing Cu-loaded algae for feeding experiments Exposure of the algae to copper above 6 µmol L -1 leads to increased Cu levels in the algae, inhibition of algal growth, and significant alterations of biochemical-physiological parameters, strongest effects being observed at highest concentration (Publication I, Figure 1, 2, and 3, Table 1). Chlorophyll contents and growth rate are the most sensitive indicators. At 5.9 µmol L -1 Cu, the observed parameters do not differ significantly from control values. 3.2. Studies of different copper exposure pathways on the freshwater mussel Anodonta anatina 3.2.1. Studies of copper uptake, distribution, bioaccumulation, and elimination Mobilization of the stable isotope 63 Cu among mussel organs reveals that Cu uptake from water occurs via the gills and mantle and from the food via the digestive gland (Publication II, Figure 4). Exogenous Cu ( 63 Cu) and total (exogenous and endogenous) Cu increase in all body compartments, highest levels being observed at day 24. Upon exposure via the water, high total Cu levels are found in the gills, mantle, digestive gland, kidney, and GHL while upon exposure via the food highest levels are found in the digestive gland and kidney (Publication II, Figure 1, 2, 3, and 4). During depuration, total and exogenous Cu decrease in all body compartments, except for total Cu in the mantle and intestines for which even further increases were observed within the first six days of depuration. 3.2.2. Studies of copper effects 3.2.2.1. Change in Ca levels Ca levels in all body compartments increase in parallel to increased Cu concentrations, reaching highest levels at day 24 (Publication III, Figure 1 and 2). Upon depuration, Ca concentrations in the body fluids decline fast, returning
10 to control values within the first six days although Cu levels are still elevated. In the organs, Ca levels tend to normalize, although not fully back to controls. 3.2.2.2. Changes in carbohydrate and protein levels Soluble carbohydrates and soluble proteins decline in all organs upon Cu exposure and in parallel to its concentrations, reaching lowest levels at day 24 (Publication III, Figure 3). When Cu administration is terminated, the levels in the studied organs start to increase, although not fully back to control within the 12 days. 3.2.2.3. Effects on metallothionein, glutathione, lipid peroxidation, and antioxidative enzymes Exposure of A. anatina to copper induces increases in metallothionein (MT) in all organs, reaching highest levels at day 24 (Publication IV, Figure 1). For glutathione (GSH), the levels decrease at similar rates in all organs, reaching lowest levels at day 24. Simultaneously with the Cu elimination, MT levels decrease in all organs, for GSH being increased slowly. Thiobarbituric acid-reactive substances (TBARS) increase strongly upon Cu exposure via the water, reaching highest levels at day 24. During depuration, TBARS levels decrease slowly. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR) are expressed in two ways, i.e. relative to protein contents and to tissue wet weight. In relation to protein contents, all enzyme activities increase reaching highest levels at day 24. Strongest effects were found in the digestive gland (Publication IV, Figure 2). In terms of tissue wet weight, activities of SOD, GPX, and GR decline in all organs, reaching lowest levels at day 24. For CAT, the activities remain unchanged for both pathways except for the kidney in which the activity is increased, reaching highest level at day 18. During depuration, most parameters tend to normalize but do not return to control values. 4. General discussion Declines in chlorophyll contents of P. kessleri upon Cu exposure suggest three possibilities, i.e. inhibition of chlorophyll synthesis, increased lipid
11 peroxidation on chloroplast membranes, and degradation of chlorophyll-a confirmed by increased pheophytin-a (Sandmann and Böger 1980; Tripathi and Gaur 2006). This condition may affect photosynthesis rates, lowering glucose synthesis and ultimately resulting in inhibition of algal growth. Decreases in carbohydrates and proteins may be a result of increased hydroxyl radical formations induced by copper via the Haber-Weiss reaction. The radicals are highly reactive, oxidizing, and breaking apart biological macromolecules (Nikookar et al. 2005). Use of the stable isotope 63 Cu can represent exogenous Cudistribution and the redistribution of endogenous Cu among body compartments. Redistribution of endogenous Cu causes pronounced alterations in total Cu in the organs, such as increases in the mantle and intestines upon exposure via the food (Publication II, Figure 2, 3, and 4). Copper elimination can occur due to the difference in gradient Cu concentration between the mussel and APW (Publication II, Figure 2 and 3). High Cu level in the kidney indicates that this organ plays an important role in elimination. Declines of exogenous 63 Cu levels during depuration indicate that the isotope is in a relatively easily exchangeable form while the endogenous Cu is more tenaciously retained (Publication II, Figure 4). Disturbance of Ca homeostasis upon Cu exposure (Publication III, Figure 1 and 2) can occur due to mobilization of CaCO 3 from the shell, most likely due to Cu-induced metabolic acidosis (Antunes et al. 2002; Faubel et al. 2008; LopesLima et al. 2008). Inhibition of Ca extrusion and of intracellular compartmentalization systems may be another complication (Pattnaik et al. 2007; Viarengo et al. 2002; Viarengo et al. 1994). Decreased protein levels (Publication III, Figure 3) may be due to increased Ca levels, activating Cadependent catabolic processes such as protein degradation (Viarengo et al. 1994). A strong decrease of carbohydrate levels is attributed to cell hypoxia caused by copper, leading to increased activities of glycolytic enzymes involved in anaerobic ATP production (Satyaparameshwar et al. 2006; Martίnez et al. 2006). Increases of MT levels in all observed organs upon Cu exposure (Publication IV, Figure 1) confirm the role of MT in copper metabolisms. Strong decreases in GSH levels within the first 6 days of exposure indicate that GSH is
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19 Viarengo, A. 1994. Heavy metal cytotoxicity in marine organisms: Effects on Ca 2+ homeostasis and possible alteration of signal transduction pathways. Advances in Comparative and Environmental Physiology 20:85-110. Viarengo, A., G. Mancinelli, M. Pertica, R. Fabbri, and M. Orunesu. 1993. Effects of heavy metals on the Ca 2+ -ATPase activity present in gill cell plasma-membrane of mussels (Mytilus galloprovincialis Lam.). Comparative Biochemistry and Physiology 106C:655-660. Watters, G.T., M.A. Hoggarth, and D.H. Stansbery. 2009. The freshwater mussels of Ohio. Ohio: The Ohio State University Press.
PUBLICATIONS
Toxicological & Environmental Chemistry Vol. 93, No. 3, March 2011, 537–548 Producing Cu-loaded algae for feeding experiments: effects of copper on Parachlorella kessleri Andhika Puspito Nugroho ab and Hartmut Frank a * a Department of Environmental Chemistry and Ecotoxicology, University of Bayreuth, 95440 Bayreuth, Germany; b Faculty of Biology, Laboratory of Ecology, Gadjah Mada University, Yogyakarta 55281, Indonesia (Received 18 September 2010; final version received 2 November 2010) Microalgae require several essential metals for optimum growth, which at elevated concentrations may interfere with biochemical and physiological processes, one of them being copper (Cu). The aim of this study is to raise Cu-loaded Parachlorella kessleri as feed for mussels. In order to spike the algae with Cu without lowering their nutritional quality, it is important to know the highest Cu-concentration at which the main parameters remain unaffected, especially in respect to proteins and polysaccharides. The dependence of growth rate, biomass, chlorophyll-a and -b, pheophytin-a, protein, and polysaccharide contents on Cu concentrations are determined. The tests show that P. kessleri is largely unchanged in its nutritional value when exposed to Cu at levels of up to 6mmol L 1 . Above 10 mmol L 1 , toxic effects become obvious, with chlorophyll contents and growth rate being the most sensitive indicators. Keywords: copper; Parachlorella kessleri; growth rate; biomass; chlorophyll-a; chlorophyll-b; pheophytin-a; proteins; polysaccharides Introduction Microalgae are fundamental constituents of food chains in almost all aquatic ecosystems, serving as food for organisms of the next trophic levels and as source of oxygen for respiration. For optimum growth, algae require a number of essential metals, some of which may be toxic above certain levels. A typical example is Cu; usually, it is present in natural fresh waters at concentrations ranging from 0.02 to 2 nmol L 1 (0.001–0.1 mgL 1 ) and in ocean waters from 0.5 to 10 nmol L 1 (0.03–0.6 mgL 1 ). To cope with its low availability, algae have mechanisms for active uptake and accumulation (Debelius et al. 2009; Lim et al. 2006; Wright and Welbourn 2002), based upon the strong complexation with functional thiol groups of the proteins involved in the active uptake of Cu (Levy et al. 2008; Nalimova et al. 2005; Stauber and Florence 1987; Yan and Pan 2002). Copper is required as a cofactor of enzymes participating in oxygen metabolism and in redox reactions, e.g., plastocyanin, polyphenol oxidase, superoxide dismutase, ascorbate oxidase, cytochrome oxidase, lysyl oxidase, and diamine oxidase (Nalimova et al. 2005; Yilmaz, Is ik, and Sayin 2005). Mollusks and other invertebrates also require Cu as a component of hemocyanine; the animals receive it from the water as well as from Cu-containing food (Amiard-Triquet et al. 2006; Company et al. 2008). *Corresponding author. Email: [email protected] ISSN 0277–2248 print/ISSN 1029–0486 online 2011 Taylor & Francis DOI: 10.1080/02772248.2010.537859 http://www.informaworld.com 20
