Chronic Effects of Dietary Selenium on Juvenile Sacramento Splittail

Oct 15, 2004 - Aquatic Toxicology Program, Department of Anatomy,. Physiology ... fish per tank, 3 tanks per diet) 7-month-old juvenile splittail were...
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Environ. Sci. Technol. 2004, 38, 6085-6093

Chronic Effects of Dietary Selenium on Juvenile Sacramento Splittail (Pogonichthys macrolepidotus) S W E E J . T E H , * ,† X I N D E N G , ‡ DONG-FANG DENG,‡ FOO-CHING TEH,† SILAS S. O. HUNG,‡ T E R E S A W . - M . F A N , §,| J E E L I U , § A N D RICHARD M. HIGASHI⊥ Aquatic Toxicology Program, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine; Department of Animal Science; Department of Land, Air & Water Resources; and John Muir Institute of Environment, University of California, Davis, California 95616, and Department of Chemistry, University of Louisville, Louisville, Kentucky 40208

The chronic effects of dietary selenium (Se) exposure in juvenile Sacramento splittail (Pogonichthys macrolepidotus) were investigated in the laboratory. A total of 960 (40 fish per tank, 3 tanks per diet) 7-month-old juvenile splittail were fed one of eight Purified-Casein diets supplemented with selenized yeast for 9 months in a flow-through system. These diets contained the following: 0.4 (control), 0.7, 1.4, 2.7, 6.6, 12.6, 26.0, and 57.6 mg of Se kg-1 dry weight. Survival, Se tissue concentration, growth, gross morphology, and liver histopathology were assessed at 5and 9-month of exposure. Mortalities occurred only in the two highest Se treatments and were accounted for 8.3 and 18.3% at 5-month and 10.0 and 34.3% at 9-month, respectively. Liver and muscle Se concentration were significantly correlated with dietary Se concentration. Fish exposed to 0.4-12.6 mg of Se kg-1 diets had reached equilibrium in liver Se concentration by 5 month. Splittail fed diets at concentrations g26.0 mg of Se kg-1 had not reached equilibrium in liver, and muscle Se concentrations and grew significantly slower (p < 0.05) at 5- and 9-month exposure. Se-induced deformities were observed in fish fed g2.7 mg of Se kg-1 diets at 5-month and in fish fed g0.7 mg of Se kg-1 diets at 9-month. Fish fed 26.0 and 57.6 mg of Se kg-1 diets had higher liver lesion scores at 5-month while fish fed 6.6 and 57.6 mg of Se kg-1 diet had higher liver lesion scores at 9-month. Results indicate that survivals, growth, changes of tissue Se concentrations, and histopathology of juvenile splittail were dose-dependent, but their response thresholds to dietary Se concentrations differed and depended on treatment concentrations and duration of exposure. Chronic exposure to 6.6 mg of Se kg-1 diet induced deleterious health * Corresponding author phone: (530)754-8183; fax: (530)752-7690; e-mail: [email protected]. † Aquatic Toxicology Program, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California. ‡ Department of Animal Science, University of California. § Department of Land, Air & Water Resources, University of California. | University of Louisville. ⊥ John Muir Institute of Environment, University of California. 10.1021/es049545+ CCC: $27.50 Published on Web 10/15/2004

 2004 American Chemical Society

effects that can potentially impact survival of juvenile splittail.

