Ecosystem Health and Sustainable 2 Agriculture

Ecology and Animal Health

Editors: Leif Norrgren and Jeffrey M. Levengood

CSDCentre for sustainable development Uppsala.  The North American Great Lakes/St. Lawrence River and Estuary

Contaminants and Health of Beluga Whales of 17 the Saint Lawrence Estuary

Daniel Martineau University of Montreal, Saint-Hyacinthe, QC, CAN

Defining the causes of mortality in wild animal popula- et al., 1994). Other factors shared by both populations and tions is difficult, largely owing to their widespread distri- that may have contributed to population bottlenecks are bution and poor spatial delineation of these populations. that both have been the object of extensive hunting in the It is especially problematic in marine mammals, because early 20th century, even being the object of bounties by the their environment is opaque to direct visual observation. corresponding government. In addition, both populations, Because of these limitations, the respective roles played because they are top predators in the food chain, have been by natural factors and human activities in mortality re- contaminated by high levels of stable lipotrophic (fat-lov- main intricate. An exception is the beluga population that ing) industrial contaminants, many of which are organo- inhabits a stretch of the SLE roughly centred on the mouth chlorine compounds that may decrease immune functions of the Saguenay River in Quebec, eastern Canada, only (which may have contributed to a severe viral epidemic 500 km from Montreal. This estuarine habitat is remi- that struck Baltic harbour seals at the end of the 1980s), niscent of that used by Arctic belugas, which enter the endocrine disruption, lower rates of reproduction (Helle estuaries of Arctic rivers in summer. The SLE drains the and Olsson, 1976; Lair et al., 1997; Martineau et al., 1987, GL, the most industrialised part of North America, and 1988; Mikaelian et al., 2000; Reijnders, 1986). thus receives a heavy input of contaminants. The slow SLE beluga, which constitute the southernmost beluga circulation of the SL water exacerbates problems of en- population worldwide, are unique by their accessibility to vironmental contamination. SLE beluga became isolated investigation and geographical isolation from the Arctic, from their original Arctic population at the end of the last the natural habitat of most beluga. The isolation of SLE glaciation, 10,000 years ago, when the region emerged beluga from Arctic populations has been confirmed by isostatically, relieved from the weight of glacial ice. genetic analysis and by the distinctly higher levels of in- This origin is similar to that of Baltic harbour seals dustrial contaminants found in their tissues (Gladden et (Phoca vitulina), which were probably imprisoned in the al., 1999). Baltic 8,000 years ago. Because both populations have Together, the position of SLE beluga at the top of the been isolated for a thousand years from Arctic popula- food chain, its high body lipid content (around 40% of tions, they show a high degree of inbreeding, expressed by body weight) and its long lifespan explain the high con- lack of genetic variation (Murray et al., 1999; Patenaude  up to 70 years according to a recent paper (Stewart et al 2006)

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Figure 17.1. Worldwide distribution of beluga whale (Delphinapterus leucas). Map by Uko Gorter for American Cetacean Society (http://www.acsonline.org/factpack/ BelugaWhale.htm). centrations of persistent lipophilic contaminants in its tis- less buoyant, which would make them more likely to sink sues. In contrast, contaminant levels in fin whales visiting and to be recovered. From 1983 to 1999, an average of the area in summer are much lower, partly because these 15 beluga carcasses were reported stranded every year animals mostly eat plankton, and thus occupy a lower po- and of these, about half were examined at the College sition in the food chain (Metcalfe et al., 2004). of Veterinary Medicine of Université de Montréal be- The SLE beluga population has dwindled from an cause they seemed in a reasonable state of preservation estimated 10,000 at the beginning of the 20th century to (Martineau et al., 2002). This probably does not represent the current estimate of anywhere between 400 and 1200 the entire mortality, because the territory is immense and animals (the maximum number of animals ever counted the shoreline is rarely visited by people in winter. Using directly is 459) (Kingsley, 1998). In 1980, SLE belu- a realistic annual 6% death rate from a population of 960, gas received the status of endangered species from the Kingsley (2002) estimated the mortality rate at 58 deaths/ Canadian Government (Sergeant, 1986). To explain the year. Thus with an average of 15 carcasses reported per decline and apparent lack of recovery, a study was initi- year, about 43 carcasses or 74% of dead animals escape ated in 1982 to carry out systematic post-mortem exami- detection. Other researchers have found higher annual nation of dead SLE beluga that drift ashore, in order to death rates in free-ranging tooth whales, closer to 10% determine the cause of death of these animals, measure (Stolen and Barlow, 2003), which would result in around tissue levels of chemical contaminants and examine the 100 beluga carcasses stranded every year, implying an hypothesis that contaminants contribute to death. even lower recovery rate. To evaluate the diseases and causes of mortality in a The three primary causes of death of SLE beluga are human population, the logical places to look are hospitals metazoan parasites, (22%), cancer (18%) and infectious and morgues. Similarly, to evaluate diseases and causes agents (bacterial, viral, or protozoan, (17%) (Martineau of death in wildlife, one has to examine animals found et al., 2002). Cancer caused the death of 27% of adults, dead in the wild. The bodies of juveniles and newborns a rate of cancer higher than in any other population of are harder to locate than those of adults, and thus some wild terrestrial or aquatic mammals with the possible ex- are likely missing from our data. There is no other obvi- ceptions of virally induced tumours in rodents and wood- ous source of bias beside emaciation. The latter could be chucks. Epithelial cancers of the gastrointestinal (GI) tract caused by chronic diseases, and would make carcasses were strikingly predominant (Martineau et al., 2002). In

