Contaminants and effects

Prepared by: Dr. Rune Dietz and Dr. Christian Sonne, PBSG members

Contaminants in Arctic biota - a clear proof of long-range transport

As Inuits are dependant on food from the Arctic marine biota and these have shown high loads of contaminant, this field has been extensively studies of contaminant loads and effect of these. Heavy metals are often studied in muscle, liver and kidney and for mercury (Hg) as well in hair and blood. Persistent organic pollutants (POPs) are generally studied in adipose tissue or blood. Effect studies on live polar bears are restricted to external examination and blood, whereas access to polar bear from the traditional Inuit hunt allows for examination of sexual organs, internal organs and bone composition as we will see below.

Age determination

Both contaminant studies and effect studies are very dependant of a precise age determination. The ages of the bears were estimated by counting annual layering in decalcified thin sections (14 μm) stained in toluidine blue in the cementum of the canine or premolar tooth, as described by Dietz et al. (1991).

Heavy metals

Ancient polar bear hides from the Candian Museum of Nature has been investigated for mercury to reveal the man-made contribution to present levelsImage: Rune Dietz

Mercury analyses were performed using hydride generation and the amalgam technique, as described by e.g. Dietz et al. (1995, 2000, 2006). Cadmium in tissue samples are in general analysed using a flame atomic absorption spectrophotometer (AAS, Perkin-Elmer 3030), or the graphite furnace technique Perkin-Elmer 3030 with Zeeman background correction at lower concentrations This latter technique was also used for selenium analyses (Dietz et al. 2000). Zinc analyses were carried out by flame AAS Perkin-Elmer 3030. For screening of other element the ICPMS technique can be used.

Persistent Organic Pollutants

Blood from from tagged polar bears is an important media to study the effects of contaminantsImage: Rune Dietz

Analysis of POPs (organochlorines and brominated flame retardants) analysis in adipose and blood plasma of polar bears has been investigated according to methods described in detail by e.g. Dietz et al., 2004, 2007, Gebbink et al., 2008a, 2008b, Muir et al., 2006, Verreault et al., 2005, 2008 (see summary of results in contaminant section). A wide variety of contaminants are often analysed including: PCBs; up to 61 congeners, OCs; 1,2,4,5-tetrachlorobenzene (TeClBz), pentachlorobenzene (PnClBz), heaxchlorobenzene (HCB), α-hexachlorocyclohexane (HCH), β-HCH, γ-HCH, octachlorostyrene (OCS), the chlordanes and metabolites heptachlor epoxide, oxychlordane, trans-chlordane, cis-chlordane, trans-nonachlor, cis-nonachlor, p,p'-DDE, p,p'-DDD, p,p'-DDT, dieldrin, photomirex, mirex and tris(4-chlorophenyl)methane (TCPM). Also in fat, 35 PBDEs are being monitored for (BDE-17, -25, -28, -54, -47, -49, -66, -77, -100, -119, -99, -116, -85, -155, -154, -153, -139, -138, -140, -180, -183, -184, -191, -187, -190, -171, -202, -201, -197, -203, -196, -207, -208, -206 and -209), as well as several other BFRs including 2,2,’,4,4’,5-pentabromobiphenyl (BB-101), 2,2’,4,4’,5,5’-hexaBB (BB-153), pentabromotoluene (PBT), hexabromobenzene (HBB), total-(α)-hexabromocyclododecane (HBCD), bis(2,4,6-tribromophenoxy)ethane (BTBPE) and pentabromoethylbenzene (PBEB). In other investigations the metabolites has been investigated as these may be more toxic to the polar bears (e.g. Verreault et al. 2005, Gebbink et al. 2008a, 2008b). Little information is available on polar bears, Dioxins, Furans and non-ortho PCBs (for methods see Riget et al. 2005).

Perfluoroalkyl contaminants (PFCs)

PFCs is in general investigated in the liver using the extraction method based on ion pairing as described by e.g. Bossi et al. (2005a, 2005b), Smithwick et al. (2005a, 2005b) and Dietz et al (200). 13C2-PFDA and 13C4-PFOS were used as surrogate standards. Instrumental analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with electrospray ionization (ESI). The extracts (20 μL injection volume) were chromatographed on a C18 Betasil column (2.1   50 mm, Thermo Hypesil-Keystone, Bellafonte, PA) using an Agilent 1100 Series HPLC (Agilent Technologies, Palo Alto, CA). The HPLC is often interfaced to a triple quadrupole API 2000 (Sciex, Concorde, Ontario, Canada) equipped with a TurboIon Spray source operating in negative ion mode. Instrument set-up, quality assurance and calibration procedures as well as the standards and reagents used are described in detail by Bossi et al. (2005).

