SARIB

Partner No. 10: The Norwegian Institute for water Research (NIVA)

The Norwegian Institute for water Research (NIVA) is a private, non-profit research foundation that performs research, development, monitoring and feasibility studies, and conveys information about water related issues at the national and international level. NIVA, which host a staff of 170 people, has its main office in Oslo (Norway) and research activities cover most fields related to fresh and marine waters, as well as adjoining fields of engineering, social sciences and computer science. The institute, which is by far the largest interdisciplinary applied water research unit in Norway, runs 3 district offices in the cities of Bergen, Grimstad and Hamar. NIVA has well-equipped laboratories with special emphasis on studies on the presence and effects of heavy metals and organic micro-pollutants in water, organisms and sediments. The institute houses laboratories for freshwater experiments using untreated water from a nearby lake, as well as the advanced marine facilities at the Solbergstrand Marine Research Station in the Oslo Fjord.
The ecotoxicological laboratory at the section of “Ecotoxicology and Risk Assessment”, which is accredited in accordance with GLP, has established a range of international standard procedures for toxicity tests with fresh water and marine organisms, and is keeping cultures of test organisms for this purpose. Such tests also include biological degradation and bioaccumulation potential of chemicals. The tests are used on a routine basis for characterisation of chemicals and complex effluents of wastewaters. In addition to standard ecotoxicology testing methods, a substantial activity of research is performed using biomarker, microscale and in vitro methods for chemical screening and environmental monitoring.

EU projects
EU-projects have since 1992 covered research within a wide range of disciplines at NIVA, some of which are listed below:
- acid rain and mitigation of its effects (EMERGE, MOLAR, ALPE1 & 2, HUMEX, HUMOR, NITREX),
- eutrophication of marine rocky shores by fertilisation (EULIT), effects of climatic change on soil and water (CLIMEX),
- migration of radionuclides in the environment (RADNUK) and physiological indicators of metals and organic contaminants (BEQUALM), and
- how migrating birds disperse aquatic plants and zooplankton and the effects on wetland biodiversity (LAKES).

Norwegian Institute for Water Research (NIVA)
P.O. Box 173 Kjelsaas, N-0411 Oslo, Norway.
Phone +47 22 18 51 00, Fax: +47 22 18 52 00
http://www.niva.no/
No of Participants in the Group: 5
Professor/Dr. scient Ketil Hylland
M.science Torsten Källquist
Dr. scient Knut-Erik Tollefsen
Dr. scient Anders Ruus
Ph. D. Merete Grung

E-mail:
ketil.hylland@niva.no
torsten.kallquist@niva
knut.erik.tollefsen@niva.no
anders.ruus@niva.no
merete.grung@niva.no

Role and Contribution
NIVA will contribute to the SARIB project with biological effect methods based on small scale/in vitro methods, caged fish and investigations of feral fish for monitoring sediments, river water and biota from the Sava River. The work will follow a tiered system, where the river water and biota component consist of 4 tiers (phase I-IV) and the sediment component consist of two tiers (phase I and phase III). The overall objective of the work is to characterise the spatial distribution of toxic pollutants, identify agents responsible for toxic activity and assess whether these pollutants may produce effect on caged and wild populations of fish:

Phase I – Spatial studies: spatial studies will be performed using a combination of time-integrated solid phase (SPE) extraction of river water, pooled sediments and sediment pore water and testing of these extracts in a range of bioassays. Semi-permeable membrane devices (SPMDs) will also be deployed in the same area for time-integrative passive sampling of organic micropollutants.

Phase II – Local and regional studies: caged fish (barbel and chub) will be placed in areas identified as “hot spots” Samples will be taken prior to deployment for some parameters (measured in blood; xenoestrogens, antioxidant status). Methods will include endpoints sensitive to major classes of contaminants. In addition to contaminant-specific methods, cellular energy allocation for selected tissues of fish will be determined to clarify effects on general metabolism. Milt and roe from caged fish will be used for in vitro fertilisation and embryonic development investigations. Methods will be developed to assay the quality of fish milt.

Phase III - Identification of toxic pollutants: a bioassay-directed fractionation and chemical identification (TiE) procedure will be used on river water and sediments at contaminant “hotspots” of the Sava river to identify the micropollutants responsible for in vitro and in vivo effects (identified in Phase I and II).

Phase IV - Effects on populations: studies on wild-caught fish will be initiated in areas with high toxic potential to clarify whether pollutants cause impacts on feral populations of fish. As for caged fish, both general and specific methods will be used, but the selection will focus on effects from the contaminants identified in Phase III.