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addition, two additional cancers involved the liver, and one case involved the salivary glands (adenocarcinoma) (Girard et al., 1991). SLE beluga are commonly affected by multisystemic infections with agents that have generally been associated with immune suppression in humans and other animals. For instance, infections with opportunistic agents such as Aeromonas sp, Edwardsiella tarda, Nocardia spp., Toxoplasma gondii, and viruses such as herpes virus and papilloma virus have all been found in SLE beluga (De Guise et al., 1994; Martineau et al., 1988; Mikaelian et al., 2000).

Persistent Organic Pollutants

Persistent Organic Pollutants (POPs) are defined as glo- bal toxic contaminants found ‘distant from sources’ for long periods, and accumulate in the fat of humans and free-ranging mammals. Many countries have decided to restrict POPS fabrication and distribution through the Stockholm Convention on Persistent Organic Pollutants and the United Nations ECE POPS Protocol to the Convention on Long-range Transboundary Air Pollution. Thus PCBs and DDT are sometimes designated ‘legacy’ contaminants because their production has stopped in Figure 17.2. Post-mortem examination of a beluga whale found dead on the the St Lawrence Estuary shoreline. College of Veterinary Medicine, many parts of the world. Many POPs, such as PCBs, University of Montreal. Source: Martineau et al., 2002. Reprinted by DDTs and PBDE, are organochlorine compounds (OCs), permission from Environmental Health Perspectives. composed of paired carbon rings with chlorine or bro- mine attachments (see chapters 13 and 16). SLE beluga are contaminated by all these compounds, as well as by loving’ properties of these compounds explain their life- other OCs such as chlordane, hexachlorocyclohexane long accumulation in lipids. (lindane insecticide (HCH)), their metabolites and heavy Most degradation-resistant chemicals find their way metals (Martineau et al., 1988; Martineau, 1987; Muir et into water. Thus it is not surprising that OCs released from al., 1996; Wagemann et al., 1990). These large halogen leaking equipment, poor storage conditions, accidental atoms protect the carbon rings from the normal enzy- spills (as for PCBs) or deliberately discharged into the ter- matic degradation (metabolism) that takes place inside restrial or aquatic environment (as for DDT) have all end- vertebrate organisms (most often in the liver and intes- ed up in water. Because the SLR drains the most indus- tine). As a general rule, the resistance to degradation is trialised area of North America, SLE belugas are among proportional to the number of halogen atoms composing the most heavily OC-contaminated marine mammals, the molecule. Together, this protection along with the ‘fat surpassing only transient killer whales of the Canadian Pacific coast and striped dolphins from the Mediterranean  http://www.pops.int/ (Martineau et al., 1987; Ross et al., 2000). SLE beluga  http://www.unece.org/env/lrtap/pops_h1.htm (United Nations Economic Commission for Europe) adipose tissues have 25 and 32 times more total PCB and