Effects of contaminants on polar bear brains

Sections of polar bear brains examined for biomarkers to reveal possible effects of mercury, POPs and PFCsImage: Rune Dietz

Recently effort has been addressed towards investigating the effects of contaminants on brain biomarkers in polar bear such as N-methyl-D-aspartate (NMDA), dopamine-2, gamma-aminobutyric acid type A, muscarinic cholinergic, and nicotinic cholinergic receptors; cholinesterase and monoamine oxidase enzymes). Similar studies have revealed negative effects on other wildlife species with high Hg burdens (Basu et al 2005a, 2005b, 2009).

Osteodensitometry - a measure of osteoporosis

An example from the bone mineral DXA scanning of a polar bear skullImage: Rune Dietz

X-ray osteodensitometry is applied to detect osteopenia (osteoporosis) by use of a Norland XR 26 X-ray bone densitometer which determines the bone mineral density (calcium-phosphate; hydroxyapatite) during a dual X-ray absorptiometry (DXA). Skulls are scanned in Research mode (speed: 60 mm/sec; resolution: 3.0 x 3.0 mm; width: 24.9 cm) and analysed in XR software revision 2.4®, which generate a picture of the bone segment and calculate bone mineral density of hydroxyapatite (BMD; g cm-2). To ensure that skull BMD represent the mineral status of the skeletal system in general, measurement of femur and vertebrae BMD is also conducted showing a high correlation. The DXA-scanner is daily calibrated using a phantom with known mineral density. In addition the precision is tested by a 10 time rescanning (mean=521.96 g cm-2, SD=0.60) which from the formula [1 – (SD/mean) x 100%] gives a precision of 99.88%. Fragmentation and loss of teeth material caused by handling and lead shot is not a problem.

Histology

Kidney hyperplasia has been detected in high frequencies in East Greenland polar bearsImage: Christian Sonne

Tissues are trimmed, processed conventionally, embedded in paraffin, sectioned at about 4 μm and stained with haematoxylineosin (HE) for routine diagnostics. Periodic acid-schiff (PAS) and periodic acid silver methenamine (PAS-M) are used to demonstrate glomerular (capillary and mesangial) and tubular changes; Van Gieson and Masson Trichrome to detect fibrous tissue (collagen) in the glomeruli (glomerulofibrosis) and in the interstitium (interstitial fibrosis); and Smorl and Perls’ prussian blue reaction to detect lipofuscin and haemosiderin pigments, respectively. Glomerular, tubular and interstitial lesions are evaluated semi-quantitatively by examinating the renal tissue in 10 randomly selected low to high power fields (5-40x magnification). Based on these observations, the lesions were graded into one of 3 groups (0: absent, 1: mild and 2: moderate).

Skull - size, assymetry and pathology

Polar bear brain biometric measurementsImage: Christian Sonne

In order to described size and growth, nine metric traits is measured on the right side of each skull using digital callipers (Mitutoyo, Mitutoyo Corporation, Japan) to the nearest 0.04 mm. Traits are measured twice on each skull, always by the same person, and never twice on the same day. Many skulls are and thus the entire range of measurements can not be performed on all skulls. The average of the two measurements were used in all further analyses ((measurement1right + measurement2right)/2). Fourteen meristic foramina traits are registered and defined by minimum inner diameter size, depending on skull site. Blunt metal pins are used to determine whether the size of the individual foramen fell within the predetermined accepted range. Observed foramina meeting the set criteria are counted on the right (R) and left (L) side of each skull. The foramina are only registered once per skull, except for a randomly chosen subsample of 61 skulls which is registered twice, in order to determine the measurement (registration) error. All registrations are conducted by the same person, and the subsample registration was done on a separate day from the rest. In addition, nine metric bilateral traits are measured in order to estimate the level of FA in the skulls. Each trait is measured on the right and left side of each skull, using digital callipers (Mitutoyo, Mitutoyo Corporation, Japan) to the nearest 0.04 mm. The traits are measured twice on each skull, always by the same person, and never twice on the same day. Skulls are examined for pathology and 7 different pathological changes are described (tooth wear, periodontitis including alveolar bone loss, adonti, tooth displacement, caries, osseous proliferations and exostosis). The prevalence of tooth breakage is often close to zero and therefore included as wear (it is often not possible to separate wear and breakage). However, only tooth wear and periodontitis prevalence often allow for quantitatively data analyses. Skulls are categorized into four groups according to the degree of tooth wear and periodontitis.