Methods description:
- Organic extraction of river water and sediments: organic pollutants such as PAH, PCB, dioxins, alkylphenols, pesticides etc. will be extracted from river water by solid phase extraction (SPE) using a mobile unit or shipping acid-conserved and frozen water to a suitable lab (partner 3). Sediments from the same localities will be sampled and shipped to the laboratory where sediment and pore water will be separated. Pore water will be frozen directly, whereas organic contaminants in sediments will be extracted by ultrasound-assisted soxhlet extraction. All extracts will be stored cold until assayed in appropriate in vitro bioassays.
- Deployment and extraction of SPMDs: see partner 3
- In vitro endpoints and assays: pore water and organic extracts from sediments and river water will be tested for various types of toxicity using an algae toxicity assay, mutatox, fish hepatocyte culture - biomarker endpoints: vitellogenin, EROD, acute toxicity, oxidative stress, DNA-damage, acetyl cholinesterase inhibition and MXR-inhibition (collaboration with partner 3)
- The health of feral and caged fish will be investigated using parallel methods to those used for in vitro assays, thereby facilitating extrapolation between levels. A range of biomarkers will be determined including phase-1 and phase-2 enzymes, responses to oxidative stress (glutatione redox-status, glutathione reductase, glutathione peroxidase, superoxide dismutase, catalase), responses to metals (metallothionein, ALA-D) and xenoestrogens (vitellogenin , eggshell Zona radiata proteins). Methods will be adapted as far as possible to enable sampling prior to exposure (i.e. measurement of blood components). Standard blood physiology will be routinely assessed for all fish using a hand-held instrument (I-STAT).
- Bioassay-directed fractionation and chemical identification (TiE) procedure: raw extracts from river water and sediments will, be subjected to RP-HPLC fractionation and individual fractions screened for toxic activity using the battery of small scale bioassays mentioned earlier. The identity of chemicals responsible for toxic activity in the most toxic fractions will be determined by a combination of high resolution GC/MS and LC/MS (TOF) analysis.

Experience of the group
The ecotoxicological group at NIVA has been active in research for the last 20 years and presently comprises 6 scientists and a technical staff of 6 people. This group has a broad expertise in ecotoxicology, marine and freshwater biology, cellular and molecular biology, and chemistry. Current activities of the group include traditional ecotoxicological tests (algae, crustaceans and fish), bioaccumulation in vertebrates and invertebrate, in vitro and small-scale bioassays, biomarkers, microbiology, and analytical chemistry. Within this area, large focus has been given environmental pollutants like metals and organic chemicals capable of producing both acute toxicity and sub-acute effects. This applies in particular to chemicals that are able to adversely affect the natural growth (and reproduction) of algae, crustaceans and fish and interfere with normal development and reproduction of fish (planare dioxin-like chemicals as PAHs, PCBs, dioxins, free-radical generators, metals, endocrine disruptors, mutagens and acetylcholineesterase inhibitors).
The use of small-scale and in vitro bioassays as well as in vivo and in vitro biomarkers have greatly facilitated high throughput testing and in-depth studies of ecologically relevant endpoints of effects and has consequently been used with success in many of NIVAs environmental monitoring programmes. Bioassays have been used in a range of applications and NIVA scientists are involved in national and international method development and quality assurance programmes. Biomarkers have become an increasingly important component of NIVAs ecotoxicological research and monitoring programmes. In recent years, methodological developments has also led to the use of small scale bioassays and various in vitro bioassays for screening of environmental samples. This applies in particular to bioassay-assisted environmental monitoring and use of bioassay-directed fractionation and chemical identification (TiE) procedures. The latter is based on a well functioning collaboration between ecotoxicologists and chemists at NIVA, leading to a viable combination of sample extraction (SPE and SPMDs) and fractionation, bioassay testing and HRGC/MS identification of unknown pollutants. Through an intimate collaboration with the Norwegian Institute for Air Research (NILU), NIVA has developed the capability to also perform HPLC/MS (TOF) analysis of polar compounds. All of these methods have been applied to various environmental compartments including studies on samples from fresh water, river water, seawater, sediments and industrial effluents. The group is presently active in development of biological and chemical methods for studies on the presence and the biological effects of both organic pollutants (PAHs, PCBs, dioxins, estrogen mimics, organotins) as well as effects of various metals (mercury, copper, cadmium, and lead).
NIVA is well equipped for performing the research activities proposed. The group has necessary infrastructure to perform effect-based studies including an algae and crustacean culture collection, marine and freshwater fish, a cell laboratory capable of running primary cultures and continuos cell lines. Instrumentation for biomarker studies include standard laboratory equipment (centrifuges, etc), a polarograph and microplate readers for fluorescence and absorbance. Instrumentation to perform bioassay-directed fractionation and chemical identification is well suited for the purpose and includes Solid phase (SPE) extraction apparatus, ultrasound-assisted soxhlet extraction, High Performance Liquid Chromatography (HPLC) equipped with a fraction collector, diode array detector and fluorescence detector, high resolution gas chromatography with high volume injectors (PTV) and a singel quadropole mass spectrometers (HRGC/MS). The laboratory is using different mass spectrometric libraries for identification of unknown compounds in the samples.
NIVA has been an active partner in international programmes and organisations, e.g. the EU-funded quality assurance programme BEQUALM (now continued as a self-funding activity). With collaborating institutes, NIVA covers most relevant biomarker analyses. NIVA has close collaboration with the University of Stockholm (DNA adducts, steroid analyses), NIFES (antioxidant responses), the University of Gothenburg (various methods), the University of Trondheim (thyroid hormones), the Norwegian Institute for Public Health (milt quality), the University of the Basque Country (histopathology, peroxisomal responses) and Biosense Laboratories as (development and use of immunochemical methods).