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total DDT, respectively, than Arctic beluga (Table 17.1). nated with PCBs in the Great Lakes (recall that the GL Concentrations found in SLE beluga are also higher than are drained by the SLE) (Campbell et al., 2003; Fox et al., those found in humans exposed to PCB during the Yusho 2007). The thyroid gland produces two major hormones, industrial accident (Letcher et al., 2000 a, b). T3 and T4, called thyroxines. These hormones are respon- sible for maintaining body temperature by regulating heat production through oxygen consumption, and maintain- ing and using adipose tissue for increased energy demand. Pathological Effects on the Endocrine The blubber of marine mammals is a thick, deep skin layer System composed of adipose tissue. Thus the thyroid glands prob- ably play a major role in the use and maintenance of blub- Many OCs and their metabolites severely damage the ber, which is critical for insulation, heat production, ener- adrenal glands in animals and people. Degenerative and gy storage and buoyancy. Thyroid functions are especially proliferative changes consistent with chronic stress and important for Arctic marine mammals whose body must dioxin-related POP (DRPOP) intoxication are commonly produce heat in cold water, a medium that would quickly observed in the adrenal cortex and medulla of SLE and suck every calorie produced if it were not for the blub- Western Hudson Bay beluga whales, and the severity of ber. The role of the thyroid gland is further complicated these lesions increases with age in both populations (Lair in marine mammals by these animals slowing down their et al., 1997). Adrenocortical cysts are also often found in metabolism in order to dive at great depths (this conflicts SLE beluga. These are rare in other marine mammals ex- with the need for high heat production levels in a cold me- cept in white-sided dolphins (Geraci and St. Aubin, 1979; dium). For all the above reasons, thyroid integrity is most Lair et al., 1997). OC metabolites may be responsible for likely central for the survival of beluga whales. Finally, these cysts. Most strikingly, a DDT derived compound, PCBS and PBDE are known to affect brain development o,p’DDD, has long been used in human and veterinary through their action on the thyroid gland and cetaceans medicine to destroy tumour or hyperplatic adrenocortical might be particularly vulnerable in that regard given their cells, when these cells secrete pathological amounts of very high relative brain size, second only to that of hu- cortisol (Cushing syndrome). In grey and harbour seals mans (Marino, 1998; Montie et al., 2009). from the Baltic Sea, adrenocortical hyperplasia has been attributed to PCB and DDT contamination based on epi- demiological data (Bergman and Olsson, 1985; Olsson, 1994; Olsson et al., 1994). Both SLE beluga and Atlantic Immunosuppression white-sided dolphins have been exposed to high levels of adrenotoxic OC metabolites for decades, suggest- PCBs have long been demonstrated as immunosuppres- ing that OCs may be responsible for the adrenal lesions sive compounds in laboratory and farm animals, and have seen in these two species (Martineau et al., l987, 2003; been associated epidemiologically with immunosuppres- McKenzie et al., 1997; McKinney et al., 2006a,b; Muir et sion in marine mammals (Bull et al., 2006; Jepson et al., al., 1996a,b; Troisi et al., 1998). 2005; Safe, 1994; Thomas et al., 1978). The finding of Proliferative and degenerative lesions (adenomatous generalised infections by microorganisms that normally hyperplasia and follicular cysts) have been found in the do not cause disease in immunocompetent animals has thyroid of SLE beluga (Mikaelian et al., 2003). Similar le- been taken as circumstantial evidence of PCB-induced sions were found in Arctic beluga but the absence of older immunosuppression (Inskeep et al., 1990; Martineau et age groups in an Arctic population precluded a compari- al., 1988). Experimental evidence has supported this hy- son between older whales from the two populations. The pothesis. Young harbour seals fed with OC-contaminated thyroid gland has long been known to be a major target fish for 2.5 years showed compromised immune func- of POPs toxicity. Thyroid hyperplasia (e.g. an increased tions when compared with a group of seals fed non-con- number of cells) has been found in fish and birds contami- taminated fish (Van Loveren et al., 2000).

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Table 17.1. Mean PCB Levels (ng/g) in blubber of male belugas from selected stocks (Modified from Andersen et al., 2001a) 1Muir et al. (1996a); 2Martineau et al. (1987); 3,4Muir et al. (1997); 5Andersen et al. (2001); 6Stern et al. (1994); 7Krahn et al. (1999); 8Muir (1994, 1996) compiled by Muir et al. (1997); 9Becker et al. (1995) compiled by De March et al. (1998); 10Muir et al. 1990; 11Krahn et al. (1999) aOnly geometric mean was available.