Due to its use as fungicide, in the building sector as roofing material, for overland highvoltage power lines, and its presence in municipal and industrial waste waters, Cu concentrations in rivers, lakes, and estuaries have increased severalfold over natural levels (Mohammed and Markert 2006; Yilmaz, Is ik, and Sayin 2005). High concentrations have been reported in rivers of Japan (1.1–3.5 mmol L 1 (0.07–0.22 mg L 1 ), Pawlik-Skowron ´ska and Skowron ´ski 2001), China (Yangtse river 0.2–0.8 mmol L 1 (15–50 mg L 1 ), Xu et al. 2000), or Brazil (Jurujuba Sound 0.08–3.3 mmol L 1 (5–210 mg L 1 ), Neto, Smith, and Mc Allister 2000). At such levels, Cu can have toxic effects to producers and consumers on the various stages of the aquatic food chain. For algae, toxicity thresholds (NOEC, no observed effect concentration) have been reported (Levy, Stauber, and Jolley 2007) to be in the range of 0.003–0.14 mmol L 1 (0.2–9 mgL 1 ), lowest observed effect concentrations (LOECs) in the range of 0.004–0.63 mmol L 1 (0.3–40 mgL 1 ), and the 72 h IC 50 between 0.009 and 8.3 mmol L 1 (0.6–530 mgL 1 ), depending on the strain. The primary toxic effects of Cu on algal cells are altering the rate of deoxyribonucleic acid (DNA) synthesis, interfering with protein and carbohydrate metabolisms, mitochondrial electron transport, and adenosine triphosphate (ATP) production and respiration, disrupting cell division, and interfering with the uptake of Ca and Mg (Arunakumara and Xuecheng 2008; Debelius et al. 2009; Markina and Aizdaicher 2006; Pawlik-Skowron ´ska and Skowron ´ski 2001; Stauber and Florence 1987; Tripathi and Gaur 2006). In Chlorella pyrenoidosa, Cu affects growth rates, photosynthesis, and content of chlorophyll-a starting at concentrations of 4 mmol L 1 (0.25 mg L 1 ), 1.6 mmol L 1 (0.1 mg L 1 ), and 1.6 mmol L 1 (0.1 mg L 1 ), respectively (Wong and Chang 1991). Yan and Pan (2002) reported the growth of Scenedesmus obliquus,C. pyrenoidosa, and Closterium lunula being inhibited at concentrations of 0.8, 1.0, and 3.0 mmol L 1 (50, 70, and 200 mgL 1 ), respectively. In this context, it should be mentioned that under laboratory culture conditions, the onset of Cu toxicity depends also on initial cell density, composition of the medium, and physical conditions (Debelius et al. 2009). The algal species Parachlorella kessleri used in this study is a common food source for herbivore consumers in freshwater ecosystems. They are easy to culture in the laboratory, often used in toxicity bioassays for predicting environmental impacts of pollutants, and known to have a remarkable ability to accumulate metals (Debelius et al. 2009; Kadukova ´ and Virc ˇı´kova ´2005; Mallick 2003). In order to raise Cu-loaded algae for feeding experiments with mussels, it is important to assess the toxicological and pathophysiological threshold of Cu which does not affect significantly the nutritional value of Cu-loaded algae compared to control algae, especially in respect to protein and carbohydrate contents. NOECs of Cu, its effects on growth rate and biomass, on chlorophyll-a and -b, in the formation of pheophytin-a, and on the protein and polysaccharide contents are determined. Materials and methods Glassware and reagents All glassware is rinsed twice with half-concentrated HNO 3 (65%; Sigma–Aldrich, Munich, Germany), deionized and bidistilled water, and sterilized in an autoclave (Certoclav CV-EL 18 O, Certoclav Sterilizer GmbH, Traun, Austria) at 120C for 15 min. Lyophilized glycogen standard (Type VII, Mytilus edulis), Coomassie blue solution, bovine serum albumin (BSA), and all other chemicals (Sigma–Aldrich) are of analytical grade. For exposure experiments, a Cu solution is prepared by dissolving 538 A.P. Nugroho and H. Frank 21
0.1 g CuCl 2 2H 2 O in bidistilled water in a 100 mL volumetric flask yielding a concentration of 5.9 mmol L 1 . Test organism, culture conditions, and toxicity testing Parachlorella kessleri is obtained from the Culture Collection of Algae (SAG) of the University of Goettingen, Germany. An algal stock culture is grown axenically in a sterilized K-medium (Kuhl and Lorenzen 1964), modified by containing the macronutrients KNO 3 , NaH 2 PO 4 H 2 O, Na 2 HPO 4 2H 2 O, MgSO 4 7H 2 O at 1.5 times, and CaCl 2 at 2.5 times increased concentrations, under omission of Cu 2þ and ethylenediaminetetraacetic acid (EDTA), and with the pH value lowered to 6.5. The algae are kept suspended by gentle shaking, maintained at 22 2C, and illuminated continuously with fluorescent tubes at a photon intensity of 48–51 mmol m 2 s 1 in 2 L Erlenmeyer flasks connected to a Drechsel gas wash bottle to distribute air and CO 2 ; the latter is filled with potassium carbonate buffer (2 mol L 1 KHCO 3 /K 2 CO 3 , 35/65 v/v). For toxicity testing, aliquots of the stock culture are added to 1.8 L modified K-medium in 2 L Erlenmeyer flasks to establish an initial cell density of 1–2 10 5 cells mL 1 . The cultures are maintained as described above. At day 3 after inoculation, Cu is added using the stock solution to establish the following exposure concentrations: 0 (control), 5.9, 11.7, 23.5, 47, and 94 mmol L 1 . The algae are grown at these levels for 96 h, i.e., from day 3 to day 7. Growth is monitored daily and, in the end of the experiment (day 7), biomass, chlorophyll-a and -b, pheophytin-a, protein, and polysaccharide contents are determined, as well as Cu accumulation. For all analyses, three aliquots are taken. Determination of growth and biomass Growth is followed by measuring optical density. Three 3 mL aliquots are taken from each culture using Pasteur pipettes connected with silicon tubing to 1000 mL micropipettors (Carl Roth, Karlsruhe, Germany) and transferred to polystyrene cuvettes with 1 cm light path. The optical density is read at a wavelength of 686 nm (OD 686 ) (UVIKON 930 Spectrophotometer, Kontron Instruments, Munich, Germany). The specific growth rate is calculated according to Mei et al. (2006), taking the rate at the highest Cu concentration as 100% inhibition. At the end of the experiment, the whole algal culture is centrifuged in six 300 mL centrifugation bottles at 10,000 rpm at 4C for 10 min (Beckman Avanti J25, rotor JA-16.50). The supernatants are discarded and the algal pellets are washed by resuspension/centrifugation, once with fresh culture medium and once with phosphatebuffered saline (PBS). The pellets are combined and the algae are resuspended in 47 mL bidistilled water, transferred to a 50 mL polypropylene centrifugation tube of known weight, and centrifuged again (Beckman Avanti J25, rotor JA-16.50); the supernatant is discarded, and the fresh weight of the biomass is calculated by subtracting the weight of the empty tube from the weight of the tube containing the algae. The pellet is frozen at 80C, freeze-dried at 40C for 72 h, and weighed again to yield the dry weight. Copper determination For Cu determination, the lyophilized algae are homogenized by acid digestion as follows: three algal samples of 10 mg each are placed in 55 mL borosilicate glass tubes, and to each Toxicological & Environmental Chemistry 539 22
tube, 5 mL of a mixture (4 : 1) of suprapure concentrated HNO 3 and suprapure concentrated HCl is added. The tubes are kept in an oven at 40C for 1 h, followed by 95C for 3 h. The digested samples are diluted with bidistilled water to 10 mL and filtered through a 0.45-mm cellulose syringe filter (Carl Roth). Copper is determined by inductively coupled plasma mass spectrometry (Agilent 7500ce, Cetac ASX-510, Agilent Technologies, Waldbronn, Germany) and expressed per kilogram dry weight. Copper concentration per kilogram wet weight is calculated by multiplying the determined concentration per dry weight with the ratio of algal wet weight versus dry weight. Determination of chlorophyll-a, chlorophyll-b, and pheophytin-a contents Chlorophyll-a and -b and pheophytin-a contents are determined according to the APHA method (APHA 1992). Lyophilized algae, 3 mg each, are suspended in 12 mL ice-cold acetone in 50 mL polypropylene centrifugation tubes and sonicated in an ice bath at 20 kHz, acoustic power 50 W (Labsonic U tip Sonicator, B. Braun Biotech International, Melsungen, Germany) for 160 s in eight 20 s periods, allowing equal time for cooling on ice. The homogenates are kept for 2 h at 4C in the dark and centrifuged at 2500 rpm at 4C for 15 min. The supernatants, 3 mL each, are transferred to 1 cm polystyrene cuvettes, and the optical densities at 750 and 664 nm (OD 750b and OD 664b ) are read (UVIKON 930 Spectrophotometer, Kontron Instruments); OD 664b value should lie between 0.1 and 1.0. Subsequently, 0.1 mL of 0.1 mol L 1 HCl is added under gentle agitation, and 90 s later, the optical densities are read again at 750 nm and, this time, at 665 nm (OD 750a and OD 665a ). The OD 664b /OD 665a ratio is calculated, and then chlorophyll-a and pheophytin-a are determined (APHA 1992): Chlorophyll-aðmg L1Þ¼26:7ðOD664b OD750bÞðOD665a ðOD750aÞ½ Pheophytin-aðmg L1Þ¼26:71:7ðOD665a ðOD750aÞðOD664b OD750bÞ½ For determination of chlorophyll-b, 3 mL of the supernatant is transferred to a 1 cm polystyrene cuvette and the optical densities at 750, 664, 647, and 630 nm are read. Chlorophyll-b is calculated according by the trichromatic method (APHA 1992): Chlorophyll-bðmg L1Þ ¼21:03ðOD647 OD750Þ5:43ðOD664 OD750Þ2:66ðOD630 OD750Þ: Protein and polysaccharide contents Lyophilized algae, 5 mg each, are placed in 2 mL Eppendorf tubes, 1 mL of PBS is added to each tube, and the samples are sonicated for 160 s in eight 20 s periods in an ice bath at 20 kHz, acoustic power 50 W, allowing equal time for cooling on ice to avoid protein denaturation. The homogenates are centrifuged at 4C for 20 min at 15,000 rpm (Beckman Avanti J25, rotor JA-16.50). The supernatants are used for determination of protein content by the dye-binding assay (Kruger 1994). Aliquots of 10 mL are filled into 1 mL disposable polystyrene cuvettes and 90 mL of bidistilled water and 1 mL of Coomassie blue solution are added. After gentle but thorough mixing, the samples are kept at room temperature for 15 min before 540 A.P. Nugroho and H. Frank 23
