Detailed project information for Study Plan Number 09060

Branch :	Conte Anadromous Fish Laboratory
Study Plan Number :	09060
Study Title :	Endocrine disruption of smolting and ion regulation in anadromous fish
Starting Date :	03/01/2002
Completion Date :	03/30/2008
Principal Investigator(s) :	McCormick, Steve
Primary PI :	McCormick, Steve
Telephone Number :	(413) 863-3804
Email Address :	steve_mccormick@usgs.gov
SIS Number :
Primary Program Element :
Second Program Element :
Status :	Active
Abstract :	The parr-smolt transformation is a critical and highly sensitive life history stage of Atlantic salmon that coincides with downstream migration and seawater entry. Restoration and conservation of Atlantic salmon may be compromised by injurious stressors experienced prior to or during this period. These stressors can include contaminants, dam passage, extreme temperature fluctuations, capture/handling, and fluctuations in water levels. Concerns about contaminant effects on Atlantic salmon survival are one of the reasons for the recent listing of Atlantic salmon as an endangered species in Maine, and are important to the growing salmon aquaculture industry. Nonylphenol (NP) and PCBs are common contaminants found in the effluent from sewage treatment plants, industrial sites, and storage facilities. Acting as an estrogen mimic, NP may have negative effects on osmoregulation by interacting with the estrogen receptor thereby down-regulating or inhibiting enzymes and hormones critical to smolting. While the developmental effects of NP have not been widely investigated, its use in pesticides in Maine and New Brunswick has been implicated in poor smolt survival and adult returns, possibly by disrupting the parr-smolt transformation (Fairchild et al. 1999). The main sub-lethal effects of PCBs in fish appear to be through their action on the hypothalamus and thyroid. Since smolting is highly influenced by these two endocrine glands, this critical developmental period is especially suitable as an integrator of the impact of PCBs. PCBs are present in relatively high concentrations in several salmon rivers in New England including the Dennys River (ME) and the Millers River (MA) (Colman, 2001) and have recently been found in commercial salmon food (BBC News). Ultimately, changes in physiology associated with smolting prepare anadromous fish for seawater entry. The ability to osmoregulate during this transition from fresh water to seawater is imperative to survival. While the effects of contaminants may vary according to timing of exposure (developmental stage), sex, species, and/or route of exposure, if a contaminant inhibits or retards physiological preparedness, smolts may die upon seawater entry or delay entry into seawater causing increased vulnerability to predation and reduced ocean survival. Atlantic Salmon Life History, Conservation and Aquaculture Historically, adult Atlantic salmon, Salmo salar, have deposited their eggs in shallow gravel depressions or redds in cold-water streams in the fall and early winter. Sac fry reside in these redds until the yolk sacs were fully absorbed. As feeding fry and later young juvenile parr, Atlantic salmon stay in their natal stream from one to three years before smolting in spring and migrating downstream, in many cases, for hundreds of kilometers to the ocean. Smolts spend one to two years in the ocean before returning to their natal stream as adults to spawn. Atlantic salmon in southern New England were extirpated over 150 years ago primarily due to dams, habitat loss, and pollution. For the past thirty years, these populations have been under active restoration, which includes stocking fish as fry and smolts. These fish can be affected by point source pollution such as from paper mills, and they must pass through some of the most polluted areas of larger rivers and estuaries. In northern New England wild stocks of Atlantic salmon on smaller rivers were unaffected by dams, but over the last twenty years have seen their populations decrease by ten-fold, resulting in their recent listing as an endangered species. Although the reason for this population decline is unknown, contaminants are one of the leading candidates. In addition to possible impacts on Atlantic salmon in the wild, contaminants are also an important issue to the salmon aquaculture industry. Recent reports of PCB’s and other contaminants in cultured salmon and commercial salmon feeds may threaten the success of this industry. The possible sub-lethal effects of contaminants, including their capacity for endocrine disruption, have only recently been appreciated. It is therefore important to evaluate the effects of low-level contamination, particularly the effects of known endocrine disruptors on key developmental stages in salmon such as the parr-smolt transformation. Smolting Smolting is a developmental process that involves a variety of morphological, physiological, and behavioral changes that prepare juvenile salmonids for migration to, and residence in, the ocean (Hoar 1976; McCormick 1998). An essential component to the parr-smolt transformation is a large increase in seawater tolerance. A key physiological parameter of this increased osmoregulatory ability is an increase