Location: Average Level Range St. Lawrence Estuary (Canada) 1,2a 78,900 SE. Baffin Island (Canadian Arctic) 4 6,794 ± 2,171 S. Hudson Bay (Canada) 4 6,768 ± 2,346 N. West Greenland (Nuussuaq) 6 5,580 ± 2,500 E. Chukchi Sea (Alaska) 7 5,200 ± 900 Svalbard (Norwegian Arctic)5 5,100 ± 1,870 Mackenzie Delta, Beaufort Sea (Canadian Arctic) 8 5,010 ± 1,618 E. Bering Sea (Alaska) 9 4,170 ± 644 Jones Sound (Canadian High Arctic) 10 2,530 ± 570 Cook Inlet (Alaska) 11 1,490 ± 700

OC-induced immunosuppression has been suspected of porpoises whose blubber total PCB concentration was to play a role in rendering harbour seals more sensitive to > 17µg/g, animals dead of infectious diseases had higher phocine morbillivirus in an epizootic that has caused more total PCBs levels than animals dead from trauma. Below than 20,000 deaths in these animals in 1988 in the Baltic that concentration, there was no correlation, suggesting Sea, whereas in contrast, grey seals were not affected by that PCB-induced immunosuppression favours infec- the epizootic even though they were infected by the virus. tions in porpoises with concentrations > 17 µg/g. Using The resistance of grey seals to morbillivirus may be ex- the same series of animals, Bull et al. (2006) showed that plained by the fact that functions of grey seal leukocytes harbour porpoises with a total blubber PCB concentration (white blood cells) are less affected by PCBs than those > 50 μg/g showed a significant, positive association be- of harbour seals (Hammond et al., 2005). These findings tween PCB levels and the number of gastric nematodes. are highly relevant for SLE beluga for several reasons: 1) In the same series of porpoises, there was a 2% increased PCB mixtures affect beluga lymphocyte proliferation and risk of being affected by an infectious disease for each 1 phagocytosis in vitro; 2) SLE beluga have no antibodies mg/kg increase in blubber, and the risk was doubled risk against morbilliviruses (Mikaelian et al., 1999); 3) pilot at around 45 μg/g lipid (Hall et al., 2006). Importantly, whales (Globicephala melaena and G. macrorhynchus) average PCB concentrations in the SLE beluga popula- occasionally enter the beluga habitat and are asymptomatic tion are higher than these putative thresholds (Table 17.1). carriers of morbilliviruses, and could thus transmit the vi- The threshold in PCB concentration might be even lower rus to immunologically naïve beluga, a role similar to that in beluga because the high lipid percentage of the beluga grey seals are thought to have played in the transmission body makes the total body burden of beluga higher than of phocine morbillivirus to harbour seals (Duignan et al., that of the leaner porpoises. 1995; Kingsley and Reeves, 1998; Mikaelian et al., 1999). One approach to study the impacts of contaminants on Based on the results from experimental exposure in immune function is to measure the in vitro response of im- aquatic mammals (minks, otters, seals) and in vitro studies mune cells from a presumably ‘normal’ population to pol- on dolphin lymphocytes, Kannan et al. (2000) estimated lutants added in concentrations identical or similar to those that marine mammals are likely to start suffering from the found in the tissues of contaminated free-ranging animals adverse effects of PCBs at a blubber concentration > 17 from the same species. Using this approach, the prolifera- µg/g. Examining harbour porpoises stranded in the UK, tive response of lymphocytes from Arctic beluga to mi- Jepson et al. (2005) supported these findings: in a series togens and their spontaneous proliferation were impaired