absorbances are read at 595 nm. Protein concentrations are determined from a calibration curve obtained with BSA. Carbohydrate is determined by the phenol–sulfuric acid assay (Masuko et al. 2005). Aliquots of the supernatants, 50 mL each, are placed in 2 mL Eppendorf tubes, and 200 mL of bidistilled water and 750 mL of concentrated sulfuric acid are rapidly added to achieve complete mixing. Immediately afterward, 150 mL of a solution of 5% phenol in water is added. After incubation for 5 min at 90C in a static water bath, the tubes are cooled to room temperature for 5 min in another water bath and wiped dry for spectrophotometric measurement at 490 nm. The concentrations of polysaccharides are determined using a calibration curve obtained with glycogen standard type VII (Sigma–Aldrich). Data analysis The data of all parameters are statistically analyzed by one-way analysis of variance (ANOVA), followed by the Duncan multiple comparison tests if significant differences are found. Data are transformed to log units before statistical analysis for homogeneity of variance and normality. Linear regression analysis is performed for evaluating the relationships between Cu concentration and physiological and biochemical parameters, followed by Pearson correlation analysis for testing the strength of linear relationships. Toxicity is expressed as NOEC, estimated using the Dunnett’s multiple comparison test after analysis by one-way ANOVA, while IC 10 and IC 50 values are determined using the inhibition concentration (ICp) approach (Version 2.0, Norberg-King 1993). Visual MINTEQ software is used to calculate Cu speciation in relation to pH of algal medium (Version 3.0, beta version; Gustaffsson 2010). Results Growth of P. kessleri is moderate and statistically, non-significantly inhibited (5% relative to control, p40.05) by Cu given between day 3 and day 7 at a concentration of 5.9 mmol L 1 . Copper exposures at 11.7, 23.5, 47, and 94 mmol L 1 cause significant decreases by 27, 34, 87, and 100% ( p50.05). Inhibition plotted on the probit scale (Figure 1) shows a linear relationship to Cu concentration with a strong, positive correlation (r¼0.96; p50.05). It should be kept in mind that at the pH of the incubation medium of 6.5, the relative percentage of free Cu 2þ is about 89–91% of the nominal concentration; decrease of pH to 6.3 at the end of exposure may increase the free Cu 2þ to about 95% (Gustaffsson 2010). Copper exposure at 5.9 mmol L 1 results in decrease in chlorophyll-a and -b contents and in OD 664b /OD 665a ratio at day 7 (Table 1) though statistically insignificant (p40.05). Reductions in chlorophyll-a and -b (42% and 32%) are found to be significant at 11.7 mmol L 1 Cu, for the OD 664b /OD 665a ratio at 23.5 mmol L 1 Cu. At the highest Cu concentration (94 mmol L 1 ), chlorophyll-a and -b contents are strongly lowered (97% and 95% relative to control), the OD 664b /OD 665a ratio moderately. Regression and correlation analysis show strong, highly negative correlations between Cu exposure concentration and chlorophyll-a (r¼0.908; p50.01), chlorophyll-b (r¼0.906; p50.01), and OD 664b / OD 665a ratio (r¼0.925; p50.01). Pheophytin-a is increased (r¼0.912; p50.01) even at the lowest Cu concentration being significantly different from control (44%, p50.05). At the highest Cu concentration, pheophytin-a is increased by 800%. Biomass is reduced by Toxicological & Environmental Chemistry 541 24
15% relative to control (p40.05) at 5.9 mmol L 1 Cu; at 11.7 mmol L 1 and above, reductions by 20% and more (p50.05) are found. Exposure of algae to Cu results in strong accumulation of the metal (Figure 2) far above the natural level, the latter being about 0.01 mmol kg 1 wet weight. On day 7 of the experiment, i.e., after 4 days of Cu exposure at 5.9 mmol L 1 , intracellular Cu reaches 2.5 mmol kg 1 wet weight, the 410-fold of its concentration in the water. At the higher Cu exposure concentrations, similar accumulation factors are found, i.e., 280to 510-fold. The polysaccharide content is raised by 32% at 5.9 mmol L 1 Cu, the level of protein is slightly and insignificantly elevated (p40.05) (Figure 3). Beyond a Cu exposure level of Figure 1. Probit plot of growth inhibition (between days 3 and 6) of P. kessleri at different CuCl 2 concentrations. Note: Identical letters indicate that differences are not significant ( p40.05). Table 1. Effects of Cu exposure on the contents of chlorophyll-a, pheophytin-a, chlorophyll-b, on OD 664b /OD 665a ratio, and on biomass in P. kessleri on day 7, i.e., after 4 days of exposure. Exposure CuCl 2 (mmol L 1 ) Effects Chlorophyll-a (mg g 1 dw) Pheophytin-a (mg g 1 dw) Chlorophyll-b (mg g 1 dw) OD 664b /OD 665a ratio Biomass (g L 1 ) 0 7.6 a 1.31 0.09 a 0.01 1.92 a 0.38 1.67 a 0.01 0.52 a 0.10 5.9 6.7 a 0.73 0.13 b 0.01 1.71 a 0.23 1.63 a 0.02 0.44 ab 0.04 11.7 4.4 b 0.37 0.15 b 0.02 1.30 b 0.05 1.59 a 0.01 0.41 b 0.05 23.5 4.0 b 0.46 0.25 c 0.03 1.00 c 0.04 1.48 b 0.08 0.38 b 0.02 47 0.3 c 0.04 0.71 d 0.05 0.14 d 0.02 1.18 c 0.01 0.30 c 0.01 94 0.2 c 0.04 0.73 d 0.03 0.10 d 0.02 1.13 c 0.06 0.25 c 0.01 Note: Means standard deviations (n¼3). Identical letters indicate that the values are statistically not different (p40.05); dw ¼dry weight. 542 A.P. Nugroho and H. Frank 25
5.9 11.7 23.5 47.0 94.0 CuCl2 concentration (mmol L–1) 0 10 20 30 40 50 60 Cu accumulation (mmol kg–1 ww) 410 280 290 340 510 Figure 2. Copper accumulation by P. kessleri after 4 days of CuCl 2 exposure. Note: Enrichment factors are given above each column. Figure 3. Concentration dependences of protein and polysaccharide contents in P. kessleri after 4 days of CuCl 2 exposure. Note: Identical letters indicate that differences in these parameters are not significant ( p40.05). Toxicological & Environmental Chemistry 543 26
hemocyanin (Birge and Black 1979) and as cofactor of the prosthetic groups of enzymes such as of cytochrome-c oxidase, tyrosinase, dopamine -hydroxylase, alcohol dehydrogenase, prolyl and lysyl oxidase, or others involved in growth regulation and development (Amiard-Triquet et al. 2006; Company et al. 2008). At higher concentrations, Cu is toxic to mussels, resulting in altered calcium (Ca) homeostasis of blood cells (Viarengo et al. 1994); the 96-hour LC 50 for mollusks ranges between 6 and 30 mmol L 1 (0.4–2 mg L 1 ) (Crompton 1998). Copper can be taken up by freshwater mussels with the water or the food. The route of uptake influences the distribution of the metal in the various organs, determines the dynamics of Cu-bioaccumulation and elimination, and has consequences on the pathophysiology of copper in the mussels (Croteau and Luoma 2005). In this work, duck mussels (Anodonta anatina) are used as model species to study the toxicological relevance of copper uptake via both pathways. The stable isotope 63 Cu is used as tracer to follow its distribution within the mussel and its elimination upon depuration. Materials and methods Algal food preparation Algae (Parachlorella kessleri) are used as food for the mussels and, when grown at a 63 Cu-concentration of 5.9 mmol L 1 (Nugroho and Frank 2010), for one experimental group as Cu-exposure source. Algae are grown in modified K-medium (Kuhl and Lorenzen 1964) for 7 days to produce normal or copper-loaded algae. Freeze-dried normal and copper-loaded algae contain 0.01 mmol kg 1 Cu (0.6 mg kg 1 Cu) and 40 mmol kg 1 Cu (2.4 mg kg 1 Cu) dry weight (dw). Isotopic Cu stock solution preparation and labware A 63 Cu stock solution (3.1 mmol L 1 , equivalent to 200 mg L 1 ) is prepared by dissolving 25 mg isotopically enriched (99%) 63 Cu oxide (Euriso-top, Saarbru ¨cken, Germany) in 1 mL suprapur HNO 3 (69%, Carl Roth, Karlsruhe, Germany) in a 100 mL glass beaker; 85 mL bidistilled water are added, and the pH of the solution is adjusted to 7.0 with aqueous ammonia (25%, VWR, Darmstadt, Germany). The solution is transferred to a 100 mL polypropylene (PP) volumetric flask which is filled to the mark with bidistilled water. Glassware and plastic equipments used for analytical purposes are rinsed twice with half-concentrated HNO 3 (65%; Sigma-Aldrich, Munich, Germany), and deionized and bidistilled water. Organisms About 70 duck mussels (A. anatina) (ZOO-Erlebnis Online Shop, Grossefehn, Germany) with shell lengths of 10–12 cm and weights of 100–200 g are brought to the laboratory in pond water. The mussels are brushed with dilute KMnO 4 solution (0.1 mg L 1 ), rinsed with tap water, and placed in 38 L aerated tap water in 45 -L glass aquaria at dim light for 7 days. During this period they are not fed; every day, half of the water is exchanged. Then the mussels are marked, weighed, and the shell lengths are measured. They are fed with freezedried Cu-free algae, 1.0 mg L 1 per day, and acclimatized for further 7 days to laboratory conditions at a temperature of 17 1C with a photoperiod of 12 h light per day, a photon flux of 13–19 mmol m 2 s 1 , in 38 L artificial pond water (APW) at pH 7.0 0.3 Toxicological & Environmental Chemistry 1839 33