Professor/Dr. scient Ketil Hylland is a senior researcher at the section for “Ecotoxicology and Risk Assessment” at NIVA and is presently an associate professor in ecotoxicology at the Department of Biology, University of Oslo. He has extensive experience in the development and use of biological effect methods in both freshwater and marine environments including most of the methods proposed. Hylland has co-ordinated several national and international projects related to environmental impact of organic and inorganic pollutants in freshwater and seawater ecosystem. He is the current chair of the ICES working group on biological effects of contaminants (WGBEC) and is involved in harmonisation of biomarker techniques through the Self-funded BEQUALM programme. He has published over 30 papers in peer-reviewed literature and proceedings of international conferences and has an active role in supervision of diploma and master students at the University of Oslo.
Torsten Källqvist (M. Sc.) is a senior researcher and acting Research Manager for section of “Ecotoxicology and Risk Assessment” at NIVA. Källqvist is a microbiologist educated at the University of Gothenburg, Sweden and has been working with micropollutants effects on single species of algae and crustaceans to community studies. Källqvist has led several projects on ecotoxicological characterisation and risk assessment of industrial wastewater for the national pollution control authority. He takes part in the international development and standardisation of ecotoxicological test methods within ISO and OECD and is currently chairman of the ISO working group for test methods with algae and aquatic plants. He is also member of the Norwegian Pesticide Board. Källqvist has published close to 50 scientific papers in peer-reviewed literature and proceedings of international conferences and approximately 350 national reports.
Dr. scient Knut-Erik Tollefsen is a researcher at the section for “Ecotoxicology and Risk Assessment” at NIVA. He is in charge of in vitro bioassays and TiE work and assist the development of biomarker analysis for chemical screening and environmental monitoring. He has a broad experience in ecotoxicology and is currently working with different sampling and extraction methods, fish bioaccumulation, ecotoxicological and bioassays testing, immunoassay development and biomarker studies. He has published fourteen papers in peer-reviewed literature and proceedings of international conferences and has had an active role in supervision of master students at the University of Oslo.
Dr. scient Anders Ruus is a scientist at the section for environmental pollutants and has his main field of research within the study of the disposition and effects of organochlorine contaminants in marine food chains, on different levels. He has worked with invertebrates and fish, as well as sea birds and marine mammals. Ruus also has experience with toxicokinetic and bioaccumulation/distribution studies involving catching, caging and exposure systems for invertebrates and fish. His previous work has given him first-hand experience of many of the methods and analysis proposed.
Ph. D. Merete Grung is a senior researcher at the section for “Organic Analytical Chemistry” and the deputy Head of NIVAs Chemistry Department. She has extensive experience in the development and use of various analytical techniques using high-resolution GC/MS and HPLC/MS and is a major contributor to establishing a TiE task-force at NIVA. Grung has published around 15 papers in peer-reviewed literature and proceedings of international conferences.

Recent & relevant publications

Hylland, K., Haux, C., Hogstrand, C., 1995. Metallothionein in marine and freshwater fish: immunological characterization. Mar. Environ. Res. 39: 111-115.

Beyer, J., Sandvik, M., Hylland, K., Fjeld, E., Egaas, E., Aas, E., Skare, J.U., Goksoyr, A. 1996. Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L) exposed by caging to polluted sediments in Sorfjorden, Norway. Aquat. Toxicol. 36: 75-98.

Hylland, K., Sandvik, M:, Skare, J.U., Beyer, J., Egaas, E., Goksoyr, A. 1996. Biomarkers in flounder (Platichthys flesus): an evaluation of their use in pollution monitoring. Mar. Environ. Res. 42: 223-227.

Beyer, J., Sandvik, M., Skare, J.U., Egaas, E., Hylland, K., Waagbo, R., Goksoyr, A. 1997. Time- and dose-dependent biomarker responses in flounder (Platichthys flesus L.) exposed to benzo(a)pyrene, 2,3,3',4,4',5-hexachlorobiphenyl (PCB-156) and cadmium. Biomarkers 2: 35-44.