Introduction Selenium (Se), in its various forms, is a natural constituent of water bodies, both freshwater and marine, and is derived from natural processes such as erosion of ore-bearing rocks. Anthropogenic activities such as burning of coal and other fossil fuels, agricultural and industrial sources, and mining constitute an increasingly important source of Se in the aquatic environment (1, 2). Se contamination of aquatic ecosystems is of particular concern and has been studied extensively in the San Francisco Estuary, the largest estuary on the west coast of the United States (3-9). This estuary is a dynamic, transitional ecosystem where nutrient-laden freshwater from two major rivers (i.e., the Sacramento and the San Joaquin) converge and are mixed with marine waters of the Pacific. The Estuary, a rich habitat supporting a diverse mixture of aquatic life, has recently undergone dramatic changes, and many fish populations are in serious decline (10-12). Bioconcentrations of Se in food chain benthic organisms have been associated with delta fish population declines (1315). Therefore, Se bioaccumulations and/or bioconcentrations in benthic invertebrates may have a significant impact on upper trophic level benthivore fish. Sacramento splittail (Pogonichthys macrolepidotus), a cyprinid endemic benthivore, once common in rivers and lakes throughout California’s Central Valley is now largely confined to the San Francisco Estuary (11, 12, 16) and was listed in 1999 (17) and later remanded in September 2003 as threatened species by the U.S. Fish and Wildlife Service (18). As an opportunistic daytime benthic forager, detritus and benthic invertebrates represent a major component of the splittail diet (19). The Asian clam (Potamocorbula amurensis), a euryhaline species of bivalve mollusk introduced to the San Francisco Estuary in the mid-1980s, has spread so prolifically that it accounts for 95% of the benthic invertebrate biomass in certain areas of the Estuary (20). In addition, Asian clams have been shown to be effective bioaccumulators of Se, and clams collected from the San Francisco Bay had tissue concentration levels as high as 20 mg of Se kg-1 dry weight (DW) (7, 9). A recent study revealed that splittail are changing their diet preference to consume a large quantity of Asian clams as their major food source (19). Therefore, there are concerns over possible growth and deleterious effects of dietary Se on the splittail populations. Although Se is an essential nutrient, chronic exposure to levels moderately higher than nutritional requirement has caused adverse health effects in fish (13, 21-26). In addition, Se toxicity is highly dependent on Se forms, exposure route, and species physiology (e.g., ability to transform and detoxify). With regard to the indigenous splittail, virtually nothing is known about these aspects. The present research was undertaken to determine the toxic effects of Se on juvenile splittail via dietary exposure to selenized yeast (abundant in proteinaceous selenomethionine). Cumulative mortality, growth, histopathology, and Se liver and muscle concentration were monitored during a 9-month dietary exposure.

Experimental Section Diet Preparation. Eight test diets containing graded levels of Se and Torula yeast were used in this experiment. The basal diets were formulated using purified ingredients. The VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Growth responses of juvenile splittail fed the test diets for 5 and 9 month. (A) Total length vs dietary Se concentrations. (B) Body weight vs dietary Se concentrations. (C) Condition factor (CF) vs dietary Se concentrations. Note that vertical bars represent the SE of each dietary treatment and different letters above or below the bars indicate significant difference among treatments, p < 0.05. O ) 5 months and b ) 9 months.

TABLE 1. Formulation of Test Diet Fed to Juvenile Splittail ingredients

g kg-1 diet as fed

vitamin-free caseina wheat glutenb egg albumina dextrina nonnutritive bulka cod liver oil:corn oil (1:1)a vitamin mixb mineral mixb choline chloridea Santoquinc Se-yeastd,e Torula yeastb,d

310 150 40 299 35 80 10 30 4 0.19 0-42 42-0

a USB Corporation (Cleveland, OH). b ICN Biomedicals Inc. (Irvine, CA). c Supplied by Monsanto Company (St. Louis, MO). d Se-yeast and Torula yeast were added into diets to obtain graded levels of dietary Se. The analytical total Se content in the test diet is 0.4 (control), 0.7, 1.4, 2.7, 6.6, 12.6, 26.0, and 57.6 mg kg-1 diet as fed. e Selenomaxs Nutrition 21 (San Diego, CA).

graded levels of dietary Se were obtained by combination of different levels of selenized yeast (Se-yeast) and Torula yeast (ICN Biomedical, Inc., Irvine, CA) (Table 1). Se-yeast con6086