(Ngo, Gerstmann, and Frank 2011) in 45 -L glass aquaria covered with transparent polypropylene lids. The aquaria are equipped with inner bio-filters and stainless steel aeration tubes. Eight kilograms glass beads are used as substrate. Two-third of the water is exchanged every two days; a complete change is conducted on every sixth day. Experimental design Of these mussels, 63 are selected to match in size and divided into three groups consisting of 21 mussels each. They are placed in three 45 L aquaria containing 38 L artificial pond water (APW). Two-third of the water is exchanged every second day; a complete change is conducted on every sixth days. A control group (1) is kept in APW. Another group (2) is exposed to 0.3 mmol L 1 (20 mgL 1 ) 63 Cu in the water using the 63 Cu stock solution; after each water change, the concentration is re-adjusted by adding appropriate volumes of the stock solution. A third group (3) receives daily 1.5 mg L 1 freeze-dried 63 Cu-loaded algae for 24 days, equivalent to a nominal copper concentration of 0.06 mmol L 1 (3.6 mgL 1 ). The mussels in the control and the exposure groups are fed with algae in amounts adjusted to their actual number. For 18 mussels, 1.5 mg L 1 of freeze-dried Cu-free (groups 1 and 2) or 63 Cu-loaded algae (group 3) are given per day. When the number of mussels is less than 18, 1.0 mg L 1 of freeze-dried Cu-free or 63 Cu-loaded algae are given daily (Ngo, Gerstmann, and Frank 2011) corresponding to a nominal concentration of 0.04 mmol L 1 (group 3). On day 24, the six mussels remaining in each group are transferred to APW-filled aquaria for 12 days of depuration, fed with 1.0 mg L 1 of freezedried Cu-free algae per day. Actual Cu concentrations in the water including the suspended algae in each group are determined every second day. On the control group, Cu concentrations in the APW during experiment are below detection limit. For the experiment involving Cu exposure via the water, after exchange of water the concentration is adjusted to 0.32 0.006 mmol L 1 , which falls to 0.03 0.01 mmol L 1 within the next 2 days. By the food pathway (group 3), the nominal Cu concentration in the beginning and after each water exchange is 0.07 0.01 mmol L 1 , falling to below detection limit within the next 2 days. For sampling, three mussels of each group are taken for analysis at days 0, 6, 12, 18, and 24 (exposure), and at days 30 and 36 (depuration). The mussels are anaesthetized with an aqueous 2-phenoxyethanol solution (4 mL L 1 ) for 30 min. Hemolymph (HML) and extrapallial fluid (EPF) are withdrawn using 5 mL syringes with 0.55 25 mm needles (B. Braun, Melsungen, Germany), transferred into 2-mL microtubes, and kept at 80C. The mussels are dissected on ice into gills, mantle, kidney, digestive gland, foot, adductors, and intestines; the remainder is collected in a combined sample (GHL), i.e., gonads, heart, and labial palps. The tissues are washed twice with bidistilled water, dried using filter paper, placed in 15 mL polypropylene (PP) tubes of known weights, weighed to obtain the wet weights (ww), and lyophilized. After lyophilization, the tubes are weighed again for dry weights (dw). Tissue fractions and body fluids of the nine mussels taken at day 0 are used to calculate the respective percentages relative to the total weight of soft body (twsb). Metal analyses Each lyophilized tissue fraction of about 10–100 mg is placed in a 55 mL borosilicate glass tubes. 5 mL of a mixture (4 þ1) of suprapure concentrated HNO 3 1840 A.P. Nugroho and H. Frank 34
(65%, Merck, Darmstadt, Germany) and suprapure concentrated HCl (30%, Merck, Darmstadt, Germany) are added to each tube. The tubes are kept in an oven at 40C for 1 h and at 95C for 3 h. The digested samples are diluted with bidistilled water to 10 mL and filtered through 0.45 mm cellulose syringe filters (Carl Roth, Karlsruhe, Germany). For the determination of Cu in HML and EPF, 0.4–1 mL of each are acidified with 0.5 mL suprapure concentrated HNO 3 in PP tubes, diluted to 10 mL with bidistilled water, and filtered through 0.45 mm cellulose syringe filters. Total Cu and its isotopes 63 Cu and 65 Cu are determined by inductively-coupled plasma mass spectrometry (ICP-MS, Agilent 7500ce, Cetac ASX-510, Agilent Technologies, Waldbronn, Germany). The detection limits for total Cu is 0.02 mmol L 1 for isotopic Cu 0.01 mmol L 1 . Total copper in each tissue fraction is calculated in mmol kg 1 ww by multiplying the analytical data with the ratio of ww versus dw. The concentration of exogenous copper C 63Cu is calculated as C 63Cu – 2.34 C 65Cu , the concentration of endogenous copper as 3.33 C 65Cu , considering the natural relative abundances of 69% 63 Cu and 31% 65 Cu. For body fluids, the Cu concentrations are given in mmol L 1 . Total and exogenous Cu-pools in the tissue fractions and body fluids are calculated in mmol kg 1 twsb by multiplying the concentration data with the weight fraction of the respective organ or body fluid. Statistical data analyses Data are transformed to log units before statistical analysis for homogeneity of variance and normality. The data for total Cu are statistically evaluated by two-way analysis of variance (ANOVA) considering exposure time and Cu exposure pathways as independent variables; if significant differences are found, those between exposure times are tested by the Dunnett multiple comparison tests, between exposure pathways and controls using the Duncan multiple comparison tests. To assess the differences in exogenous Cu between exposure pathways, the independent t-test is performed. Results Exposure of A. anatina to Cu via the water results in rapid increases (Figure 1) of the concentrations of total Cu (solid lines) in the hemolymph (HML) and the extrapallial fluid (EPF) within the first 12 days, followed by slower increases until concentrations of 0.38 mmol L 1 are reached at day 24, about the 2.5-fold of control level. From the food, increases are more moderate, reaching about 0.25 mmol L 1 , the 1.7-fold of controls. In respect to exogenous Cu, the concentrations in both body fluids (dotted lines) increase similarly upon exposure via the water and the food within the first 6 days although the nominal concentration per liter water volume in food is considerably lower. Later on, exposure via the water entails faster uptake, especially in the HML between days 6–12 to reach 0.14 mmol L 1 , continuing until 0.17 mmol L 1 at day 24. Overall, increases during the first days are faster for the EPF than for the HML. Within the 12 days of depuration, total Cu concentrations decline rapidly in EPF and in HML, in HML of animals having received the metal by the water pathway to about 50% over control; when having been exposed via the food, the Cu concentrations decline almost fully back to control values. For exogenous Cu, the concentrations in the HML and EPF of waterand food-exposed animals decline in similar relative rates. At the end of the depuration, the fraction of exogenous Cu, i.e., the excess of 63 Cu over the natural Toxicological & Environmental Chemistry 1841 35
abundance of this isotope, represents between 10% (food pathway) and up to 30% (water pathway) of total copper. In the organ and tissue fractions (Figures 2 and 3), concentrations of endogenous Cu at day 0 are highest in the kidney and the digestive gland (63 and 58 mmol kg 1 ww). In the other organs, initial Cu levels are much lower, i.e., in mantle, intestines (both 15 mmol kg 1 ww), gills, and foot (both 12 mmol kg 1 ww). The mixed fraction of the gonads, heart, and labial palps (GHL) (20 mmol kg 1 ww) shows a fairly high initial copper level although nothing can be said about the distribution between the tissues contained in it. The adductors have the lowest concentration (7 mmol kg 1 ww), but this is still much higher than in HML and EPF (Figure 1, 0.17 mmol L 1 ). The development of the total copper concentrations is quite diverse for the various tissues/organs over time (Figures 2 and 3; solid lines) in relative and absolute terms. Upon uptake via the water, strongest relative increases are seen for the gills, the mantle, and the digestive gland, especially within the first 6 days. When 63 Cu is administered via the food, an almost equal increase of 63 Cu as via water is found for the digestive gland, although its nominal initial concentration is only a fifth of the concentration in the water in dissolved form. For other organs, uptake from food leads to moderate rise in the mantle, kidney, intestines, and GHL, almost none in the gills, adductors, and foot. In the digestive gland, highest concentrations, i.e., 120–140 mmol kg 1 , are reached within 24 days irrespective of exposure pathway. For other organs, exposure via water results in peak concentrations in the gills of 75 mmol kg 1 (6.5fold relative to control), 70 mmol kg 1 in the mantle (4.2-fold), and 70 mmol kg 1 in the mixed fraction GHL (3.5–fold); moderate to low relative increases are seen in the foot, intestines, adductors, and kidney (2.8-, 1.7-, 1.6-, and 1.4-fold). Upon depuration, Cu concentrations fall immediately and strongly in most Figure 1. Concentrations of total (solid lines; ˙¼via water, #¼via food; N¼control) and exogenous (dotted lines; ¼via water, h¼via food) Cu in hemolymph (HML) and extrapallial fluid (EPF) of A. anatina during exposure (days (d) 0–24) and depuration (days 24–36). Significant differences in comparison to control within each group are indicated by o . The same letters indicate that differences of Cu concentrations are not significant among groups at each time sampling (day) while the different letter indicate p50.05. Significant differences between concentrations of exogenous Cu via food or water are indicated by þ . 1842 A.P. Nugroho and H. Frank 36
organs, except for the mantle and the intestines; for these even further increases are observed within the first 6 days of depuration. In respect to exogenous 63 Cu (Figures 2 and 3; dotted lines), exposure to 63 Cu via water leads to rapid increases in the gills, mantle, digestive gland, and GHL within the first 6 days. In some organs, i.e., digestive gland, gills, and mantle, the increases continue until day 24 to reach a maxima of about 50 mmol kg 1 ww. Exogenous copper in the kidney, Figure 2. Concentrations of total (solid lines; ˙¼via water, #¼via food; N¼control) and exogenous (dotted lines; ¼via water, h¼via food) Cu in the gills, mantle, digestive gland, and kidney of A. anatina during Cu exposure via water and food and during depuration. Significant differences in comparison to control within each group are indicated by o . The same letters indicate that differences of Cu concentrations are not significant among groups at each time sampling (day (d)) while the different letter indicate p50.05. Significant differences between concentrations of exogenous Cu via food or water are indicated by þ . Total and exogenous Cu are calculated by multiplication of the analytical data with the ratio of dry weight versus wet weight. Toxicological & Environmental Chemistry 1843 37