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in vitro by exposure to p,p’-DDT and PCB 138 concentra- explaining why the PAH family includes members such tions similar to those found in SLE beluga tissues (PCB as benzo[a]pyrene (BaP), which are powerful carcino- 138 is one of the most abundant PCB congeners present in gens based on their demonstrated carcinogenicity in ro- SLE beluga tissues) (De Guise et al., 1998). Because bel- dents (IARC) (Fitzgerald et al., 2004). uga and other marine mammals are not contaminated with The SLE waterway receives the effluent from much one or two pollutants but rather with a complex mixture of of northeastern North America, one of the most indus- congeners and distinct compounds, De Guise’s laboratory trialised regions of the world. In addition, 60 miles (100 has also explored the effects of mixtures of different OC km) upstream and upwind of the SLE beluga habitat, on (Levin et al., 2004, 2005a, b, c; Mori et al., 2006). the Saguenay River shore, is a large complex of alumin- ium smelters developed in that region as early as 1926 because of the access to the sea and the availability of vast and cheap amounts of hydroelectricity, two condi- Temporal Trends in Contaminant Loads tions required for aluminium production. Because of this rare conjunction of characteristics, 4% of world alu- The use of PCB and DDT was banned in Europe and minium production, takes place in the Saguenay region. North America in the mid-1970s. PCBs are still in use in Aluminium is produced by the electrolysis of alumina Russia, however and γ-HCH (lindane) is in restricted use (e.g. aluminium oxide) using the Söderberg method, a in North America. In addition, legacy compounds such as process that releases huge amounts of PAHs into the at- toxaphene, DDT and PCBs continue to leach into waters mosphere. Alumina itself is chemically extracted on-site from contaminated soils, explaining the continued elevat- (which generates abundant liquid residues) from bauxite, ed concentrations of these contaminants in marine mam- a rock absent from Canadian soil. Because the Saguenay mals tissues (Braune et al., 2005). In SLE beluga, levels River provides direct access to the sea, bauxite is shipped of most well known OC have decreased at least 2-fold directly from Australia, Brazil, Guinea and Jamaica to the between 1987 and 2002 with the important exceptions Saguenay smelter. In addition, electricity is inexpensive of chlordane, Mirex and γ-HCH. DDT metabolites have for the aluminium smelters because of generous discounts not decreased. In Arctic Canadian beluga, PCB levels de- provided by the Quebec state-run power company, and clined 1.7 to 2.8 fold depending on PCB congeners con- because the aluminium smelter company has operated sidered from 1982 to 2002. In contrast to concentrations its own private hydroelectric dams for almost a century. of the parent compounds, temporal trends in tissue levels The Söderberg process, used by the smelter until 2004 for of PCB metabolites have not been monitored in Canadian extracting aluminium from alumina, is highly polluting beluga populations (Lebeuf et al., 2007), a significant gap because electrolysis involves the passage of strong elec- in our knowledge, since the toxicity of PCB metabolites trical current through an anode. This is a compact mass may be higher than that of the parent compounds. of tar which burns when the current passes through, re- leasing large amounts of PAHs into the atmosphere (other metallurgical processes, not only the Söderberg process, produce PAH, but generally in lesser amounts). Polycyclic Aromatic Hydrocarbon Contamination  The Saguenay fjord is a 104-km long inland extension of the SLE, SLE beluga are heavily contaminated by polycyclic aro- 200-300 m deep, having strong tidal currents at its mouth. It is the only fjord on the North American mainland. matic hydrocarbons (PAHs), a large family of compounds  63% of Canada’s production produced by the incomplete combustion of organic mol-  The production of 1 tonne of aluminium requires 13 kilowatts. The ecules (Martineau et al., 1988). PAHs, once metabolised aluminium company in the Saguenay region produces 1950 megawatts by cytochrome oxidase enzymes, are degraded in metab- annually using its own dams, that is, roughly enough electricity for 3 medium-sized cities (a medium-sized city of 100,000 inhabitants con- olites that are unstable, strongly reactive and mutagenic, sumes about 600 megawatts/year).