in gill sodium-potassium-activated adenosine triphosphatase (Na+, K+,-ATPase) activity at the time of smolting. The increase in gill Na+, K+,-ATPase activity is directly related to an increase in size and number of gill chloride cells, specialized cells that are responsible for salt secretion. Because of this relationship, the measurement of gill Na+, K+,-ATPase activity has been utilized extensively to monitor smolt physiology using a non-lethal gill biopsy method (McCormick, 1993). While the parr-smolt transformation typically occurs during the first or second spring of the fishes life, it is recognized that smolting is a reversible process. Smolts retained in fresh water in the laboratory exhibit a decrease in hypoosmoregulatory capacity and concurrent decreases in gill Na+, K+,-ATPase activity (Zaugg and McLain, 1970; Wedemeyer et al., 1980). Several endocrine glands are activated during smolting, but much emphasis has been placed on the role of the pituitary-thyroid axis. Because of its important role in controlling development and growth at the embryonic and larval stages of many fishes (Norris, 1983; Tagawa and Hirano, 1987), as well as its influence on the physiological process associated with smolting in salmonids (Folmar and Dickhoff, 1980; Leatherland, 1982), a major focus has been placed on the activity of thyroid hormones. The presence of thyroid hormones in fish eggs was first reported in coho salmon (Kobuke et al., 1987) and has been identified in several teleosts since that time (Sullivan et al., 1987; Tagawa, 1996). Thyroid hormones have been clearly shown to have a role in the morphological changes such as silvering that occurs during smolting (Johnston et al., 1994), and have been hypothesized to have a role in the imprinting process (see McCormick 1998, for review). The role of thyroid hormones in osmoregulation is less certain. Most studies have found that exogenous thyroid hormones do not promote salinity tolerance in salmonids (McCormick 1995). Nonetheless, Madsen and Korsgaard (1989) found that long-term administration of thyroxine (T4) during smolting increased gill Na+, K+,-ATPase activity and gill chloride cell numbers in Atlantic salmon smolts. Similarly, feeding triiodothyronine (T3), considered the active form of the hormone converted from T4 at target tissues, increased seawater tolerance and growth in pre-smolt Atlantic salmon (Refstie, 1982). However, Shrimpton and McCormick (1998) found that exogenous T3 did not significantly increase gill Na+, K+,-ATPase activity in this species. Evidence is available to demonstrate that thyroid hormones have a role in the behavioral changes that occur during smolting. Iwata (1995) found that T4 treatment results in decreased aggression and increased migratory activity in Pacific salmon. Following T4 treatment, pre-smolt coho salmon (Oncorhynchus kisutch) showed a behavioral preference for salinity of 14 ppt (Iwata et al., 1990). Cortisol is both a stress hormone and a mineralocorticoid in teleost fish and has a direct role in promoting osmoregulatory changes during smolting (Specker, 1982). Plasma levels of cortisol exhibit a prolonged increase during smolting and coincide with an increase in saltwater tolerance in Atlantic and coho salmon, (Specker and Schreck, 1982; Virtanen and Sovio, 1985; McCormick et al. 2000). Exogenous cortisol has been shown to stimulate gill Na+, K+,-ATPase activity in several salmonids (Richman and Zaugg, 1987; Björnsson et al., 1987; Madsen, 1990; Pelis and McCormick, 2001). In addition, researchers have found that administration of exogenous cortisol in salmonids increases chloride cell numbers (Richman and Zaugg, 1987; Madsen, 1990) and size (Madsen, 1990). The effectiveness of exogenous cortisol treatment to increase gill Na+, K+,-ATPase activity varies with developmental differences in coho and Atlantic salmon (McCormick et al., 1991). It has been postulated that this variation may be due to changes in abundance or availability of cortisol receptors at different developmental stages (McCormick, 1995). Independent of its function in growth, growth hormone (GH) has been implicated in having a role in seawater acclimation of salmonids (see Sakamoto et al., 1993 for review). Exogenous GH treatment has resulted in increased gill Na+, K+,-ATPase activity and chloride cells in several salmonids (Boeuf et al., 1990; Madsen, 1990). An increase in salinity tolerance has also resulted from GH treatment (Komourdjian et al., 1976; Bolton et al., 1987; Madsen, 1990). Following ovine growth hormone (oGH) treatment, pre-smolt coho salmon (Oncorhynchus kisutch) showed a behavioral preference for salinity (14 ppt) (Iwata et al., 1990). While information on the role of insulin-like growth factor I (IGF-I) in the parr-smolt transformation is limited, it has been found that IGF-I mRNA increases in the gill and kidney of rainbow trout following exposure to seawater (Sakamoto and Hirano, 1993). In addition, administration of exogenous IGF-I treatment has resulted in increased salinity tolerance (McCormick et al., 1991). The mechanisms of action for these effects are not well known and to our knowledge, no studies have been conducted to investigate the effects of IGF-I on the seawater