foot, intestines, and GHL shows maximum concentrations at day 12, followed by declines until the end of exposure. Via the food, exogenous 63 Cu initially increases in the digestive gland as fast as via the water, followed by slight further increase to reach a maximum of 20 mmol kg 1 ww on day 24. In the gills, mantle, adductors, and foot, after slight increases during the first 12 days of exposure, exogenous Cu remains relatively unchanged until the end of the experiment. During depuration, in the gills, mantle, and digestive gland, the levels of exogenous copper drops within the first 6 days by 85, 70, and 60%. For animals having received the metal via food, similar patterns of decrease are observed for the kidney and GHL, only the levels being lower, i.e. about a third. The isotope ratios of 63 Cu/ 65 Cu and its deviation from the natural ratio (2.33) are also monitored (Figure 4). Complementary to Figures 2 and 3, this allows to follow the movement of exogenous Cu within the body. When 63 Cu is administered via water, the relative abundance of 63 Cu in the body fluids HML and EPF rise up to 4.0 at day 12, then remain constant. Upon depuration, the ratio declines to about 3.0. In the kidney, GHL, foot, and intestines, peaks of 63 Cu are reached at day 12, while in the adductors, digestive gland, mantle, and gills, maximum isotope ratios are found at the end of exposure at day 24. During depuration, the relative abundance of 63 Cu in all organs declines, but not Figure 3. Concentrations of total (solid lines; ˙¼via water, #¼via food; N¼control) and exogenous (dotted lines; ¼via water, h¼via food) Cu in the GHL (gonads, heart, and labial palps), intestines, foot, and adductors of A. anatina during Cu exposure via water and food and during depuration. Significant differences in comparison to control within each group are indicated by o . The same letters indicate that differences of Cu concentrations are not significant among groups at each time sampling (day (d)) while the different letter indicate P50.05. Significant differences between concentrations of exogenous Cu via food or water are indicated by þ . Total and exogenous Cu are calculated by multiplication of the analytical data with the ratio of dry weight versus wet weight. 1844 A.P. Nugroho and H. Frank 38
totally back to the natural ratio remaining about 25–45% higher than before exposure. When 63 Cu is administered via food, the increase in the isotope ratio is pronounced for the digestive gland, while all the organs show only small increases. Calculating the Cu-pools in the body compartments gives interesting insights (Figure 5). Although HML and EPF together constitute about 70% (33 5% and 37 4%) of the total soft body volume (Figure 5, A), both are insignificant as Cu-pools. The mantle, the gills, and the intestines are the largest solid organs; together they represent about 18% (6.4 0.5, 5.9 0.6, and 5.4 0.8%) twsb. Smaller body fractions are the adductors (2.9 0.2% twsb), the digestive gland (2.8 0.3% twsb), the foot (2.2 0.4% twsb), the mixed fraction GHL (3.9 0.6% twsb), and the kidney (0.5 0.06% twsb). In the beginning (Figure 5, B), the total pool of Cu (endogenous Cu) is about 6 mmol kg 1 twsb, the largest being in the digestive gland, followed by the mantle, gills, intestines, and GHL (Figure 5, B). Upon exposure via water (W), the total Cu-pools increases, continuing until day 24 to reach a maximum of 25 mmol kg 1 twsb, i.e., the four-fold of the initial pool size; uptake via the food (F) entails a total Cu-pool of only 9 mmol kg 1 twsb at day 24, i.e., slightly less than double the control, the largest pool being in the digestive gland. The exogenous 63 Cu-pool increases in parallel to total Cu-pool upon exposure via the water, reaching a maximum of about 12 mmol kg 1 twsb at day 24. For the food pathway, it increases only slightly, the maximum level being at about 1.5 mmol kg 1 twsb (Figure 5, C). During the 12 days of depuration, all the pools are rapidly emptied, particularly the gills. The mantle and the digestive gland retain the Cu-pools relatively long (as also reflected in Figures 2 and 3), in the latter most tenaciously. Similar patterns are found for exogenous Cu. Discussion The experiments show that 63 Cu is highly available to A. anatina (Figures 2 and 3, a and b), both in water-dissolved form or from 63 Cu-loaded algae. Calculation of Cu speciation Figure 4. Isotope ratio of 63 Cu/ 65 Cu in organs and body fluids of A. anatina during exposure to 63 Cu via water (˙) or food (#), and following depuration (GHL ¼gonads, heart, and labial palps). The relative increases of 63 Cu in percent over the natural ratio upon exposure via water are given for day (d) 36. Toxicological & Environmental Chemistry 1845 39
in artificial pond water (APW) at a pH of 7.0 and at 17C shows that the metal is completely in the free Cu 2þ ionic form, ready for uptake (Gustaffsson 2010). By the food pathway, the low nominal Cu concentration in the APW may be the main factor responsible for the low Cu accumulation in the mussel in absolute terms, but in relative terms it is obviously even more efficient. During the 24 days of water-borne Cu exposure, exogenous 63 Cu levels in the organs increase differently, strongest in the gills to represent about 70% of total Cu (Figures 2 and 3, a and b). There is evidence of mobilization and re-distribution of endogenous Cu among the organs derived from the time pattern of the 63 Cu/ 65 Cu isotope ratio (Figure 4). The ratios are highest in the gills, mantle, and digestive gland (both pathways) at the sixth day of exposure, showing that the exogenous 63 Cu is initially taken up into these organs. The peaks of highest ratios at day 12 in the kidney, GHL, foot, and intestines indicate that these organs first receive fairly high amounts of exogenous Cu but – as the exposure continues – become recipients of endogenous copper mobilized from the other organs, presumably mobilized by exogenous 63 Cu. Later, exogenous and mobilized endogenous Cu is mainly stored in the digestive gland, gills, adductors, and mantle, the latter serving as transient recipient even beyond the exposure phase. The observation of copper being particularly strongly retained in the mantle is noteworthy as it is one of the most important organs for regulating the calcium household and for building the protective shell of the bivalve (Lopes-Lima et al. 2008). In the body fluids, the isotope ratio remains relatively constant at about 4.0 during days 12–24, reflecting the roles of HML and EPF as transitory exchange and transport compartments, being small as pools (Figure 5). Upon depuration the isotope ratios tend to fall strongly, indicating that a large fraction of exogenous 63 Cu remains in a relatively easily exchangeable form while the endogenous Cu is more tenaciously retained. Figure 5. A: Percentages of total weight of soft body (twsb) (EPF ¼extrapallial fluid; HML ¼hemolymph; GHL ¼gonads, heart, and labial palps), and B: total and C: exogenous Cu-pools (right ordinate) in A. anatina during Cu exposure via food (F) or water (W) and during depuration. Significant differences in comparison to control (day (d) 0) within each group (water ¼ o ; food ¼*), and between Cu exposure via food and water are indicated by þ . The total Cu-pools in the body fractions are calculated by multiplication of the concentration data (Figure 2) with the respective percentages. 1846 A.P. Nugroho and H. Frank 40
Nevertheless, at the end of the depuration, the relative abundance of 63 Cu taken up via the water pathway is between 25% and 45% higher than in the beginning in various body compartments, indicating that about a third of the functional Cu pool has been exchanged for exogenous 63 Cu. Distribution of Cu in the mussel’s body allows to assess the relative importance of the various copper pools (Figure 6). From the water it is mainly compartmentalized into the mantle (30%), the gills (24%), and the digestive gland (22%), altogether three quarters of the total Cu-pool. The former two organs have large surface areas and interact directly with the water coming into mantle cavity during filtration (Marigo ´mez et al. 2002); the digestive gland is the major receiving organ for the hemolymph pathway. The mantle has a high secretor epithelium lined with acid mucopolysaccharides for digestion of trapped small particles (Machado 2011). By the food pathway, the digestive gland and the intestines are the major Cu-recipients. In addition, the former organ secretes high amount of digestive mucus to facilitate Cu storage (Machado 2011). High Cu levels in GHL suggest a role of the heart as ion recipient and its close anatomical relation to the intestines and the kidney (Gosling 2003; Machado 2011). Figure 6. Scheme illustrating the distribution of the total Cu burden (Cu-pools) among the body fractions (EPF ¼extrapallial fluid; HML ¼hemolymph; GHL ¼gonads, heart, and labial palps; LP ¼labial palps; F ¼feces; U ¼urine) of A. anatina after exposure to Cu via water (a. black arrow (routes to the heart) and grey arrow (routes from the heart to the other organs) or food (b. white arrow (routes to the heart) and grey/heavy-lined (routes from the heart to the other organs)) (per kg of total weight of soft body). White/heavy-lined arrows show the routes of both Cu exposure pathways. The figure is adopted from Marigo ´mez et al. (2002). Toxicological & Environmental Chemistry 1847 41