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Thus, the light weight of aluminium, often promoted that beluga competed with fisheries) (Vladykov, 1946). as an energy saver for motor vehicles, came in fact with Worms found in their stomachs were identified, weighed a high environmental cost until very recently, when the and counted. Twenty-five to nearly 1,400 worms were Söderberg process was replaced (partly) by a different present in individual stomachs; individual worms weighed process using prebaked anodes. Note that the amount of 1.3 to 9.8 g as determined from worms collected live in PAHs currently released into the environment has been the sediments of that region. Thus roughly, the stomach of monitored solely by the smelter since 1998, e.g. there is a single beluga could contain up to 14 kg of worms. On the no independent monitoring by the government or by any average, Nereis contains 0.68 μg/kg of BaP. Thus 14 kg of independent organisation since 11 years. Two chemists at worms contain roughly 9.5 μg of BaP. Canada’s Department of Fisheries and Oceans estimated It has been estimated that 5.6 μg of BaP is the maxi- that the local aluminium smelters have released 40,000 mum daily amount of BaP that could be ingested by a 70- tons of PAHs downwind into the Saguenay river watershed kg human adult to ‘safeguard human health’ (Fitzgerald since 1926 (Smith and Levy, 1990). PAHs released by the et al., 2004). Based on this estimation, the maximum smelters have contaminated the Saguenay river sediments amount of BaP ‘safely’ ingested daily by a 900-kg beluga along with beluga, and the aquatic and terrestrial animals would be 72 μg, an amount about 8 times larger than that that live in that region (Blondin et al., 1992; Lun et al., contained in 14 kg of worms. However, the local smelter 1998; Martel et al., 1986; Martineau et al., 1988; Shugart claims that PAHs emissions into the Saguenay River et al., 1990; Smith and Levy, 1990; White et al., 1997). have decreased about 8-fold since the 1970s, implying Beluga are among the rare species of tooth whales that that BaP concentrations in worms were 8-fold higher feed significantly on invertebrate animals living in sedi- at that time. Thus the BaP dose ingested by a 900 kg ments, particularly on annelids (worms), and these an- beluga in 14 kg of worms is near the estimated maximal nelids have been contaminated by PAHs. Unlike PCBs, daily dose ‘safely ingested’ by a beluga (although it is PAHs are biodegraded quickly once released into the not known over what period this amount of worms would environment but they may accumulate in benthic anne- have been eaten). lids because some annelid species are naturally deprived Local aluminium workers are affected by high prev- of CYP enzymes (Rewitz et al., 2006). The metabolites alences of urinary bladder and lung cancers, and these resulting from CYP-mediated degradation of PAHs are have been epidemiologically related to PAH exposure often more toxic or carcinogenic than the parent com- within the smelter. Because the epidemiological relation- pounds. Within the cells (generally but not exclusively ship between PAH exposure and these cancers is particu- liver cells) of contaminated animals/human, many pollut- larly strong, workers affected by urinary bladder cancers ants, among which PAHs and PCBs, induce the prolifera- and exposed for more than 20 years are now compensated tion of smooth endoplasmic reticulum, the cell organelle by the Quebec Worker Compensation Board (Tremblay in which CYPs reside. Thus the generation of carcino- et al., 1995). Increased cancer rates have also been ob- genic PAHs metabolites is accelerated by the ingestion of served in the rest of the human population living in that PAHs and PCBs. area, where drinking water is taken mostly from surface It is likely that beluga become contaminated with PAHs waters, and is also contaminated by PAHs (Martineau et by ingesting contaminated annelids. PAHs have been de- al., 2002) (Figure 17.3). tected in Nereis virens, a benthic annelid living in the sedi- The aluminium industry has argued that there is no ments of the SLE beluga habitat (E. Pelletier, manuscript proof that PAHs cause cancer in beluga whales. This ar- in preparation). The daily amount of fish ingested by an gument ignores the historical and, more importantly, the adult beluga is equivalent to 2 to 3 % of its body mass, i.e. between 13 and 20 kg of fish (Robeck et al., 2005). A series  Also in Vladykov “Although beluga of both sexes eat Nereis, lactating of beluga was dissected in the 1930s to determine whether or pregnant females show a marked preference for this type of food”  beluga eat significant amounts of commercial fish species Aluminium workers in the Saguenay region have up to 42 times more urinary bladder cancer than the general population (Tremblay et al., (the government was responding to fishermen’s complaints 1995; Theriault et al., 1984)