Sandvik, M., Beyer, J., Goksoyr, A., Hylland, K., Egaas, E., Skare, J.U. 1997. Interaction of benzo[a]pyrene, 2,3,3',4,4',5-hexachlorobiphenyl (PCB-156) and cadmium on biomarker responses in flounder (Platichthys flesus L.). Biomarkers, 2: 153-160.

Hylland, K., Haux, C. 1997. Effects of environmental oestrogens on marine fish species. Trends Analyt. Chem. 16: 606-612.

Knudsen, F.R., Schou, A., Wiborg, M.L., Mona, E., Tollefsen, K.E., Stenersen, J.V., Sumpter, J.P. 1997. Increase in plasma vitellogenin in rainbow trout (Oncorhynchus mykiss) exposed to effluents from oil refinery treatment works and municipial sewage. Bull. Environ. Contam. Toxicol. 59: 802-806

Hylland, K., Nissen-Lie, T., Christensen, P.G., Sandvik, M. 1998. Natural modulation of cytochrome P4501A and metallothionein in flounder, Platichthys flesus. Mar. Environ. Res. 46: 51-55.

Tollefsen, K-E., Ingebrigtsen, K., Olsen, A.J., Zachariassen, K.E., Johnsen, S. 1998. Acute toxicity and toxicokinetics of 4-heptylphenol in juvenile Atlantic cod (Gadus morhua L.) Environ. Toxicol. Chem. 17: 740-746.

Berntssen, M., Hylland, K., Wendelaar Bonga, S., Maage, A., 1999. Accumulation of Cu i Atlantic salmon following exposure to Cu through diet. Aquat. Toxicol. 46: 87-99.

Nas, K., Hylland, K., Oug, E., Förlin, L., Ericson, G. 1999. Accumulation and effects of aluminium-smelter generated PAHs in soft bottom invertebrates and fish. Environ.Contam.Toxicol. 18: 2205-2216

Hylland, K. 1999. Biological effects of contaminants: quantification of metallothionein in fish. ICES Tech. Mar. Environ. Sci. (TIMES), No. 26, 18 p.

Berntssen, M.H.G., Aspholm, O.O., Hylland, K., Wendelaar Bonga, S.E., Lundebye, A.-K., 2001. Tissue metallothionein, apoptosis and cell proliferation responses in Atlantic salmon (Salmo salar L.) parr fed elevated cadmium. Comp. Biochem. Physiol., 128C: 299-310.

Ruus A, Skaare JU, Ingebrigtsen K. 2001. Disposition and depuration of lindane (?-HCH) and polychlorinated biphenyl-110 (2,3,3’,4’,6-pentachlorobiphenyl) in cod (Gadus morhua) and bullrout (Myoxocephalus scorpius) after single oral exposures. Environ. Toxicol. Chem. 20: 2377-2382.

Scott, A.P., Hylland, K. 2002. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques for the measurement of marine fish vitellogenins. ICES Tech. Mar. Environ. Sci. (TIMES), No. 31, 21 p.

Ruus, A., Sandvik, M., Ugland, K.I, Skaare, J.U. 2002. Factors influencing activities of biotransformation enzymes, concentrations and compositional patterns of organochlorine contaminants in members of a marine food web. Aquat. Toxicol. 61: 73-87.

Ruus, A., Ugland, K.I., Skaare, J.U. 2002. Influence of trophic position on organochlorine concentrations and compositional patterns in a marine food web. Environ. Toxicol. Chem. 21: 2356-2364.

Tollefsen, K.-E. 2002. Interaction of estrogen mimics, singly and in combination, with plasma sex steroid-binding proteins in rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 56: 215-22.

Tollefsen, K.-E., Mathisen, R., Stenersen, J. 2002. Estrogen mimics bind with similar affinity and specificity to the hepatic estrogen receptor in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 126: 14-22.

Tollefsen, K.-E., Meyes, J.F.A. Frydenlund, J., Stenersen, J. 2002. Environmental estrogens interact with and modulate the properties of plasma sex steroid-binding proteins in Atlantic salmon (Salmo salar). Mar. Environ. Res. 54: 697-701.

Källqvist, T. and Svenson, A. 2003. Assessment of ammonia toxicity in tests with the microalga, Nephroselmis pyriformis, Chlorophyta. Water Research 37: 477-484.

Tollefsen, K.-E., Mathisen, R., Stenersen, J. 2003. Induction of vitellogenin synthesis in an Atlantic salmon (Salmo salar) hepatocyte culture: a sensitive in vitro bioassay for (anti)estrogenic activity of chemicals. Biomarkers. (In press)