In relation to the organ pools of A. anatina, the size of the respective volumes is not directly related to Cu burden (Figure 5, A, B, and C). Binding to specific compounds and compartmentalization within the organs, and physiological and metabolic functions of the organs may play some roles (Otchere 2003). In any case, the organs which serve as the primary sites for uptake, i.e., gills, mantle, and digestive gland, tend to concentrate the copper. During depuration, Cu is eliminated fairly fast from the body (Figures 2 and 3, a and b), due to the large differences in gradient Cu concentration between the mussel and APW. Han et al. (1993) reported that the initial rapid elimination can be caused by desorption of loosely bound, unassimilated copper, whereas slower elimination reflects the loss from pools (endogenous Cu) where copper is more tightly bound to tissue components. Rapid elimination is also observed in the gills and digestive gland of the marine clam R. decussatus within the first 10 days of depuration (Serafim and Bebianno 2009). In respect to Cu elimination from the digestive gland, the level in A. anatina drops to about 60% over control within 6 days (Figures 2 and 3, a). A similar pattern is observed in the marine mussels Mytilus galloprovincialis exposed to Cu at 0.63 mmol L 1 (40 mgL 1 ) via water for 3 days (Viarengo et al. 1981). This confirms that the digestive gland is the main organ for metal elimination in bivalves (Marigo ´mez et al. 2002). According to Marigo ´mez et al. (2002), the release of metals from mussel body can occur via the digestive tract as a component of feces or via the kidney together with excretory concretions as a component of urine (Figure 6). Copper accumulation in A. anatina during exposure via water or food represents two different processes, i.e., bioconcentration (water) and biomagnification (food). Calculation of the bioconcentration and biomagnification levels allows to assess the relative importance of exposure via water or food. Bioconcentration can be expressed as enrichment factor (EF), i.e., the ratio of the concentration of exogenous Cu kg 1 twsb (Figure 5, C) to the concentration in the water. Biomagnification is normally assessed as transfer factor (TF). The enrichment factor in the mussel at the end of the exposure (day 24) is about 43, by the food pathway a TF of 25 is reached (Cu concentration in the APW-added algal food is equivalent to 0.06 mmol L 1 , exogenous Cu-pools in the mussel ¼1.5 mmol kg 1 twsb; Figure 5, C). Thus, exposure via water is more effective from this point of view. In respect to biomagnification, the TF is lower than the EF for algae which is about 400-fold (Nugroho and Frank 2010), indicating only weak biomagnification of copper along the food chain from the algae to the mussel. Overall, distribution and accumulation of copper in A. anatina are the results of exposure time, exposure pathways, and physiological functions of the respective organs. Food uptake is more efficient taking the five-fold lower nominal concentration of copper in these experiments into consideration. These experiments will help understand the risks associated with copper exposure of freshwater mussels. Copper accumulation may promote the situation of metabolic acidosis leading to the dissolution of CaCO 3 deposits, inducing the increase of Ca concentration in the EPF (Antunes et al. 2002; Faubel et al. 2008; Lopes-Lima et al. 2008). Interference with Ca homeostasis by the inhibition of Ca-ATPase by Cu (Santini et al. 2011) may lead to physiological stress. These factors together with the involvement of copper in the formation of reactive oxygen species (Company et al. 2008) may be a contributory factor in the overall Europe-wide observed decline of freshwater bivalves. Conclusions Exposure of A. anatina to Cu via the water or via the food leads to enrichment of the transition metal in the mussel. Copper is mainly stored in the digestive gland, gills, 1848 A.P. Nugroho and H. Frank 42
increases were more moderate due to the lower Cu-intake, i.e., about the 1.2-fold of control at day 24. Ca levels in HML and EPF were correlated to total Cu concentration (rfood (HML) ¼0.85, r-water (HML) ¼0.90, r-food (EPF) ¼0.61, r-water (EPF) ¼0.73; p50.05). Upon depuration, Ca concentrations in the body fluids declined fast, returning to control values during the first six days although Cu was still elevated. In the other organs and tissues (Figure 2), highest Ca concentrations at day 0 were found in the mantle (90 mmol kg 1 ww), the gills, the digestive gland, the mixed fraction containing gonads, heart, and labial palps (GHL) (all about 80 mmol kg 1 ww), and in the intestines (70 mmol kg 1 ww); much lower were the Ca-levels in the kidney, the adductors (both 20 mmol kg 1 ww), and the foot (10 mmol kg 1 ww). Upon Cu exposure via water or food, Ca levels increased in all organs except for the adductors. Highest Ca levels were found in the gills, mantle, and digestive gland upon exposure via the water, reaching about 140–160 mmol kg 1 ww (2-fold control) at day 24, highest relative increase being found in the kidney (4-fold, 80 mmol kg 1 ww). For GHL and the intestines, maximum Ca levels were at 110 (1.3-fold of control) and 100 (1.4-fold) mmol kg 1 ww, respectively. In the foot, Ca reached about the 4-fold (40 mmol kg 1 ww) of control at day 24 upon Cuexposure via the water. When Cu-exposure took place via the food with its nominally lower Cu-levels per liter APW, Ca concentrations in the digestive gland, the intestines, the Figure 2. Concentrations of Ca (right ordinate, dotted lines; S¼upon exposure via water, h¼upon exposure via food, D¼control) and Cu (left ordinate, solid lines; ^¼via water, #¼via food; N¼control) in the various organs of A. anatina during Cu exposure (E) and depuration (D) (GHL ¼gonads/heart/labial palps). Significant differences in comparison to control within each group are indicated by o . Similar letters indicate that differences of Ca concentrations are not significant among groups at each time sampling (day, d) while different letters indicate p50.05. Concentration of Ca is calculated by multiplication of the analytical data with the ratio of dry weight versus wet weight. Concentrations of copper are the same as in Nugroho and Frank (2011b). Toxicological & Environmental Chemistry 103 49
kidney, and the GHL increased almost equally as in the animals having received Cu via the water. In the adductors, Ca levels remained almost unchanged, as also found for Cu. In the gills, mantle, digestive gland, and kidney upon exposure via the water or the food, Ca concentrations were strongly and positively correlated to the Cu levels in the respective organs (r40.7; p50.05). Upon depuration, Ca levels declined slowly, except for the kidney with fast elimination of excessive Ca. Upon the 12 days of depuration, Ca in the kidney, adductors, intestines, and GHL returned almost fully back to control values while in the gills, mantle, digestive gland, and foot the Ca levels declined to about 20–70% above control values. Soluble carbohydrates and proteins in all organs were lowered upon Cu exposure and in parallel to its concentrations (Figure 3), strongest effects being observed when Cu was supplied via the water. Carbohydrate levels decreased drastically until the end of exposure with water-dissolved Cu, i.e. by 80% (gills) and 70% (kidney). Exposure to food-contained Cu had moderate effects except for the digestive gland and the kidney; these two organs showed only little differences between the two exposure pathways. For all other solid organs (not shown in Figure 3), soluble carbohydrates were decreased by 5–10%. Carbohydrates in HML and EPF at day 24 were lowered by only 5–10% upon Cu exposure via the food, the effects again being slightly stronger when Cu was taken up from the water (8–12%). Correlation analyses confirmed strong negative relationships between Cu and carbohydrate (r40.6; p50.05) in the gills, mantle (water pathway), digestive gland, and kidney (both pathways). Figure 3. Contents of soluble carbohydrates and proteins (N¼control; #¼via food; ^¼via water) in the gills, mantle, digestive gland, and kidney of A. anatina during Cu exposure (E) and depuration (D). Significant differences in comparison to control within each group are indicated by o . Similar letters indicate that differences of Ca concentrations are not significant among groups at each time sampling (day, d) while different letters indicate p50.05. 104 A.P. Nugroho and H. Frank 50
Soluble proteins in the gills, mantle, digestive gland, and kidney declined to between 20% and 45% of control levels at day 24. For the digestive gland and the kidney, the effects were almost equal for both exposure pathways while for the gills and the mantle great differences were found. Soluble protein levels in all other tissue compartments were decreased by not more than 5–10% (not shown in Figure 3). In the HML and EPF, levels of soluble proteins at day 24 were lowered by only 5–10% (HML: 550 40 mg L 1 ; EPF: 390 40 mg L 1 ;n¼3) for both pathways. Significant relationships of Cu and Ca with soluble protein levels (r40.6; p50.05) existed in the gills, mantle (water pathway), digestive gland, and kidney (both pathways). Upon depuration, soluble carbohydrate and protein levels in the studied organs started to increase, although not fully back to control within the 12 days. Discussion Exposure of A. anatina to copper via water or food causes increases of Ca levels in all body compartments, by the water pathway being stronger than via the food pathway. Nevertheless, when considering the nominally five-fold lower Cu concentration contributed by the algal suspension in the APW, the effects of the latter exposure pathway on the digestive gland and the kidney are surprisingly strong. Increase of Ca in all organs (Figure 2) suggests that Cu not only affects the enzymes regulating the internal Ca balance but also the overall Ca burden is strongly increased; such a flooding of the organism with excess Ca, mainly of the gills, mantle, and digestive gland, can only result from mobilization of CaCO 3 from the shell, most likely due to Cu-induced metabolic acidosis (Antunes et al. 2002; Faubel et al. 2008; Lopes-Lima et al. 2008). This is confirmed by the increase of Ca in the EPF between days 6 and 24. Interference of Cu with Ca homeostasis by affecting the mechanisms of Ca extrusion across cellular membranes may be another complication (Viarengo et al. 1994; Viarengo, Burlando, and Bolognesi 2002; Pattnaik, Chainy, and Jena 2007). Increase of Ca in the HML has also been reported by Viarengo (1994) for exposure of Mytilus edulis to Cu at 0.5–2 mmol L 1 . A strong decrease of carbohydrate levels in A. anatina upon Cu exposure indicates that this is another sensitive toxicological endpoint associated with the disturbance of Ca homeostasis. A similar pattern is found in the gills and mantle of the freshwater mussel Lamellindens marginalis under copper stress at 2 mmol L 1 (133 mgL 1 ) for 3 days. This has been attributed to cell hypoxia (Satyaparameshwar, Reddy, and Kumar 2006) leading to increased activities of glycolytic enzymes involved in anaerobic ATP production (Martı´nez et al. 2006). Further decrease observed between days 12 and 24 (Figure 3) suggests that at lower carbohydrate levels gluconeogenesis is stimulated. Strong correlation between increased Ca and low protein levels illustrate the key role of the electrolyte as intracellular signaling factor. According to Viarengo et al. (1994), elevated cytosolic Ca levels activated protein degradation. Decrease in protein levels was also found in the freshwater mussel Anodonta woodiana after exposure of Cu at 0.9 mmol L 1 (0.06 mg L 1 ) for 4 weeks (Kurnia, Purwanto, and Mahajoeno 2010). Similar to our findings, only minor effects on the soluble protein levels in HML and EPF were observed with the freshwater mussel Anodonta cygnea upon exposure to CuSO 4 at 10 6 mol L 1 for 1 month (Moura, Vilarinho, and Machado 2000). Overall, Cu exposure at environmentally relevant levels leads to increased Ca levels in all body compartments of A. anatina, indicating that Cu interferes with Ca homeostasis. Dissolution of Ca from the shell upon Cu-induced metabolic acidosis (Antunes et al. 2002; Toxicological & Environmental Chemistry 105 51