Sexual organs - size and morphology

Testes length and weight is recorded as an average of left and right testes before the tissue are trimmed, processed conventionally, embedded in paraffin, sectioned at about 4 μm and stained with Haematoxylin (Al-Haematein)-Eosin (HE) for routine diagnostics. In males, all slides are examinated consecutively from seminiferous tubules to the caput of epididymidis. The maximal diameter (µm) of 5 randomly chosen seminiferous and epididymal tubules is measured as well despite in individuals with immature closure and/or suboptimal fixation. The presence of elongated spermatids, spermatozoa and fibrosis/atrophy/hyalinisation/inflammation of seminiferous tubules and interstitium is recorded together with the sperm morphology in the individuals exhibiting accumulation of these in the epididymal. Penis and baculum are investigated macroscopically for gross pathology before recording of weight and length and X-ray osteodensitometry is also applied to all bacula for detecting osteoporosis. In females, dimensions of clitoris, uterine corpus (diameter) and each uterine horn (diameter at mid-point and length) and ovaries (length, weight) are measured. Data are given as the average values of the measurement of the left and right organs. Each ovary is sliced longitudinally in four sections. The number of follicles in four size categories (<1 mm; 1-2 mm; 2-3 mm and 3-6 mm) are counted macroscopically in each section. For summary statistics an average of the number from 4 longitudinal sections is used. The presence of corpus luteum is detected and histology is conducted as described above.

Further reading

Bancroft J.D., Stevens, A.,1996. Theory and Practice of Histological Techniques. Churchill Livingstone, New York.

Basu N, Klenavic K, Gamberg M, O'Brien M, Evans RD, Scheuhammer AM, Chan HM. 2005a. Effects of mercury on neurochemical receptor binding characteristics in wild mink. Environ Toxicol Chem 24:1444-1450.

Basu N, Scheuhammer AM, Grochowina NM, Klenavic K, Evans RD, O'Brien M, Chan HM. 2005b. Effects of mercury on neurochemical receptors in wild river otters (Lontra canadensis). Environ Sci Technol 39:3585-3591.

Basu, N., Scheuhammer, A.M., Sonne, C., Letcher, R.J., Born, E.W., Dietz, R. 2009. Is dietary mercury of neurotoxicological concern to wild polar bears (Ursus maritimus), Environmental Toxicology and Chemistry, vol. 28 nr. 1:133-140.

Bechshøft TØ, Rigét FF, Sonne C, Wiig Ø, Dietz R Letcher RJ. In press. Skull foramina asymmetry in East Greenland and Svalbard polar bears (Ursus maritimus). Annal Zool Fenn.

Bechshøft TØ, Rigét FF, Wiig Ø, Sonne C. Fluctuating asymmetry in metric traits; a practical example of calculating asymmetry, measurement error and repeatability. Annal Zool Fenn 2008b; 45:32-38.

Bechshøft TØ, Sonne C, Rigét FF, Wiig Ø, Dietz R. 2008c. Skull growth, size and sexual dimorphism in East Greenland and Svalbard polar bears (Ursus maritimus). Polar Biol 31:945-958.

Bechshøft TØ, Wiig Ø, Sonne C, Rigét FF, Dietz R, Letcher RJ, Muir DCG. Temporal and spatial variation in metric asymmetry in skulls of polar bears (Ursus maritimus) from East Greenland and Svalbard. Annal Zool Fenn 2008a; 45:15-31.

Bossi, R., F. F. Riget, R. Dietz, C. Sonne P. Fauser, M. Dam, & K. Vorkamp 2005a. Preliminary screening of Perfluoroctanate (PFOS) in fish, mammals and birds from Greenland and Faroe Islands. Environ Pollut 136(2005): 323-329.

