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Liver and renal histopathology ofNorth Atlantic long-finned pilot whales(Globicephala melas) contaminatedwith heavy metals and organochlorinecompoundsChristian Sonne a , Maria Dam b , Pall S. Leifsson c & Rune Dietz aa Department of Arctic Environment, National EnvironmentalResearch Institute, Section for Contaminants, Effects and MarineMammals, Aarhus University, Frederiksborgvej 399, P.O. Box 358,DK-4000 Roskilde, Denmarkb Environment Agency, Research, Traðagøta 38, P.O. Box 2048,FO-165 Argir, Faroe Islandsc Department of Disease Biology, Faculty of Life Sciences,University of Copenhagen, Bülowsvej 17, DK-1870, Frederiksberg,Denmark
Version of record first published: 30 Apr 2010.
To cite this article: Christian Sonne, Maria Dam, Pall S. Leifsson & Rune Dietz (2010): Liver andrenal histopathology of North Atlantic long-finned pilot whales (Globicephala melas) contaminatedwith heavy metals and organochlorine compounds, Toxicological & Environmental Chemistry, 92:5,969-985
To link to this article: http://dx.doi.org/10.1080/02772240903187221
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Toxicological & Environmental ChemistryVol. 92, No. 5, May 2010, 969–985
Liver and renal histopathology of North Atlantic long-finned pilot
whales (Globicephala melas) contaminated with heavy metals and
organochlorine compounds
Christian Sonnea*, Maria Damb, Pall S. Leifssonc and Rune Dietza
aDepartment of Arctic Environment, National Environmental Research Institute,Section for Contaminants, Effects and Marine Mammals, Aarhus University,Frederiksborgvej 399, P.O. Box 358, DK-4000 Roskilde, Denmark; bEnvironment Agency,Research, Tra�agøta 38, P.O. Box 2048, FO-165 Argir, Faroe Islands; cDepartment ofDisease Biology, Faculty of Life Sciences, University of Copenhagen, Bulowsvej 17,DK-1870, Frederiksberg, Denmark
(Received 6 June 2009; final version received 7 June 2009)
Long-finned pilot whales (Globicephala melas) from the Faroe Islandsare known to be heavily polluted with contaminants, such as mercury (Hg)and organochlorine compounds (OC). This is postulated to exert adversehealth effects on whales as well as the human population who rely onits meat and blubber as food sources. It was therefore decided to conduct ascreening pilot study to determine contaminant concentrations andhistopathology of liver and renal tissues in a total of 14 specimens fromthis subpopulation. In blubber, the mean
POC concentration was
31,887 ng g�1 lw (range: 18,170–47,425 ng g�1 lw) of whichP
PCB concen-tration was 23,416 ng g�1 lw (range: 13,947–34,543 ng g�1 lw; n¼ 3). In liver,mean Hg concentration was 138mg�1 ww (range: 54–351 mg g�1ww; n¼ 7)and mean cadmium (Cd) concentration 15mg g�1ww (range: 7–31mg g�1 ww; n¼ 7). In the kidney, the mean Cd concentration was36mg g�1 ww (range: 23–47mg g�1ww; n¼ 7). Of the liver Hg concentra-tions, two were at the suggested toxic threshold levels of 60 mg g�1 ww andfive were 2–6-fold above. Liver selenium (Se) :Hg was on average 1.11(range: 0.97–1.41; n¼ 7) indicating that Se was in excess, which decreasedthe risk of acute Hg poisoning. Histopathological examinations showedhigh prevalence (435%) of renal glomerular arteriosclerosis, glomerularcapillary dilatation, dilatation and hyalinization of Bowman’s space/capsuleand tubular hyaline casts. In liver tissue, high prevalence was found forportal cell infiltrates, lipid granulomas, hepatocytic lipid accumulation, bileduct proliferation, lipid-filled Ito cells, and focal necrosis. In a single juvenilemale, 4 of 8 renal and 3 of 7 liver lesions were present. Cadmiumconcentrations increased significantly in the presence of glomerular arterio-sclerosis and a similar trend was found for tubular hyaline casts. Based onthese findings, as well as the nature of the lesions, data indicate that thehistopathological changes were a result of age and that contaminants arelikely to be the co-factors in the development in at least three renal and fourliver lesions.