Lopes-Lima et al. 2008) and distribution of the electrolyte throughout the other body compartments are likely to contribute to its elevated levels. This can lead to the activation of Ca-dependent catabolic processes such as lipid hydrolysis, DNA fragmentation, and protein degradation, ultimately leading to cell death (Viarengo et al. 1994). Decreased carbohydrate and protein levels, being strongly inversely correlated to Ca levels, suggest that the mussels may not have sufficient energy and essential nutrients for normal reproduction, growth, and development. At the same time, carbohydrates and proteins are important components of the organic matrix which controls CaCO 3 polymorphism, size, and shape of the crystallites (Marin and Luquet 2004). These strong pathophysiological responses to environment-like levels of Cu may be one of the many factors involved in the presently observed decline of many European freshwater bivalves, including the freshwater pearl mussel Margaritifera margaritifera (Bauer 1986). Conclusions Copper exposure results in increases of Ca levels in all body compartments, accompanied by decreases in the levels of soluble proteins and carbohydrates. These effects may result in disturbance of mussel’s reproduction, growth and development, and shell formation, leading to population decline. Acknowledgments We would like to thank Prof. Dr. Jorge P. Machado (Laboratory of Applied Physiology, University of Porto, Portugal) for helpful discussion. Financial support by the Directorate General of Higher Education, Ministry of National Education of the Republic of Indonesia, is highly appreciated. References Albert, B., D. Bray, J. Lewis, M. Raff, K. Robert, and J.D. Watson. 1994. Molecular biology of the cell. 3rd ed. New York: Garland Publishing. Antunes, C., T. Magalha ˜es-Cardoso, G. Moura, D. Gonc¸ alves, and J. Machado. 2002. Effects of Al, Ni, Co, Zn, Cd, and Cu metals on the outer mantle epithelium of Anodonta cygnea (Unionidae). Haliotis 31: 71–84. Bauer, G. 1986. The status of the freshwater pearl mussel Margaritifera margaritifera in the south of its European range. Biological Conservation 38: 1–9. Besser, J.M., C.G. Ingersoll, and J.P. Giesy. 1996. Effects of spatial and temporal variation of acidvolatile sulfide on the bioavailability of copper and zinc in freshwater sediments. Environmental Toxicology and Chemistry 15: 286–93. Birge, W.J., and J.A. Black. 1979. Effects of copper on embryonic and juvenile stages of aquatic animals. In Copper in the environment. Part II. Health effects, ed. J.O. Nriagu, 373–99. New York: John Wiley & Sons. Demayo, A., and M.C. Taylor. 1981. Guidelines for surface water quality. Vol. 1: Inorganic chemical substances – Copper. Ottawa: Water Quality Branch, Inland Waters Directorate, Environment Canada. Faubel, D., M. Lopes-Lima, S. Freitas, L. Pereira, J. Andrade, A. Checa, H. Frank, T. Matsuda, and J. Machado. 2008. Effects of Cd 2þ on the calcium metabolism and shell mineralization of bivalve Anodonta cygnea.Marine and Freshwater Behaviour and Physiology 41: 93–108. Honkoop, P.J.C., J.V. der Meer, J.J. Beukema, and D. Kwast. 1999. Reproductive investment in the intertidal bivalve Macoma balthica.Journal of Sea Research 41: 203–12. 106 A.P. Nugroho and H. Frank 52
Julshamn, K., E.K. Torpe, C. Børnes, L.J. Sæthre, and A. Maage. 2001. Cadmium, lead, copper and zinc in blue mussels (Mytilus edulis) sampled in the Hardangerfjord, Norway. Journal of Environmental Monitoring 3: 539–42. Kruger, N.J. 1994. The Bradford method for protein quantitation. In Methods in molecular biology: Basic protein and peptide protocols, Vol. 32, ed. J.M. Walker, 9–15. New Jersey: Humana Press, Inc. Kurnia, A.I., E. Purwanto, and E. Mahajoeno. 2010. Exposure copper heavy metal (Cu) on freshwater mussel (Anodonta woodiana) and its relation to Cu and protein content in the body shell. Bioscience 2: 48–53. Lopes-Lima, M., R. Bleher, T. Forg, M. Hafner, and J. Machado. 2008. Studies on a PCMA-like protein in the outer mantle epithelium of Anodonta cygnea: Insights on calcium transcellular dynamics. Journal of Comparative Physiology B 178: 17–25. Marie, B., G. Luquet, J.P.D. Barros, N. Guichard, S. Morel, G. Alcaraz, L. Bollache, and F. Marin. 2007. The shell matrix of the freshwater mussel Unio pictorum (Paleoheterodonta, Unionoida): Involvement of acidic polysaccharides from glycoproteins in nacre mineralization. FEBS Journal 274: 2933–45. Marin, F., and G. Luquet. 2004. Molluscan shell proteins. Comptes Rendus Palevol 3: 469–92. Martı´nez, M.L., C. Landry, R. Boehm, S. Manning, A.O. Cheek, and B.B. Rees. 2006. Effects of long-term hypoxia on enzymes of carbohydrate metabolism in the Gulf killifish, Fundulus grandis. The Journal of Experimental Biology 209: 3851–61. Masuko, T., A. Minami, N. Iwasaki, T. Majima, S. Nishimura, and Y.C. Lee. 2005. Carbohydrate analysis by a phenol–sulfuric acid method in microplate format. Analytical Biochemistry 339: 69–72. Moura, G., L. Vilarinho, and J. Machado. 2000. The action of Cd, Cu, Cr, Zn, and Pb on fluid composition of Anodonta cygnea (L.): Organic components. Comparative Biochemistry and Physiology B 127: 105–12. Nugroho, A.P., and H. Frank. 2011a. Producing Cu-loaded algae for feeding experiments: Effects of copper on Parachlorella kessleri.Toxicological and Environmental Chemistry 93: 537–48. Nugroho, A.P., and H. Frank. 2011b. Uptake, distribution, and bioaccumulation of copper in the freshwater mussel Anodonta anatina. Toxicological and Environmental Chemistry 93: 1838–50. Pattnaik, S., G.B.N. Chainy, and J.K. Jena. 2007. Characterization of Ca 2þ -ATPase activity in gill microsomes of fresheater mussel, Lamellidens marginalis (Lamarck) and heavy metal modulations. Aquaculture 270: 443–50. Santini, O., N. Chahbane, P. Vasseur, and H. Frank. 2011. Effects of low-level copper exposure on Ca 2þ -ATPase and carbonic anhydrase in the freshwater bivalve Anodonta anatina. Toxicological and Environmental Chemistry 93: 1826–37. Satyaparameshwar, K., T.R. Reddy, and N.V. Kumar. 2006. Study of carbohydrate metabolism in selected tissues of freshwater mussel, Lamellidens marginalis under copper sulphate toxicity. Journal of Environmental Biology 27: 39–41. Streit, B., and S. Winter. 1993. Cadmium uptake and compartmental time characteristics in the freshwater mussel Anodonta anatina.Chemosphere 26: 1479–90. Viant, M.R., J.H. Walton, P.L. TenBrook, and R.S. Tjeerdema. 2002. Sublethal actions of copper in abalone (Haliotis rufescens) as characterized by in vivo 31 P NMR. Aquatic Toxicology 57: 139–51. Viarengo, A. 1994. Heavy metal cytotoxicity in marine organisms: Effects on Ca 2þ homeostasis and possible alteration of signal transduction pathways. Advances in Comparative Environmental Physiology 20: 85–110. Viarengo, A., B. Burlando, and C. Bolognesi. 2002. Cellular responses to copper in aquatic organisms. In Handbook of copper pharmacology and toxicology, ed. E.J. Massaro, 417–27. New Jersey: Humana Press, Inc. Toxicological & Environmental Chemistry 107 53
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Toxicological & Environmental Chemistry Vol. 94, No. 5, May 2012, 918–929 Effects of copper on lipid peroxidation, glutathione, metallothionein, and antioxidative enzymes in the freshwater mussel Anodonta anatina Andhika Puspito Nugroho ab and Hartmut Frank a * a Environmental Chemistry and Ecotoxicology, University of Bayreuth, D-95440 Bayreuth, Germany; b Laboratory of Ecology, Faculty of Biology, Gadjah Mada University, Yogyakarta 55281, Indonesia (Received 20 August 2011; final version received 8 March 2012) Copper is an essential element to all animals. At elevated concentrations, it is toxic and can participate in the formation of reactive oxygen species, leading to cellular damage. In this study, the ecotoxicological relevance of copper was investigated with freshwater mussels, Anodonta anatina. When the mussels were exposed to copper at environmentally realistic concentrations, either via the water (0.3 mmol L 1 Cu) or fed with Cu-loaded algae (equivalent to 0.06 mmol L 1 Cu), the level of thiobarbituric acid-reactive substances rose and glutathione decreased. This was associated with the induction of metallothionein and, relative to total protein, of glutathione reductase and the antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase. But, since the overall protein-synthetic capacity was hampered by the copper insult, the activities of the enzymes relative to tissue weight and copper concentrations were depressed. During depuration, most parameters started to normalize although not returning to control values within 12 days. Keywords: copper; Anodonta anatina; thiobarbituric acid-reactive substances; glutathione; metallothionein; antioxidative enzymes Introduction Metals are brought to the earth’s surface by mining for a multitude of agricultural, industrial, and technological applications. One of the technologically important metals is copper (Cu), used for electrical power installations and in the building sector, as animal feed additive or fungicide, as part of machineries, vehicles, electric appliances, and in many other consumer products. During its use, it is released by corrosion and/or abrasion, mobilized as particulate matter and dry or wet deposited, to some extent ending up in the sediments of freshwater ecosystems (Smolders et al. 2003). In non-contaminated freshwater ecosystems, its concentrations range from 0.02 to 0.3 mmol L 1 (1–20 mgL 1 ) (Momc ˇilovic ´2004). Close to mining activities, aquatic copper pollution can reach levels of up to 30 mmol L 1 (1.7 mg L 1 ) (Smolders et al. 2003). Mussels live at the interface of free-flowing waters and sediments and may be chronically exposed to copper for long time periods or intermittently at fluctuating levels, depending upon temporary hydrological conditions and extent of sediment oxygenation *Corresponding author. Email: [email protected] ISSN 0277–2248 print/ISSN 1029–0486 online ß2012 Taylor & Francis http://dx.doi.org/10.1080/02772248.2012.675156 http://www.tandfonline.com 55