preference of salmonids. While many experimental investigations in the laboratory interpret results based on the administration of one or two hormones, the endogenous system is considerably more complex. There is substantial evidence indicating that thyroid hormones, cortisol, growth hormone, and IGF-I work in conjunction to produce the physiological and behavioral changes that occur during smolting. There is a clear cooperation between growth hormone and cortisol to induce increased salinity tolerance in Atlantic salmon (Madsen, 1990; McCormick 1995). Additionally, Shrimpton and McCormick (1998) have shown that T3 acts additively with GH in rainbow trout to increase corticosteroid receptor concentration in the gill, which is a likely site of action for the osmoregulatory actions of cortisol during smolting. IGF-I has been demonstrated to increase the stimulatory effects of cortisol on gill Na+, K+,-ATPase activity (McCormick, 1995). It has been postulated that IGF-I may play a permissive role in the action of cortisol and growth hormone to stimulate gill Na+, K+,-ATPase activity and chloride cells (McCormick, 1995). Iwata et al (1990) found that while T4 or GH alone increased salinity preference, the combination further increased this effect. A likely conclusion from the evidence is that each of these hormones plays a role in smolting and that their interaction is a key component of the control of this developmental process. If this is true, then the removal of any single factor could be detrimental to the physiological and behavioral ability of smolts to survive. Contrary to the stimulatory effects of thyroid hormones, cortisol, IGF-I, and growth hormone, sex steroids have been shown to be inhibitory to the development of salinity tolerance that occurs during smolting (McCormick and Naiman, 1985; Ikuta et al., 1987; Schmitz and Mayer, 1993). Administration of exogenous Estradiol (E2) significantly reduced circulating thyroid hormone levels in rainbow trout (Leatherland, 1985) and decreased gill Na+, K+,-ATPase activity, chloride cell density, degree of silvering and salinity tolerance in Atlantic salmon (Madsen and Korsgaard, 1989; Madsen et al., 1997). While the mechanisms of action of sex steroids on osmoregulatory physiology are unknown, this inhibitory role may be related to the anadromous life history of salmon in which maturation and the attendant increases in sex steroids accompany movement into fresh water. Although it seems likely that increased circulating sex steroids will decrease salinity preference, this has yet to be examined. Contaminants Major sources of aquatic pollutants include effluents from sewage treatment plants, industrial sites and storage facilities. Nonylphenol (NP) is a degradation product of nonylphenol ethoxylate, which is used widely in detergents, plastics, emulsifiers, and other commercial cleaning products. It is also present in pesticides used in Maine and New Brunswick and has been implicated in poor smolt survival, possibly by disrupting the parr-smolt transformation (Fairchild et al., 1999). Water chemistry analysis from Fairchild et al., (1999) indicates that concentration ranges of NP within 4 hours of pesticide spraying were 0.26-88.4 g/l in lotic environments and 20.67-860.6g/l in lentic environments. A direct consequence of the wide spread use of NP is its presence in rivers and estuarine systems (Arukwe et al., 2000). Typical NP concentrations in waters receiving contaminant discharge are less than or equal to 2 g/l, but values of 325-1,000 g/l have been reported (see Liber et al., 1999). It is known that environmental estrogens such as NP can interact with the native estrogen receptor and adversely affect reproductive development (Knudsen and Pottinger, 1999; Yadetie et al., 1999). There is also evidence that other estrogen mimics (some pesticides, alkylphenolic chemicals, pthalates, bisphenol A) including NP have negative effects on osmoregulation by interacting with the estrogen receptor (see Jobling et al, 1998). Investigating NP as a potential estrogen mimic, Madsen et al. (1997) and McCormick (unpublished; fig. 1) found that repeated doses of NP (150 g/g) or estradiol (E2; 0.5g/g) decreased gill Na+, K+, -ATPase and reduced salinity tolerance in Atlantic salmon smolts. The possible sub-lethal alterations in development due to PCBs in fish have not been widely examined, although PCBs are a widespread aquatic contaminant. The majority of lab studies examining PCBs have focused on reproductive and developmental toxicity (with lethality as a common endpoint in the latter). In fish, the lethal effects for larvae occur at concentrations as low as 5 g/g (Monosson, 1999), which suggests that sub-lethal effects may occur at still lower concentrations. These effects may vary according to timing of exposure (developmental stage), sex, species, and/or route of exposure (see Monosson, 1999). Some effects of PCBs in fish include reproductive and developmental toxicity, liver damage, neurobehavioral effects, thyroid hormone effects, and cytochrome P450 induction (e.g., Fingerman and Russell, 1980; Fisher et al., 1994; Stegeman and Hahn, 1994). A major site of action of PCBs in fish appears to be at the level of the hypothalamus, thyroid, and