*Corresponding author. Email: [email protected]
ISSN 0277–2248 print/ISSN 1029–0486 online
� 2010 Taylor & Francis
DOI: 10.1080/02772240903187221
http://www.informaworld.com
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Keywords: cadmium; chlordane; DDTs; Faroe Island; Globicephala melas;�-HCH; HCB; histopathology; kidney; liver; long-finned pilot whale;mercury; Mirex; PCB; toxaphene
Introduction
The heavy metals mercury (Hg) and cadmium (Cd) produce adverse effects in variousmammals (MacLachlan and Cullen 1995; Merrill, Morton, and Soileau 2001;Ratcliffe, Swanson, and Fischer 1996; Sweet and Zelikoff 2001). Liver and renaltissues accumulate, depending on exposure route and chemical composition, highconcentrations of either inorganic or organic forms of these metals (Endo et al. 2005).In liver and renal tissues, inorganic Hg toxicity is mediated through a variety ofcytosolic pathways including the high affinity to a variety of microsome andmitochondria enzymatic SH-groups (co-enzyme inhibitor) (Goyer and Clarkson2001). In the case of Hg, environmental concentrations have been increasing due toanthropogenic inputs dramatically in wildlife during the past 100 years (Dietz et al.2006). If this rise continues it is likely that Se (selenium):Hg ratio becomes51 andthereby exerts even greater adverse effects than observed earlier (Ralston, Blackwell,and Raymond 2007). In addition, organic neuro-endocrine pollutants such aspolyfluorinated compounds (PFC) and polybrominated diphenyl ethers (PBDE) havebeen increasing over recent decades, whereas most of the conventional organochlorinecompounds (OC) have stabilized (AMAP 2004; Dietz et al. 2004, 2007, 2008;Meironyte, Noren, and Bergman 1999). In wildlife and humans, it is often the case thatthese individuals are contaminated with a mixture of heavy metals and organicpollutants such as OC (Dietz et al. 2004, 2006, 2007, 2008; Petersen et al. 2008), soseparating heavy metal and OC contaminant toxicity is not always possible whenassessing environmental toxicology. Despite the fact that Hg is a naturally occurringelement, inorganic Hg liver and renal toxicity has been described in wildlife (Laveryet al. 2009; Nichols, Bradbury, and Swartout 1999; Rawson et al. 1993; Rosa et al.2008; Woshner et al. 2002) and humans (Goyer and Clarkson 2001; Merrill, Morton,and Soileau 2001; Sweet and Zelikoff 2001). However, Se at a certain level is known toprotect against Hg toxicity and the same is the case for metallothionein which is acysteine (sulfur)-rich low molecular weight intra-cellular protein that binds metalssuch as Zn, Cu, Hg, and Cd (Felley-Bosco and Diezi 1992; Ohta and Cherian 1995;WHO1992). Similar relationships betweenOC and histopathology were established inmarine wildlife species, such as harbor seals, Baltic seal species, and polar bears(Bergman et al. 2001; Neale et al. 2005; Sonne et al. 2005, 2006, 2007a, 2008a) as well asmodel species such as sledge dogs and arctic foxes (Sonne et al. 2007b, 2008b, c).
At the Faroe Islands, the long-finned pilot whale is known to be heavilycontaminated with heavy metals and OC and is, together with the killer whale(Orcinus orca), one of the most polluted species in the world (AMAP 2004, 2005;Dam and Bloch 2000). Due to these circumstances this species is at a risk of sufferingfrom health impact on internal organs, such as the liver and kidneys. Furthermore,the local Faroe Island human population relies on pilot whales as an aboriginal andimportant wildlife food resource, which make it a key species from a wildlife andhuman health perspective. It was therefore decided to initiate a pilot study ofhistopathology on formaldehyde preserved liver and renal tissues and concomitantcontaminant concentrations in internal organs and blubber of 14 hunted and killedpilot whales caught in July 2007.
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Materials and methods
Sampling
All samples were taken by scientists in Torshavn at the Faroe Islands (62�N, 6�500W)on July 3, 2007 (Figure 1). A randomly chosen single renal lobe and tissue fromthe periphery of a right liver lobe were obtained for histological examination andfixed in a phosphate buffered formaldehyde/alcohol solution (3.5% formaldehyde,96.5% H2O). In addition, tissue samples for metal and persistent organic pollutantsanalyses were collected and stored frozen (ca �20�C) in separate polyethylene(Minigrip�) bags until subsampling for chemical analyses. All samples were takenapproximately 4 h post mortem.