(Bhaduri et al. 2000; Poot, Gillissen, and Koelmans 2007). Copper is an essential element of their circulatory oxygen carrier hemocyanin (Momc ˇilovic ´2004) and plays a role as cofactor of a number of enzymes such as cytochrome oxidase, superoxide dismutase (SOD), alcohol dehydrogenase, dopamine hydroxylase, tyrosinase, and lysyl oxidase (Serafim and Bebianno 2009). However, at excessive concentrations copper can participate in the formation of reactive oxygen species (ROS) through a Haber–Weiss cycle, producing hydroxyl radicals ( OH) from hydrogen peroxide (H 2 O 2 ) and superoxide (O 2) (Bigot et al. 2011; Company et al. 2008). ROS may cause cellular damage by lipid peroxidation when the antioxidative defense systems of aquatic animals are overwhelmed, leading to inactivation of membrane enzymes, destruction of proteins (Remmer et al. 1989), and changes in the DNA structure (Company et al. 2008; Lackner 1998; Serafim and Bebianno 2009). Mussels can cope with moderately elevated copper in various ways (Serafim and Bebianno 2009). In the cytosol, glutathione (GSH) and metallothionein (MT), the latter a family of cysteine-rich proteins (Ivankovic ´et al. 2010), provide protection against increased concentrations through binding the copper ions to the thiol groups of their cysteine residues (Company et al 2008; Freedman, Ciriolo, and Peisach 1989). Other strategies against copper-induced oxidative toxicity is the induction of enzymes such as SOD, catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR) (Isani et al. 2003). Copper has been observed in high concentrations in the tissue of freshwater pearl mussels Margaritifera margaritifera (Frank and Gerstmann 2007) and other European freshwater mussel species (Tallandini et al. 1986). Their populations are strongly affected Europe-wide and some are threatened with extinction (Cuttelod, Seddon, and Neubert 2011). Understanding the potential involvement of Cu in this phenomenon is the major motivation for this study. In this work, Anodonta anatina is used as model species. In previous publications, it has been shown that A. anatina can accumulate copper from the water or by feeding on copper-containing algae (Nugroho and Frank 2011b). This article focuses on the effects of copper on MT and GSH and on antioxidative enzymes as response to oxidative stress, signaled by increased levels of thiobarbituric acid-reactive substances (TBARS). Materials and methods Chemicals Isotopically enriched (99%) 63 Cu oxide (Euriso-top, Saarbru ¨cken, Germany) was used. Concentrated HNO 3 (69%) and concentrated HCl (30%) were of suprapure grade (Merck, Darmstadt, Germany); other chemicals (Carl Roth, Karlsruhe, Germany; Sigma-Aldrich, Munich, Germany) were of analytical grade. Cleaning of labware and preparation of the Cu 2þ stock solution are described in a previous publication (Nugroho and Frank 2011b). Animals and experimental design Seventy duck mussels (A. anatina) (ZOO-Erlebnis Online Shop, Grossefehn, Germany) with shell lengths of 10–12 cm and weights between 100 and 200 g were brought to the laboratory in pond water. Mussel handling, acclimatization, and experimental design Toxicological & Environmental Chemistry 919 56
(Nugroho and Frank 2011b) as well as the preparation of normal and Cu-loaded algae have been described earlier (Nugroho and Frank 2011a). The mussels were divided into three groups consisting of 21 mussels each. The first group was kept in artificial pond water (APW); the second one was exposed to 0.3 mmol L 1 (20 mgL 1 ) 63 Cu 2þ in the water; the third group received daily 1.5 mg L 1 freeze-dried 63 Cu-loaded algae (40 mmol 63 Cu per kg dry weight) for 24 days, equivalent to a nominal concentration of 0.06 mmol (3.6 mgL 1 ) 63 Cu per liter APW. For sampling, three mussels of each group were taken for analysis at days 0, 6, 12, 18, and 24 (exposure), and at days 30 and 36 (depuration). The mussels’ soft bodies were dissected on ice into gills, mantle, kidney, and digestive gland. Two aliquots of every tissue fraction, about 5–10 mg each, were placed in separate 2-mL microtubes of known weight. The first aliquot was used for the determination of MT and the second one for the determination of TBARS, GSH, enzyme activities, and proteins. All microtubes were kept in a freezer at 80C until further analysis. The remainders of the tissues were placed in 15-mL polypropylene (PP) tubes of known weights and were lyophilized for copper determination. Analytical methods Sample preparation Frozen tissue samples in microtubes were thawed and immediately mixed with 500 mL sucrose (0.5 mol L 1 )/Tris-HCl (20 mmol L 1 ; pH 8.6) buffer, to which leupeptine (6 mmol L 1 ) and phenylmethanesulfonylfluoride (PMSF) (0.5 mmol L 1 ) were added as anti proteolytic agents and -mercaptoethanol (0.01%) as reducing agent. The mixtures were sonicated in an ice bath with 12 strokes of a sonicator (Labsonic U tip sonicator, B. Braun Biotech International, Melsungen, Germany) at 20 kHz, acoustic power 50 W. The homogenates were centrifuged at 4C for 30 min at 10,000 g(Heraeus Multifuge 1L-R, Thermo Scientific, Osterode, Germany). Supernatants were used for MT determination. For the determination of TBARS, GSH, enzyme activities, and proteins, frozen tissue samples of 5–10 mg were thawed and immediately mixed with 500 mL phosphate buffer (50 mmol L 1 ; pH 7.4) containing 150 mmol L 1 KCl, 1 mmol L 1 ethylenediaminetetraacetic acid (EDTA), 1 mmol L 1 dithiothreitol (DTT), and 0.01% (w/v) PMSF. The samples were homogenized in an ice bath with 12 strokes of a sonicator at 20 kHz, acoustic power 50 W, and centrifuged at 4C for 30 min at 10,000 g. The supernatants were used for analysis. Total copper Total copper in lyophilized tissues and freeze-dried algal food, and – every second day – the actual copper concentrations in APW were determined by inductively-coupled plasma mass spectrometry. Details have been described previously (Nugroho and Frank 2011b). Lipid peroxidation Lipid peroxidation was determined following the method of Buege and Aust (1978) by measuring TBARS, expressed as malondialdehyde (MDA) equivalents. Absorbances of samples were read at 535 nm with a microplate reader (Biotek Synergy HT, Bad Friedrichshall, Germany). TBARS levels were estimated using a standard curve obtained 920 A.P. Nugroho and H. Frank 57
with 1,1,3,3-tetramethoxypropane (99%; VWR, Darmstadt, Germany) as stable precursor of MDA and expressed as mmol kg 1 tissue wet weight (tww). Glutathione GSH was determined according to Anderson (1985). Absorbances of samples were measured at 412 nm with a microplate reader. The GSH content was estimated using a standard curve obtained with reduced GSH and expressed as mmol kg 1 tww. Metallothioneins MT concentrations were determined by the spectrophotometric method of Viarengo et al. (1997) modified by Verlecar, Jena, and Chainy (2008). Absorbances of samples were read at 412 nm with a microplate reader. The MT content was determined using GSH (Carl Roth, Karlsruhe, Germany) as standard, assuming that 1 mmol GSH is equivalent to 0.055 mmol MT. Concentrations of MT were expressed as mmol kg 1 tww. Enzyme activities SOD activities were determined by the procedure of Beauchamp and Fridovich (1971), based on the inhibition of nitrotetrazolium blue reduction and measuring sample absorbances at 560 nm. CAT activities were assayed spectrophotometrically according to Rao, Paliyath, and Ormrod (1996) by monitoring the decrease in the absorbance of H 2 O 2 at 240 nm. GPX activities were determined according to Paglia and Valentine (1967) and GR activities according to Massey and William (1965) in the presence of GSSG, in both cases following the rate of NADPH oxidation at 340 nm. Absorbances were measured with a microplate reader; enzyme activities were calculated in units per milligram protein and per gram tww. Proteins Proteins were determined by the dye-binding assay (Kruger 1994). Absorbances of the samples were read at 595 nm with a microplate reader. The concentrations were determined using BSA (96%; Sigma-Aldrich, Munich, Germany) for calibration. Statistical data analyses The variability of the observed parameters and of total Cu concentration in the different organs were tested by two-way analysis of variance (ANOVA) considering exposure time and copper exposure pathways as independent variables, followed by the Duncan multiple comparison tests ( p<0.05) if significant differences were found. Data were transformed to log(Xþ1) units before statistical analysis for the homogeneity of variance and normality. Linear regression analysis was performed for evaluating the relationship between Cu concentration and the observed parameters, followed by Pearson correlation analysis for testing the strength of linear relationship. Toxicological & Environmental Chemistry 921 58
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67 DECLARATION I hereby declare that this submission is my own account of my own research and that, to the best of my knowledge and belief, it contains neither material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of a university or any other institute of higher learning, except where due acknowledgment has been made in the text. ERKLÄRUNG Hiermit erkläre ich, dass ich die Arbeit selbstständig verfasst und keine anderen als die angegebenen Hilfsmittel verwendet habe. Weiterhin erkläre ich, dass ich nicht anderweitig mit oder ohne Erfolg versucht habe, eine Dissertation einzureichen oder mich einer Doktorprüfung zu unterziehen. Bayreuth, den 9 November 2011 ____________________________ Andhika Puspito Nugroho