interrenal. This is in agreement with findings in mummichogs (Fundulus heteroclitus) suggesting behavioral and hormonal effects are related to thyroid hormones (Zhou et al., 1999) and in humans, indicating that PCBs alter serum thyroid hormone levels (Cheek et al., 1999). In addition, laboratory exposure to the PCB mixture A1254 has resulted in reduced levels of plasma cortisol (Sivarajah, et al., 1978). Since smolting is highly influenced by these endocrine glands, this critical developmental period is especially suitable as an integrator of the impacts of PCBs and other potential endocrine disruptors. PCBs are present in relatively high concentrations in several salmon stocked rivers in New England including the Dennys River, Maine and the Millers River, Massachussetts, where levels were 0.5-8 mg/kg in fish (Colman, 2001). In addition to environmental contamination, PCBs have recently been found in commercial salmon food (BBC News). Velpar, also known as hexazinone, is an herbicide sprayed to support the blueberry industry in Maine. Atlantic salmon abundance has declined to a level that is difficult to determine possible correlation between Velpar application and salmon recruitment based on epidemiological criteria and approaches. Nevertheless, it is possible to develop an experimental approach to assess potential impacts of Velpar on wild salmon. This approach should address at least two critical questions in order to decipher a causal relationships between Velpar spray and salmon abundance and return rates. First, are wild Atlantic salmon exposed to Velpar at levels that could result in toxicological impacts that decrease long-term survival? And second, what are the physiological consequences of this exposure that could affect smolt survival and adult returns? Biomarkers for exposure to Velpar and PCB’s need to be developed to assess the exposure and toxic responses of salmon captured in rivers. Velpar is mainly composed of hexazinone, a systemic herbicide in the triazine class of pesticides. Although limited information is available about hexazinone and its metabolism in exposed organisms, toxicity studies have shown that s-triazine pesticides (eg. 1,3,5-s-chlorotriazines), which is similar to hexazinone (6-dimethylamino-1 methyl-1,3,5-triazine), are inducers of aromatase (CYP19) and some other P450 enzymes, most notably CYP2C. Both of these genes have been cloned in numerous animals. CYP19 in particular has been cloned in many fish species. Therefore it is feasible to develop markers for Velpar exposure based on CYP19 and CYP2C. A quantitative PCR method for Atlantic salmon CYP1A, which is useful for assessing the exposure and toxic responses to dioxin and PCB’s, has recently been developed (Rees, Li and McCormick, in preparation). This method will be modified so that only non-lethal biopsies of gill tissues will be applied to fish in the Dennys River in which the Eastern Surplus Company EPA superfund site in Meddybemps, Maine is suspected as a source of PCB contamination. A similar method can be developed for CYP19 and CYP2C, and used to compare Atlantic salmon smolt from several downeast Maine rivers with known levels of Velpar, and from the Penobscot River and Green Lake National Fish Hatchery where levels of hexazinone are undetectable. This will determine whether fish in contaminated rivers have been exposed to hexazinone in sufficient amounts to result in a toxicological response. Atrazine is a widely used insecticide present in many rivers of the United States, Canada and Europe. Although generally considered to have low toxicity, recent studies suggest that this contaminant may affect fish and wildlife populations through sublethal pathways. Atrazine can cause feminization of male frogs, and a recent survey of ponds in the United States has found a strong relationship between the levels of atrazine and the absence of fertile males (Tyrone Hayes, U.C. Berekely, unpublished results). In Atlantic salmon, atrazine can compromise smolt development and is especially potent when exposed along with estrogenic compounds such as nonylphenol (Andrew Moore, Marine Lab, Lowestoft, personal communication). The mechanisms by which atrazine may affect the parr-smolt transformation, either alone or in combination with estrogenic compounds, is currently unknown. Acid precipitation has been responsible for the loss of Atlantic salmon populations in Nova Scotia and Norway, and is suspected in having a role in declines of Atlantic salmon in Maine (Parrish et al. 1998). Many of the toxic aspects of low pH are caused by increased aluminum and its transition to a more toxic form. The smolt stage has been shown to be especially sensitive to acid and aluminum exposure, and even short term exposure (24 hours) to moderate levels of acid and aluminum will reduce salinity tolerance of Atlantic salmon smolts (Staurnes et al. 1996). These short term exposures to acid and aluminum may be typical of rivers in downeast Maine which have reduced but nonlethal acid levels, but are poorly buffered and receive substantial acid snow melt and precipitation when smolts are normally migrating downstream. Several aspects of these short-term effects therefore require further investigation. These