Age estimation
Age estimation from cement growth layer groups (GLG) was not conducted.Individuals were categorized into adult females (AF), adult males (AM), and
Figure 1. Location of Torshavn (62�N, 6�500W) where renal and liver samples were takenfrom 14 Faroe Island Mink whales on July 3, 2007.
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a juvenile male (JM) according to the length and maturity of sexual organs asdescribed by Dam and Bloch (2000).
Histopathology
The liver tissue was trimmed, processed conventionally, embedded in paraffin,sectioned at about 4 mm, and stained with hematoxylin-eosin (HE) for routinediagnostics (Bancroft and Stevens 1996; Lyon et al. 1991). Kidney tissue wastrimmed, processed conventionally, embedded in paraffin, sectioned at about 4 mm,and stained with periodic acid–Schiff (PAS) to demonstrate glomerular (capillaryand mesangial), tubular, and interstitial changes (Bancroft and Stevens 1996; Lyonet al. 1991). All slides were evaluated in low (50�) to high (400�) power fields in aLeica DMLB microscope. Seven histological liver changes were found: (1) portal cellinfiltrates, (2) lipid granulomas, (3) bile duct proliferation, (4) portal fibrosis,(5) hepatocytic lipid accumulation, (6) lipid-filled Ito cells, and (7) focal necrosis.These histological changes were grouped as either absent or present. Eight renalhistopathological changes were found: (1) glomerular arteriosclerosis, (2) glomerularcapillary dilatation, (3) glomerular capillary thickening, (4) glomerular sclerosis,(5) dilatation and hyalinization of Bowman’s space and capsule, (6) interstitiallymphocyte infiltration, (7) tubular hyaline casts, and (8) tubular hyalinization,atrophy, necrosis, and interstitial fibrosis. The changes were categorized as eitherabsent or present.
Analyses of heavy metals
Mercury and Cd in liver and kidney were analyzed at the lab of Food and VeterinaryAgency of the Faroe Islands. Cadmium was analyzed with atom absorptionspectrophotometry using flame (Perkin Elmer 2380; m. ISO 11047 led, 5961(2)2nd ed.). Mercury was analyzed with the FIMS 400 (Mercury analysis system;Atomic spec. 94V.15 No.4, mod.). The lab is accredited (DANAC reg. no. 303)to the performed analyses of Hg and Cd. Selenium was analyzed at the Centrefor Toxicology in Quebec, Canada using ICP-MS following sample digestion withconcentrated nitric acid. The metal analyses were part of another program withdifferent sample selection criteria than the present pilot study, and thus onlysix specimens in the present study were analyzed so far for Cd, Hg, and Se.
Analyses of organochlorine compounds
Persistent organic pollutants were analyzed at the Centre for Toxicology in Quebec,Canada. The compounds quantified were 15 congeners of PCB (nos. 28, 52, 99, 101,105, 118, 128, 138, 153, 156, 163, 170, 180, 183, and 187), p,p-DDE and p,p-DDT,5 congeners of toxaphene (Parlar nos. 26, 32, 50, 62, and 69), 5 compounds of thechlordane family (�- and �-chlordane, cis- and trans-nonachlor, and oxychlordane)as well as Mirex, hexachlorobenzene and �-HCH. All compounds were analyzedusing gas chromatography with mass spectrometry (GC Agilent #6890, MS Agilent5973 Network; E-448). The lab participates in the inter-lab comparison programsNorthern Contaminant Program and QUASIMEME. The OC analyses were part ofanother program with different sample selection criteria from a larger sample size
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than the present pilot study. Thus, only three specimens in the present study wereanalyzed so far.
Statistical analyses
The statistical analyses were performed with the SAS statistical software package(SAS V9 and enterprise guide V4) and the level of significance was set at p� 0.05while 0.055p50.1 was considered a trend. Data were log-transformed (base e) priorto the analyses in order to meet the assumption of normality and homogeneity ofthe variance (Zar 1984). Kruskal–Wallis test was performed to test for differencesin the mean contaminant concentrations between age/gender groups, between tissues(liver, kidney), and between groups of histopathology (present vs. not present).Then, in order to test for the relationships between length and contaminant concen-trations, Pearson’s correlation analyses were applied. Finally, chi-square tests wereperformed to test for histopathology prevalence (present vs. not present) betweenage/gender groups.