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Figure 17.3. Distribution of beluga in the SLE and incidence of standardised rate ratio for digestive system cancer in (A) men and (B) women in the Saguenay River area, Québec. Source: Martineau et al., 2002. Reprinted by permission from Environmental Health Perspectives. experimental evidence provided by laboratory rodent pound is the active component of antifouling paints used models and in vitro cellular and molecular models used to protect ship hulls. It is immunotoxic, hepatotoxic, and a to demonstrate PAH carcinogenicity. These results have powerful endocrine disruptor (Ogata, 2001). The toxicity been traditionally extrapolated to humans because peo- of butyltin metabolites remains to be determined. In the ple (like rodents and beluga whales) are mammals and as Saguenay River, gastropods naturally exposed to butyl- such, share most cellular machineries with other mam- tin had significant sexual changes (‘imposex’). Annelids mals, including beluga (Fitzgerald et al., 2004). recovered from the Saguenay sediments contained an av- That argument – roughly that the absence of evidence erage of 289 ng Sn/g of total Bt compounds, implying is equivalent to the evidence of absence – is also reminis- the possible ingestion of 4 mg of these compounds (in 14 cent of that used by tobacco companies, which have long kg of worms) by a beluga (see PAHs discussion; Viglino invoked the absence of proof regarding whether tobacco et al., 2006). For a 1,000-kg beluga, this represents a 4 smoke causes lung cancer, despite overwhelming evi- μg/kg dose, which is 16 times higher than the maximum dence supporting the carcinogenicity of tobacco smoke in dose tolerable in humans, based on immunological toxic- animal and in vitro models (the weight of evidence now ity (0.25 μg/kg) (Penninks, 1993). Liver concentrations points at PAHs as the major culprit for the carcinogenic- measured in SL beluga approach those found to be lethal ity of tobacco smoke; Alberg et al., 2005). The role of for the lymphocytes (white blood cells) of Dall’s porpoise PAHs has also been demonstrated in the aetiology of GI in vitro (100 ng/g). In addition, liver lesions caused by tract cancers in chronic carcinogenicity studies in rodent Bt in rats are consistent with those found in SL beluga models (reviewed in Martineau et al., 2002). (Martineau et al., 2003). As PBDEs are used to reduce the risk of fire (in poly- urethane foams, computer casings, textiles used in fur- niture and automobiles), they must be quickly released Emerging and Recently-Emerged from burning material to rapidly make oxygen unavail- Contaminants able to flames. Thus these compounds are only mixed with, not chemically bound to, plastic. Consequently, Butyltin (Bt) and its metabolites have been found in they readily leach out from these materials, ending up in SL beluga (Saint-Louis et al., 1999). Pelletier’s group the environment. Canada’s regulations on PBDE produc- also recently detected butyltin in invertebrates living in tion and use are lagging, however (Health Canada, 2007). Saguenay sediments (Viglino et al., 2006). This com- Thus it is not surprising that the concentration of these

146 The North American Great Lakes/St. Lawrence River and Estuary

compounds in wildlife is increasing, in contrast to other they bind to proteins, because of their polarity. The lack OC such as PCBs, the production of which has generally of lipophilicity also explains that generally, and in con- been banned. trast to PCB, mammalian males and females have similar PBDE are found in SL beluga tissues at lower con- tissue levels. PFOA and PFOS have been found in SLE centrations than PCBs but their concentrations are rap- beluga and show similar temporally increasing trends idly increasing (Hobbs et al., 2003; Lebeuf et al., 2004; (M. Lebeuf, pers comm.). PFOS has been found in the Letcher et al., 2000a; McKinney et al., 2006a; Metcalfe et liver of Baltic Sea seals (Kannan et al., 2002). So far al., 1999). Between 1988 and 1999, concentrations have porpoises from the UK show the highest concentrations doubled in males and females every 3 and 2 years respec- among marine mammals (Law et al., 2008). PFCs have tively (Lebeuf et al., 2004). In contrast to PCB levels been detected even in the Arctic fauna. Their concentra- (males have higher PCB levels than females), males and tions have increased exponentially over recent years in females have similar PBDE levels, most likely because of polar bears, which are the most contaminated marine the relatively recent massive input of these contaminants mammals in the Arctic (Bossi et al., 2005; Butt et al., in the beluga’s environment, which has not given suffi- 2008; Sonne et al., 2008; Martin et al., 2004; Tomy et cient time for females to transfer their load to newborns. al., 2004). In Arctic beluga, the recent temporal increase in PBDE Because the molecular structure of PFCs resembles levels has been similar (6.5 fold from 1982 to 1997, 1.6 that of endogenous fatty acids, PFCs disturb lipid metab- fold between 1998 and 2001 (de Wit et al., 2006). In olism (laboratory rodents become hypocholesterolaemic Arctic ringed seals, levels increased 9-fold between 1981 and show fat atrophy (Xie et al., 2003)). In humans, low and 2000 (Ikonomou et al., 2002). birth weight has been recently associated with high levels The toxicity of PBDEs probably adds to that of PCBs, of PFOA in maternal plasma, perhaps because the dis- because both groups of compounds affect the develop- turbed lipid metabolism deprives the developing foetus ment of the nervous system and lower vitamin A and of lipid input, resulting in low weight at birth (Fei et al., thyroxin serum levels. In by-caught Baltic and North 2007). Such disturbance in lipid metabolism may have a Seas porpoises, thymic atrophy has been associated with strong impact on Arctic mammals considering the cen- elevated PBDE levels (Beineke et al., 2005). The rapid tral role played by fat in energy storage, buoyancy and increase in PBDE levels in beluga tissues along with the heat insulation in these animals. PFCs also downregulate current use and production of these compounds and the the expression of genes involved in inflammation and lack of regulations almost guarantee that PBDE concen- immune functions in rodents (Guruge et al., 2006). This trations will continue to increase (Lebeuf et al., 2004). feature might amplify PCB-induced immunosuppression Among fluorinated organic compounds (perfluoroalkyl in beluga and in other marine mammals: this hypothesis compounds (PFC)), two have been studied in some detail: is supported by the epidemiological association between PFOA, a major ingredient in the manufacture of Teflon, high PFOA tissue levels and infectious diseases in sea ot- composed of a 8-carbon chain ornate with fluorine atoms; ters (Kannan et al., 2006). and PFOS, very commonly used as a surfactant in fab- ric stain repellents (3M Scotchgard). In 2000, worldwide production of PFOS by the US 3M company was around 3700 tonnes. In 2001, 3M discontinued PFOS produc- Future Research tion. At the time of writing, however, the compound was still being produced in Germany, Switzerland, Russia and Cancer has been observed in SLE beluga but not in other Japan (OSPAR, 2006). SLE marine mammals or in marine mammals of the Baltic Although PFCs are more polar than PCBs, and there- Sea or the Arctic. This observation indicates that SLE fore much less lipotrophic (fat-loving), they are biomag- beluga are specifically exposed to chemical or biologi- nified from fish to odontocetes. They do not accumulate cal mutagens or both, possibly through unique ecologi- in fat but rather build up in liver and kidneys because cal features such as feeding on benthic invertebrates. Out