Bossi, R., F.F. Riget, R. Dietz 2005b. Temporal and Spatial trends of Perflourinated Compounds in Ringed Seal (Phoca hispida) from Greenland. Environ Sci & Technol. 39: 7416-7422.

Dietz, R. F. Riget and E.W. Born 2000b. Geographical differences of zinc, cadmium, mercury and selenium in polar bears (Ursus maritimus) from Greenland. The Science of the Total Environment 245: 25-48.

Dietz, R., C. Overgaard Nielsen & M. Munk Hansen and C.T. Hansen 1990. Organic mercury in Greenland birds and mammals. The Science of the Total Environment 95: 41-51.

Dietz, R., E. W. Born, C.T. Agger & C.O. Nielsen 1995a. Zinc, cadmium, mercury, and selenium in polar bears (Ursus maritimus) from East Greenland. Polar Biology 15: 175-185.

Dietz, R., F. Riget, C. Sonne, D.C.G. Muir, S. Backus, E.W. Born M. Kirkegaard & R.J. Letcher 2007. Age and Seasonal Variability of Polybrominated Diphenyl Ethers in Free-Ranging East Greenland Polar Bears (Ursus maritimus). Environmental Pollution 146 (1): 177-184.

Dietz, R., F. Riget, E.W. Born, C. Sonne, P. Grandjean, M. Kirkegaard, M.T. Olsen, G. Asmund H. Baagøe & C. Andreasen 2006. Trends in Mercury in hair from Greenland Polar Bears (Ursus maritimus) during 1892-2001. Environmental Science & Technology 40(4): 1120-1125.

Dietz, R., F. Riget, E.W. Born, C. Sonne, P. Grandjean, M. Kirkegaard, M.T. Olsen, G. Asmund H. Baagøe & C. Andreasen 2006. Trends in Mercury in hair from Greenland Polar Bears (Ursus maritimus) during 1892-2001. Environmental Science & Technology 40(4): 1120-1125.

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Dietz, R., R. Bossi, F.F. Riget, C. Sonne & E.W. Born 2008. Increasing Perfluorinated Acids in East Greenland Polar Bears (Ursus maritimus) - a new toxic threat to the Arctic bears. Environ Sci & Tech 42: 2701-2707

Gebbink, W.A., C. Sonne, R. Dietz, M. Kirkegaard, E.W. Born, D.C.G. Muir & R.J. Letcher in 2008b. Target Tissue Selectivity and Burdens of Diverse Classes of Brominated and Chlorinated Contaminants in Polar Bears (Ursus maritimus) from East Greenland. Environ. Sci. Technol. 2008, 42: 752–759.

Gebbink, W.A., C. Sonne, R. Dietz, M. Kirkegaard, F.F. Riget, E.W. Born, D.C.G. Muir & R.J. Letcher 2008a. Tissue-Specific Congener Composition of Organohalogen and Metabolite Contaminants in East Greenland Polar Bears (Ursus maritimus). Environmental Pollution in press. Environmental Pollution 152: 621-629.

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Muir D.C.G, R.J. Norstrom, I. Stirling, A.T. Fisk, G.W. Gabrielsen, A.E. De¬rocher, T. Evans, R. Dietz, C. Sonne, G.M. Sandala, M.K. Taylor, J. Nagy and R.J. Letcher 2005. Chlorinated hydrocarbon contaminants and metabolites in polar bears (Ursus maritimus) from Svalbard, East Greenland, Alaska and the Canadian Arctic during 1999−2002. Sci. Total Environ. 351: 369-390 Sp. Iss. SI, DEC 1 2005

Muir, D.C.G., Backus, S., Derocher, A.E., Dietz, R., Evans, T.J., Gabrielsen, G.W., Nagy, J., Norstrom, R.J., Sonne, C., Stirling, I., Taylor, M.K., Letcher, R.J. 2006. Brominated Flame Retardants in Polar Bears (Ursus maritimus) from Alaska, the Canadian Arctic, East Greenland, and Svalbard. Environ. Sci. Technol. 40(2); 449-455.