Results
Contaminant concentrations
Metal concentrations were measured in four adult males and three adult females andas such the sample was relatively homogenous (Table 1). Despite the size differencesbetween males and females, the length versus metal concentration relationships wasnot significant. Renal Cd concentrations were in general low but significantly higherthan the corresponding liver tissue concentrations (Table 1). The mean liver Cdconcentration was 15 mg g�1 ww (range: 7–31 mg g�1 ww; n¼ 7) while mean kidney Cdconcentration of 36 mg g�1 ww (range: 23–47 mg g�1 ww; n¼ 7) were in the lower endof the range of potential dysfunction for liver (20–200 mg g�1 ww) and kidney (50–400 mg g�1 ww). However, liver Hg concentrations were considerably higher and fiveindividuals were 2–6-fold above the suggested toxic threshold levels of 60 mg g�1 wwfor marine mammals. Only Cd in liver tissue showed gender differences with adultfemales being significantly higher than adult males. Regarding the Se:Hgmolar ratio itwas on average 1.11 (range: 0.97–1.41) and only in individual #24 the Se :Hg of 0.97indicating that Hg on a molar basis was in excess relative to Se, which decreased therisk of acute Hg poisoning. Organochlorine concentrations were measuredin only three individuals (two adult females and one juvenile male; Table 1).In general the concentrations were increasing in the order �-HCH5Mirex5HCB5chlordane5toxaphene5DDT5PCB. The analyses showed that for all PCB,chlordane, DDT, Mirex, and toxaphene, the juvenile male was up to 3-fold higher thanadult females. In the case of �-HCH and HCB the juvenile male concentrations wereintermediate (Table 1).
Renal lesions
Renal lesions were categorized as glomerular, tubular, and interstitial (Figure 2,Table 2). Glomerular arteriosclerosis, glomerular capillary dilatation, and diffuse cap-illary thickening (similar to membranous glomerulonephritis) were found in 44–50,55–75, and 25–44% of adult pilot whales, respectively (Figure 3a, Table 2).
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Table
1.MetalandOHC
concentrationsin
liver,renal,andblubber
tissues
from
10FaroeIslandpilotwhalessampledin
June2007.
Idno
Age/
sex
Length
(cm)
Pregnant
Liver
(mgg�1ww)
Kidney
(mgg�1ww)
Blubber
(ngg�1lw)
Selenium
Cadmium*
Mercury
Cadmium**
�PCB
�CHL
�DDT
�-H
CH
HCB
Mirex
�TOX
4AF
480
Yes
58
24
128
36
7AF
390
Yes
13947
712
2510
4140
110
748
8AF
380
No
21758
1763
3650
21
430
100
2342
11
JM475
34543
2433
6920
19
260
150
3100
13
AF
455
Yes
63
13
139
42
14
AF
435
Yes
150
31
351
36
15
AM
505
30
11
54
23
20
AM
560
50
12
126
47
24
AM
550
42
7110
39
25
AM
540
30
957
32
Notes:CHL:chlordanes.TOX:Toxaphenes.AF:adultfemale;JM
:juvenilemale;andAM:adultmale.
*Significantlyhighestin
adultfemaleswhen
comparedto
adultmales(K
ruskal–Wallis:p¼0.03);
**Significantlyhighestin
kidney
when
comparedto
liver
(Kruskal–Wallis:p¼0.004).
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From Figure 3a, it is also seen that glomeruli displayed dilatation and hyalinizationof Bowman’s space and capsule in 67–75% of the individuals (Figure 3a, Table 2).Glomerular sclerosis, including dense PAS-positive total fibrous obliteration ofthe glomeruli, was found in 22–25% of the adult females and males, respectively,but not in juvenile individuals. This effect was independent of the occurrence ofinterstitial fibrosis (Figure 3c, Table 2). Regarding interstitial lymphocyteinfiltrations, these were fibrotic in nature and thereby induced tubular
Table 2. Prevalences (%) of renal and liver lesions in 14 Faroe Island pilot whales sampled inJune 2007.