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tained approximately 21% of the Se in selenomethionine and proteinaceous Se forms. The test diets were prepared by mixing dry ingredients with oil and water in a Hobart mixer (Troy, OH) and pelleted with a California Laboratory pellet mill (27). The wet pellets were fan-dried overnight at room temperature, crumbled, sieved into various particle sizes, and then stored at -20 °C until use. Analyzed levels of Se in the diets were as follows: 0.4 (no Se-yeast) and 0.7, 1.4, 2.7, 6.6, 12.6, 26.0, and 57.6 (no Torula yeast) mg kg-1 diet. Fish and Experimental Conditions. Splittail juveniles (7month-old) were reared from the natural spawning of captive broodstocks at the Center for Aquatic Biology and Aquaculture, University of California, Davis. The initial average body weight was 6.8 ( 0.71 g (mean ( SE, n ) 24). The experiment was conducted in an indoor flow-through system with 24 individual tanks (40 fish per tank, 66 cm in diameter, 90 L water in volume). Prior to the initiation of the experiment, 40 fish were randomly distributed into each tank and acclimated with 0.4 mg of Se kg-1 control diet for 4 weeks. Water temperature was maintained at 23 ( 1 °C during the first 6 months of exposure but dropped to 18 ( 1 °C for the rest of the experiment due to the failure of the water heating system. The flow rate of individual tanks was 4 L min-1. The

system was kept under a natural photoperiod. Test diet was randomly assigned to three replicate tanks. Fish were fed twice daily (9:00 a.m. and 4:00 p.m.) with a daily feeding rate of 3% body weight (BW) in the first 5 months and adjusted to 2% BW per day thereafter. To minimize Se leaching from diet to water, rations were divided into two halves during each feeding. Half of the amount of feed was provided first followed by the remaining half 15 min later. Feces and uneaten feeds in the tanks were cleaned by siphon 30 min after feeding, and mortality was recorded daily. Sample Collection for Gross Morphology, Selenium, and Histopathologic Analyses. Five fish from each tank were randomly collected at 5 and 9 months of exposure. Each fish to be measured was individually netted, examined for gross deformities, and euthanized with an overdose of 3-aminobenzoic acid ethyl ester (MS-222; Sigma, St. Louis, MO). Next, fish were blotted gently on paper towels, measured with a standard ruler to the nearest 1.0 mm, placed in a tared beaker of water, and weighed to the nearest 0.1 mg. Livers were surgically removed, weighed, and divided into a larger and a smaller portion. The larger portion was frozen in liquid nitrogen for selenium analysis, and the smaller portion was fixed in 10% neutral buffered formalin for histopathologic examination. In addition, white muscle was also collected for selenium analysis. Total dietary Se was analyzed by incubating pulverized diet in perchloric:nitric acids (1:5) and heated stepwise from 50, 100, 140, and 160 to 205 °C until the digest volume was reduced to 0.05) below 12.6 mg of Se kg-1 dietary treatments and were significantly increased at or beyond 12.6 mg of Se kg-1 dietary treatments 6088

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(Figure 2A). In all treatments, Se concentrations in liver were higher than those in muscle by 9-month (cf. Figure 2A,B), but muscle Se concentrations were significantly increased at a lower dietary Se concentration (1.4 mg of Se kg-1 diet) (Figure 2B) and continued increasing significantly at every treatment beyond this level. This indicated that muscle Se concentrations reflected a more distinct dose-response to dietary Se concentrations than that of liver Se concentrations

FIGURE 5. Multiple deformities in a juvenile splittail fed 6.6 mg of Se kg-1 diet for 9 months. 1 ) facial deformity showing deformed lower and upper jaws, soft operculum, and protruding eye; and 2 ) fin deformity showing underdeveloped pectoral fin and folded tail fin.

TABLE 2. Gross and Microscopic Pathology of Juvenile Splittail at 5 and 9 Months of Dietary Exposure to Selenized Yeast diet concentrations (mg of Se kg-1)a 0.4 Liver macrophage aggregate glycogen depletion single cell necrosis fatty vacuolar degeneration eosinophilic protein droplets sum of mean lesion scores

Histopathologyb

0.7

1.4

2.7

(Mean Lesion Scores, N ) 15) at 5 Months 0.07 0.20 0.27 0.27 0.07 0 0.07 0.07 0 0 0 0.13 0 0 0.20 0.27 0 0 0 0 0.14 0.20 0.54 0.74