147 The North American Great Lakes/St. Lawrence River and Estuary

of many other fish species from the approximate same seems to counterbalance the reduction of thymus weight habitat, cancer has been found in only a single fish spe- by tributyltin – or the interactions may be negative – for cies, lake whitefish. This is the only salmonid that feeds instance, PCBs induce CYP, which increases the forma- significantly on benthic invertebrates. Thus, two aquatic tion of carcinogenic PAHs metabolites. PCB congeners vertebrates, phylogenetically very distant, may be affect- have no or little effects on lymphocyte proliferation in ed by cancer because they share unique feeding habits vitro when tested individually, whereas they significantly within their respective taxonomic groups. Because cancer reduce this immune function when mixed together (De is an ultimate but rare consequence of chemical mutagen- Guise et al., 1998). Characterising these interactions will esis, the epidemiological association of xenobiotics with remain a major challenge. Biopsies (samples taken from carcinogenesis requires the examination of large numbers live animals) will occupy an increasingly important role of animals exposed to environmental concentrations of in monitoring contamination. Already, CYP induction has carcinogens. To demonstrate the role of xenobiotics in been visualised by immunohistochemistry in skin biop- carcinogenesis in SL beluga or in any other free-rang- sies taken from free-ranging cetaceans, and has been cor- ing mammal, convincing statistics would require the ex- related with OC levels measured from the same biopsies amination of much larger numbers of beluga and/or the (Branchi et al., 2002). follow-up of SLE beluga for many more decades. In ad- dition, the observation of high prevalences of cancer in other populations of marine mammals similarly exposed to carcinogens would strengthen an etiologic relationship with chemical carcinogenesis. Few odontocetes species feed on benthic invertebrates: the Amazon River (Inia geoffrensis); Franciscana (Phocoena blainvillei); Susu (P. gangetica); and Irrawaddy (Orcinus brevirostris) dolphins (Ridgway et al., 1989). Because these species generally inhabit rivers that are often more contaminated than the open ocean, they might also be affected by high rates of cancers of the GI tract. Several chemical carcin- ogens leave a signature on the host genome by causing mutations at specific sites in genes involved in cell pro- liferation. The finding of the same signature in tumours of SLE beluga, fish and people would strongly support the aetiological role of contaminants, especially PAHs, in carcinogenesis,. New experimental approaches for demonstrating the chronic effects of OC on cetaceans are emerging. For in- stance, the immune systems of several cetacean species have been reconstituted in SCID mice. These ‘cetacean- ised’ mice have been used successfully to demonstrate the validity of a morbillivirus vaccine (De Guise and Levin, 2004). OC immunotoxicity can now be tested on these mice, a task not feasible in vivo. There are complex interactions between the numer- ous pollutants and the associated metabolites which contaminate SLE beluga. Interactions between contami- nants may be positive – for instance in rats, p,p’-DDE

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