Organ lesionJuvenilemale
Adultfemales(n¼ 9)
Adultmales(n¼ 4)
All(n¼ 14)
KidneyGlomerular arteriosclerosis 0 44 50 43Glomerular capillary dilatation 0 55 75 57Glomerular capillary thickening 0 44 25 36Glomerular sclerosis 0 22 25 21Dilatation and hyalinization of Bowman’sspace and capsule
100 67 75 71
Interstitial lymphocyte infiltration 100 11 0 14Tubular hyaline casts 100 22 50 36Tubular hyalinization, atrophy, necrosisand interstitial fibrosis
100 11 25 21
LiverPortal cell infiltrates* 100 44 100 64Portal fibrosis 0 33 25 29Bile duct proliferation 0 44 25 36Focal necrosis 0 67 50 57Lipid-filled Ito cells 100 44 50 50Hepatocytic lipid accumulation 100 33 75 50Lipid granulomas 0 44 25 36
Notes: *Significantly highest prevalence in adult males (chi-square: p¼ 0.04).
01020304050607080
Glom
erular
arte
riosc
lerosis
Glom
erular
capilla
ry d
ilata
tion
Glom
erular
capilla
ry th
icken
ing
Glom
erular
scler
osis
Dilat./
hyalin
. of B
owman
's sp
ace/c
apsu
le
Inte
rstit
ial ly
mphocy
te in
filtra
tion
Tubular h
yalin
cast
s
Tubular h
yalin
., atro
phy and n
ecro
sis
Portal c
ell in
filtra
tes
Lipid
gra
nulom
as
Bile d
uct p
rolif
erat
ion
Portal fi
brosis
Hepat
ocyt li
pid ac
cum
ulatio
n
Lipid
-fille
d Ito ce
lls
Necro
sis
Figure 2. Prevalence (%) of renal and liver lesions in 14 Faroe Island pilot whales sampled inJune 2007.
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hyalinization, atrophy, and necrosis (Figure 3b). The prevalence of corticalmononuclear cell infiltration was recorded in 11% of adult females but not males.Tubular cylindrical hyaline casts (protein) were found in the medulla of 22% ofthe adult females and 50% of the adult males and indicated protein loss. PAS-positive hyalinization of the tubular basement membrane accompanied by tubulardilatation, atrophy, necrosis, and interstitial fibrosis was found in 11–25% of theadult animals (Table 2). Surprisingly, changes in Bowman’s space and capsule,interstitial lymphocyte infiltration, tubular changes, and hyaline casts as well asinterstitial fibrosis were also found in the juvenile male indicating that thesechanges were not age-related. The other effects were lacking in this juvenile male.Furthermore, there was no significant differences in the prevalence of renal lesionsbetween adult females and adult males.
Liver lesions
Liver changes could be divided into parenchymal and portal (Figures 2 and 4,Table 2). Portal cell infiltrates, fibrosis, and bile duct proliferation were found in
Figure 3. Renal histopathology. (a) Left glomeruli show arteriosclerosis (AS) and glomerularcapillary dilatation and thickening (arrows) while right glomeruli show dilatation andhyalinization of Bowman’s space and capsule (arrowheads) in an adult female long-finnedpilot whale. Periodic acid–Schiff staining, magnification� 20, bar¼ 50mm. (b) Tubularhyalinization, atrophy, necrosis and interstitial fibrosis (arrows) in an juvenile male long-finned pilot whale. Periodic acid–Schiff staining, magnification� 20, bar¼ 50mm. (c)Glomerular sclerosis (arrows) and interstitial lymphocyte infiltration (arrow) in an adultfemale long-finned pilot whale. Periodic acid–Schiff staining, magnification�20, bar¼ 50 mm.
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25–100% of the individuals depending on gender (Figure 4a). Focal necrosis wasfound in 50–67% of the individuals and different stages are seen in Figure 4b. Visibleand lipid-filled Ito cells, located in the narrow space of Disse between hepatocytes,was found in 44–50% of the individuals and seemed to be located preferablycentroacinary. Furthermore, 33–75% of the whales showed hepatocytic foamycytoplasm (lipids) in the periacinary zones 2 and 3. Finally, granulomas were foundin 25–44% of the adult whales (Figure 4c, Table 2). Similarly, the juvenile maleexhibited lesions. This time including portal cell infiltrates, hepatocyte lipidaccumulation, and lipid-filled Ito cells indicating that environmental factors wereco-factors in the development of these lesions. Furthermore, portal cell infiltrateswere the only liver lesions that showed gender differences with prevalence beinghighest in males.