include the time required for recovery to acid and aluminum, the endocrine effects of this exposure, and determination of gill levels of aluminum which would directly determine whether smolts in downeast Maine rivers are being impacted by exposure to acid/aluminum. Stress During the course of migration throughout New England, Atlantic salmon smolts pass through numerous dams traveling for hundreds of kilometers from their natal streams to the ocean. It is unknown if passage through each dam is perceived as a discrete event, or if the effect of each passage event builds upon the previous events to produce a cumulative physiological response. Recent studies indicate that a number of immune functions of fish are suppressed by chronic exposure to crowding or other stressors (Pickering 1993). The effects of acute stress (seconds to minutes) are more equivocal, resulting in transient decreases or increases in immune function or disease resistance at various intervals after exposure to a single acute stressor (Maule et al. 1988). It is not known whether cumulative stress responses to repeated stressors experienced by juvenile salmonids during migration such as passage through or around multiple dams, encounters with predators, or exposure to toxic chemicals, could temporarily suppress or reverse normal physiological processes or functions of both specific and innate immune systems. Activation of chronic diseases by stress, or increased susceptibility to new pathogens encountered in the river, estuary, or marine environments, could be a major cause of mortality for Atlantic salmon smolts during early marine life. It has been demonstrated in the laboratory that repeated exposures to acute handling can accumulate to produce a stress response that is of greater severity than that observed following a single handling (Barton et al., 1987). In a field study, it was found that salmonid migrants exhibited a cumulative stress response (progressively elevated plasma cortisol) that corresponded to their progression through a dam bypass system (Schreck et al., 1985). Increasing the magnitude of the stress response also appeared to extend the period needed for recovery. It has also been demonstrated that repeated handling stress in rainbow trout results in a significant reduction in gill corticosteroid receptor numbers, which in turn reduced the gill responsiveness to cortisol (Shrimpton and McCormick, 1999). Stress is known to delay smolting in salmonids (Patiño, et al., 1986), and could delay seawater entry, thus increasing the amount of time salmonid migrants spend in the estuary. These experiments, however, have not examined the effects of combinations of stressors over the course of development leading to smolting and migration. Atlantic salmon in Maine have recently been listed as an endangered species, yet the reason for this decline is unknown. Contaminants and their impact on the parr-smolt transformation have been suggested to be involved in the decline in Atlantic salmon populations in Maine and the limited success of restoration efforts in southern New England. While there is increasing knowledge on the sublethal impacts of contaminants on reproductive processes in fish, there is very little scientific information on the impacts of contaminants on other hormone driven developmental events such as the parr-smolt transformation. The studies outlined here will determine whether contaminants can act as endocrine disruptors of smolting and the freshwater-seawater transition in anadromous fish. OBJECTIVES: 1. Investigate the developmental effects of the environmental contaminants nonylphenol (NP), polychlorinated biphenyls (A1254), hexazinone, atrazine, and acid/aluminum on Atlantic salmon (Salmo salar) by integrating physiological and behavioral indices. These studies will attempt to answer the following principle objectives: A. To determine the capacity of contaminants to act as endocrine disruptors of the parr-smolt transformation by adversely affecting salinity preference and tolerance of smolts following exposure at different developmental stages. B. To determine how contaminant exposure affects smolt capacity to cope with stressful events (e.g., swimming fatigue, dam passage, and capture or handling) C. To investigate the mechanisms by which contaminant exposure affects seawater survival. 2. Monitor physiological changes in wild and hatchery-reared Atlantic salmon juveniles during the parr-smolt transformation and ocean entry to determine if smolting is being compromised by exposure to contaminants. 3. Develop non-lethal methods for determining whether Atlantic salmon have been exposed to contaminants (gene expression and levels of CYP1A, CYP19 and CYP2C; gill levels of aluminum). 4. Determine hormones and transporters involved in the freshwater-to-sea water transition of other Anadromous fishes (American shad, (Alosa sapidissima); Atlantic sturgeon (Acipenser oxyrinchus) and striped bass (Morone saxatilis)) and determine whether these are affected by exposure to environmental contaminants. The contaminants to be tested will be determined following analysis of the results of experiments conducted on Atlantic salmon.
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