Muir, D.C.G., Backus, S., Derocher, A.E., Dietz, R., Evans, T.J., Gabrielsen, G.W., Nagy, J., Norstrom, R.J., Sonne, C., Stirling, I., Taylor, M.K., Letcher, R.J. 2006. Brominated Flame Retardants in Polar Bears (Ursus maritimus) from Alaska, the Canadian Arctic, East Greenland, and Svalbard. Environ. Sci. Technol. 40(2); 449-455.

Riget, F., J. Vikelsøe, R. Dietz 2005. Levels and Temporal Trends of Dioxins, Furans and non-ortho PCBs in Ringed seals from East Greenland. Marine Pollution Bulletin: 1523-1529

Sandala, G. M., C. Sonne, R. Dietz, D. C. G. Muir, K. Valters, E. R. Bennett, E. W. Born and R. J. Letcher 2004. Hydroxylated and Methyl Sulfone PCB Metabolites in Adipose and Whole Blood of Polar Bear (Ursus maritimus) From East Greenland. The Science of the Total Environment 331(1-3): 125-141

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Smithwick M., D.C.G. Muir, S. A. Mabury, K. Solomon, J.W. Martin, C. Sonne, , E.W. Born, R. Dietz, A.E. Derocher, Tom Evans, G.W. Gabrielsen, J. Nagy, I. Stirling and M.K. Taylor 2005b. A Circumpolar Study of Perfluoroalkyl Contaminants in Polar Bear Hepatic Tissue (Ursus maritimus). Environ Sci & Technol. 39: 5517-5523.

Smithwick M., D.C.G. Muir, Scott A. Mabury, K. Solomon, J.W. Martin, C. Sonne, E.W. Born, R. Letcher, R. Dietz 2005a: Perfluorinated acids in hepatic tissue from East Greenland polar bears (Ursus maritimus) 1999-2001. Environ Toxicol and Chem 24(4): 981-986

Sonne C, Bossi R, Dietz R, Leifsson PS, Rigét FF, Born EW. The Potential Correlation between Perfluorinated Acids and Liver Morphology in East Greenland Polar Bears (Ursus maritimus). Toxicol Environ Chem 2008a ; 90:275-283.

Sonne C, Dietz R, Born EW, Rigét FF, Kirkegaard M, Hyld¬strup L, Letcher RJ, Muir DCG. Is bone mineral composition disrupted by organo¬chlorines in East Greenland polar bears (Ursus mari¬timus)? Environ Health Perspect 2004; 112:1711-1716.

Sonne C, Dietz R, Born EW, Rigét FF, Leifsson PS, Bechshøft TØ, Kirkegaard M. Spatial and temporal variation in size of polar bear (Ursus maritimus) sexual organs and its use in pollution and climate change studies. Sci Total Environ 2007b; 387:237-246.

Sonne C, Dietz R, Leifsson PS, Asmund G, Born EW, Kirkegaard M. Are liver and renal lesions in East Greenland Polar Bears (Ursus maritimus) associated with high mercury levels? Environ Health 2007; 6:11.

Sonne C, Dietz R, Leifsson PS, Born EW, Kirkegaard M, Letcher RJ, Muir DCG, Rigét FF, Hyldstrup L. Are organohalogen contaminants a co-factor in the development of renal lesions in East Green¬land polar bears (Ursus maritimus)? Environ Toxicol Chem 2006b; 25:1551-1557. 

Sonne C, Dietz R, Leifsson PS, Born EW, Kirkegaard M, Rigét FF, Letcher RJ, Muir DCG, Hyldstrup L. Do Organohalogen Contaminants Contribute to Liver Histopathology in East Greenland Polar Bears (Ursus maritimus)? Environ Health Perspect 2005a; 113:1569-1574.

Sonne C, Leifsson PS, Dietz R, Born EW, Letcher RJ, Hyldstrup L, Rigét FF, Kirkegaard M, Muir DCG. Xenoendocrine Pollutants May Reduce Size of Sexual Organs in East Greenland Polar Bears (Ursus maritimus). Environ Sci Technol 2006a; 40:5668-5674.

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Sonne C, Rigét FF, Dietz R, Wiig Ø, Kirkegaard M, Born EW. Gross Skull Pathology in East Greenland and Svalbard Polar Bears (Ursus maritimus) during 1892 to 2002 in Relation to Organohalogen Pollution. Sci Total Environ 2007e; 372:554-561.

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