Contaminant concentrations and histological lesions
In general it was hard to evaluate the relationship between prevalence ofhistopathological changes and contaminant concentrations due to the low samplesize not the least for OC analyses (n¼ 3) (Table 1). These analyses were henceconducted only for Cd and the results are shown in Table 3. It can be seen that the
Figure 4. Liver histopathology. (a) Portal cell infiltrates (arrowhead), fibrosis (arrow) and bileduct proliferation (arrow) in an adult female long-finned pilot whale. HE staining,magnification�20, bar¼ 50 mm. (b) Focal necrosis (arrows) and Ito cells (arrowheads) in anadult female long-finned pilot whale. HE staining, magnification�10, bar¼ 25 mm. (c) Lipidgranuloma in an adult female long-finned pilot whale. HE staining, magnification�20,bar¼ 50mm.
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mean concentrations of renal Cd were significantly higher in individuals withglomerular arteriosclerosis compared to individuals without glomerular arterioscle-rosis (n¼ 6) and a similar trend was found for tubular hyaline casts (n¼ 6).
Discussion
Contaminant concentrations
Among whale species, the toothed ones such as pilot and killer whales mayaccumulate extremely high concentrations of both heavy metals such as Hg as well asOC (AMAP 2004, 2005; Dam and Bloch 2000). Earlier investigations of pilot whalesfrom the Faroe Islands, covering years 1977–1987, detected high liver Hg levels of upto 280 mg g�1 ww, which increased with age (AMAP 1998; Julshamn et al. 1987). Inconnection with the Arctic Monitoring and Assessment program on the FaroeIslands, liver and renal tissues from 50 pilot whales composed of mainly adultssampled during 1 July 2001–2007 were analyzed for Hg and Cd (Hoydal and Dam2009). Among these individuals, the median liver Hg concentration was 75 mg g�1 wwwith a median Se :Hg molar ratio of 0.99� 4.82, with 50% of the individuals havinga Se :Hg ratio 51.0. The highest liver Hg concentration was 574 mg g�1 ww in anadult female whose Se :Hg ratio was 0.62, which indicated that Hg, on a molar basis,reduced Se and thereby increased risk of Hg toxicity (Ralston, Blackwell, andRaymond 2007). In these same 50 individuals, the median kidney Hg concentrationwas 64.6 mg g�1 ww with the highest concentration at 188 mg g�1 ww in an adult male.Investigations of other pilot whale subpopulations in the Pacific Ocean also reportedhigh liver Hg concentrations around 400 mg g�1 ww (Endo et al. 2004). Independentof the whale subpopulations investigated, all pilot whale liver Hg concentrations areabove suggested threshold levels for adverse health effects for marine mammalsat 30–60 mg g�1 ww including those of the present study (Dam and Bloch 2000;Endo et al. 2004; Hoydal and Dam 2009; Thompson 1996).
Kidney Cd concentrations in the present pilot whales were not especiallyhigh when compared to toothed whales and seals in the North Atlantic andNorth West Greenland (AMAP 1998, 2005; Dietz et al. 1998a, b, 2004; Hansen et al.1990; Sonne-Hansen et al. 2002). The concentrations were also below suggested Cdkidney cortex toxicity threshold levels at 50–400mg g�1 ww for animals and humans(Elinder and Jarup 1996; WHO 1992). It is worth noting that the renal Cdconcentrations found in the present study were lower than that reported earlier fromthis subpopulation of pilot whales (AMAP 1998, 2005; Hoydal and Dam 2005).Studies from 1999 to 2001 showed that median kidney Cd concentrations were
Table 3. Significant results from the analyses of relationships between histology (presence vs.absence) and pollutant levels in 3/7 Faroe Island pilot whales sampled in June 2007. In all thefour cases, individuals with lesions had the highest concentrations.
Age group Pollutant (tissue) Histologic change p (n; F; R2)
All Cadmium (kidney) Glomerular arteriosclerosis 0.04 (7; 7.3; 0.59)All Cadmium (kidney) Tubular hyaline casts 0.05 (7; 6.1; 0.55)
Notes: The calculations of differences in contaminant concentrations between individuals withand without lesions are based on Kruskal–Wallis test.
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132 mg g�1 ww in adult females (n¼ 29) and 110 mg g�1 ww in adult males (n¼ 17) andwithmaximal concentrations in the two groups of 239 and 215 mg g�1 ww, respectively,which all exceeded toxic threshold levels of 50–400mg g�1 ww for adverse renal effects.
The OC concentrations of the Faroe Island pilot whales were high and similar tothose reported from earlier investigations of this subpopulation (AMAP 2004; Damand Bloch 2000; Hoydal and Dam 2005, 2009). That is not surprising as toothedwhales in general are high in blubber OC levels compared to, for example polar bearswith high metabolic hepatic CYP 1A activity (Dietz et al. 2004; Muir et al. 1999;Verreault et al. 2005), whereas this metabolic pathway may be slower in pilot whales(White et al. 2000). In comparison with toxic threshold levels for
PPCB, the pilot
whales are in a range that exceeds those of neuroendocrine, reproductive, andimmune effects for marine and terrestrial mammals ranging from 500 to120,000 ng g�1 lw (AMAP 1998, 2004). From a health perspective, this lowermetabolic capacity has advantages and disadvantages. It may be positive if thewhales have a restricted capacity to metabolize the original compounds into highlyendocrine disrupting metabolites (Colborn 2004; Colborn, Saal, and Soto 1993; vanDuursen et al. 2003). On the other hand, the whales keep accumulating OC blubberconcentrations, as they cannot excrete the compounds, and when the whales then gointo periods of fasting and become leaner due to their reliance on their blubber layer,the compounds become bioavailable at high concentrations that exert potential acuteneuroendocrine and immune toxicity. The female situation is different once theybecome reproductively active as this enables trans-generational transfer of OC bodyburdens (Borrell, Bloch, and Desportes 1995).
Histopathology – kidney
In general, most histopathological lesions are nonspecific in nature. The glomerular,tubular, and interstitial lesions found in the present pilot whales are similar tothose reported from terrestrial and marine mammals (Bergman, Bergstrand andBignert 2001; Sonne et al. 2005, 2006, 2007a; Woshner et al. 2002), and domesticanimals (Sonne et al. 2007b). For example, thickening of glomerular capillary wallsare often incorporations of immune-complex deposits at the epithelial side ofthe glomerular capillary basement membrane due to immune challenges from, forexample, micropathogen infections. In the case of Hg-induced nephrotoxicity, it isa two-step mechanism that firstly includes glomerular basement membrane suscep-tibility and secondly an immune-complex deposition similar to auto-immunity.Furthermore, a tubular and renal blood MeHg re-uptake results in an infection ofthe entire juxta-glomuerular apparatus, including a proximal convoluted tubularpeptide-bound Hg re-uptake. Only three of eight renal lesions found in the pilotwhales were age-related. These were all glomerular (arteriosclerosis, capillarydilatation, and general sclerosis) and found to be age-related in other wildlifespecies (Bergman, Bergstrand, and Bignert 2001; Sonne et al. 2005, 2006, Sonne-Hansen et al. 2002; Woshner et al. 2002). Glomerular sclerosis was not exacerbatedby interstitial fibrosis in the present pilot whales although this is often the case forage-related changes (Bergman, Bergstrand, and Bignert 2001; Woshner et al. 2002).Interstitial nephritis (mononuclear cell infiltrations) was found only in the juvenilemale and a single adult male. The reason for that is unknown and may be ascribed tothe low sample size. Higher prevalence was found in other marine species, such as
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seals (Bergman, Bergstrand, and Bignert 2001; Sonne-Hansen et al. 2002), polarbears (Sonne et al. 2006, 2007a), beluga (Delphinapterus leucas), and bowhead(Balaena mysticetus) whales as well as Australian bottlenose dolphins (Lavery et al.2009; Rosa et al. 2008; Woshner et al. 2002) probably due to different metal/OCconcentrations and exposure to micro-pathogens and thereby incidences of chronicrecurrent infections.
As five of eight renal lesions were present in the juvenile male, factors other thanage may be co-factors in the development of these. Among environmental factorscould be mentioned micro-pathogens, such as parasites, bacteria, fungi, and virusesthat are all known to induce renal histopathological changes. Beside these,environmental metal and OC pollutants in similar concentrations were shown asco-factors in previous studies of renal histopathology which is important to keep inmind as juvenile whales receive large trans-lactation contaminant burdens for up to22 months (Ridgway and Harrison 1998). For example, the histopathological lesionsfound in glomeruli, tubules, and interstitium were similar to those reported in Balticgray seal and ringed seal heavily polluted with heavy metals and OC between theyears 1977 and 1996 (Bergman, Bergstrand, and Bignert 2001) as well as polar bearand Australian bottlenose dolphins polluted with heavy metals and OC in similar orhigher magnitudes (Lavery et al. 2009; Rosa et al. 2008; Sonne et al. 2006, 2007a).The fact that Cd was higher in individuals with glomerular arteriosclerosis may beascribed to both age and exposure to this specific heavy metal as well as otherpollutants and micro-pathogens (Elinder and Jarup 1996; WHO 1992). In the hyalinecase, the relationship was not significant while the interpretation is the same.
Histopathology – liver
Three of seven liver lesions were found in the juvenile male indicating that only lipidgranulomas, portal fibrosis, bile duct proliferations, and focal necrosis were age-related. All lesions were non-specific and might be inflammatory reactions to micro-organisms and/or injury of local blood vessels from toxic compounds (Kelly 1993;MacLachlan and Cullen 1995). The lipid accumulation in Ito cells functions as themajor accumulation and storage site for the lipophilic vitamin A (Kelly 1993;Leighton et al. 1988; MacLachlan and Cullen 1995; Senoo et al. 1999, 2001). Thezoonary hepatocyte macrovesicular lipid may be due to lipid hyperphagia,starvation, excessive dietary intake of carbohydrates or exposure to toxic substances,such as Hg and OC (Kelly 1993; Leighton et al. 1988; MacLachlan and Cullen 1995).Such zonary pattern is characteristic during periacinar subcellular damage due tolow oxygen gradient which could sensitize the liver parenchyma to metabolicdisorders subsequently resulting in lipid accumulation (Kelly 1993; MacLachlan andCullen 1995). Regarding bile duct proliferation, it is known to be induced by toxicinjury, parasitism, and periductular fibrosis while portal fibrosis most often is ageassociated as seen in the present pilot whales (Hori et al. 1982; Kelly 1993;MacLachlan and Cullen 1995).
Taken together, these findings support the postulation that Hg and OC areco-factors in the development of liver lesions in pilot whales. Studies of histologyand Hg exposure in free-ranging Atlantic bottlenose dolphin (Tursiops truncatus)(Rawson et al. 1993), Arctic beluga whale (Woshner et al. 2002), and Bowheadwhales (Rosa et al. 2008) showed similar changes, such as fat globules, necrosis, and
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mononuclear cell infiltrates. Further, studies of histopathology in lab rodents andother mammals support Hg and OC induced liver toxicity in Faroe Island pilotwhales (Al-Saleh et al. 2003; Bergman et al. 1992; Bruckner Khanna, and Cornish1974; Chu et al. 1998; Jonsson et al. 1981; Kelly 1993; Kimbrough, Gaines,and Linder 1971; MacLachlan and Cullen 1995; Sonne et al. 2005, 2006, 2007a, b,2008a, b, c). In a single specimen the liver Se :Hg molar ratio was below 1 indicatingthat some of the total Hg was in an ionic and potentially toxic form. In a study ofpolar bears, Dietz et al. (2000) showed that liver Se :Hg molar ratio in polar bearswas below 1 for some individuals indicating potential toxicity. As up to 94% ofHg found in East Greenland polar bears is of anthropogenic origin and asconcentrations continue to increase (Dietz et al. 2006); it likely demands that selenidHgSe tiaminite-complex and metallothionein complexes are present in order to adaptto and detoxify high Hg concentrations (AMAP 1998, 2005; Rawson et al. 1993;Sonne-Hansen et al. 2002).
Considerations
Despite low samples size, the present study shows high prevalence of histopatho-logical changes in liver and renal tissues of Faroe Island pilot whales and thatage, heavy metal, and OC contaminants may be important factors in pathologydevelopment. The clinical impact at the individual level from the present histo-pathological lesions is unknown. However, it is likely a stress factor as Hg andP
PCB toxicity thresholds for adverse health effects were exceeded in all individualswhere such information is available. Such impacts are recommended to beinvestigated in a larger future study to elaborate further on the toxic health andpopulational effects on this whale species.
Acknowledgments
The authors thank Katrin Hoydal, Johanna Olsen, and Thea Dam for assistance in the fieldand in the sample preparation. Funding for tissue contaminants analyses were made availableby the Environmental Protection Agency, Denmark via the Faroe Island AMAP core programon OC and heavy metals. Laboratory technicians are acknowledged for processing slides forhistology. A conflict of interest was not reported.
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