Gross Pathology (No. of Deformities, N ) 15) at 5 Months facial deformities (eye, jaw, and mouth) 0 0 0 1 skeletal deformities (kyphosis, lordosis, scoliosis) 0 0 0 1 prevalence of deformity (%) 0 0 0 13.3 macrophage aggregate glycogen depletion single cell necrosis fatty vacuolar degeneration eosinophilic protein droplets sum of mean lesion scores

Liver Histopathologyb (Mean Lesion Scores, N ) 15) at 9 Months 0.13 0.07 0.20 0.27 0 0 0.20 0 0 0 0 0.07 0 0 0 0.20 0 0 0 0 0.13 0.07 0.4 0.54

Gross Pathology (No. of Deformities, N ) 15) at 9 Months facial deformities (eye, jaw, and mouth) 0 1 0 1 body deformities (kyphosis, lordosis, scoliosis) 0 0 4 2 prevalence of deformity (%) 0 6.7 26.7 20.0

6.6

12.6

26.0

57.6

0.07 0 0 0.27 0 0.34

0.20 0 0.07 0.07 0 0.34

0.13 0.27 0.07 0.53 0.60 1.60

0.53 0.67 0.07 0.2 0.40 1.87

0 1 6.7

0 1 6.7

0.20 0 0.07 0.20 0.07 0.54

0.85 1.38 0.46 0.08 0.85 3.62

0 1 6.7

0 0 0

2 1 20.0 0.40 0.40 0.13 0.53 0 1.46 5 3 53.3

1 3 26.7 0.20 0.20 0 0.07 0 0.47 3 1 26.7

a Diet dry weight. b Liver lesion severity scoring were based on 0 ) not present or infrequently observed, 1 ) mild (affected less than 10% of the liver), 2 ) moderate (affected greater than 10% but less than 50% of liver, and 3 ) severe (affected greater than 50% of the liver). Macrophage aggregate is characterized as a cluster of macrophages packed with coarsely granular yellow-brown pigment. Glycogen depletion is characterized by decreased size of hepatocytes, loss of the “lacy”, irregular, and poorly demarcated cytoplasmic vacuolation typical of glycogen and increased cytoplasmic basophilia (i.e., blue coloration). Single cell necrosis is characterized by cells having eosinophilic (i.e., pink coloration) cytoplasm with nuclear pyknosis and karyorrhexis. Fatty vacuolar degeneration or lipidosis is characterized by excess lipid appears as clear, round, and welldemarcated cytoplasmic vacuoles. Eosinophilic protein droplets are characterized by the presence of proteins appearing as refractile, eosinophilic (pink coloration), round, and well-demarcated cytoplasmic vacuoles.

(Figure 2A). Therefore, muscle Se concentrations were selected to establish a dose-response relationship between the tissue Se burden and the growth parameter body weight (Figure 2C), which was identified to be more responsive to the dietary Se concentrations than CF (Figure 1B,C). The dose-response curve showed that body weight linearly decreased with the increases of muscle Se concentrations. In comparison with liver Se concentrations between 5and 9-month exposure, there were no significant differences at the same dietary treatments (Figure 2A), indicating that Se accumulation in the liver reached their equilibrium levels after 5-month. However, liver Se concentrations kept increasing with the increases of dietary Se concentrations by 5- and 9-month. The similar trend had shown in muscle Se concentration as well after 9-month exposure. Gross Morphology and Histopathology. Gross morphological examination revealed several types of deformities (Figures 3-5) in fish exposed to 2.7-57.6 mg of Se kg-1 diets at 5-month and 0.7-26.0 mg of Se kg-1 diets at 9-month (Table 2). No deformity was observed in fish fed 0.4 mg of

Se kg-1 control diet (Table 2). Prevalences of deformities were higher in fish fed 6.6 and 12.6 mg of Se kg-1 diets than in fish fed other diets for both 5- and 9-month exposure (Table 2). The dose-response relationship between gross deformity and dietary exposure to Se did not follow expectations (Table 2). Except for the significant smaller size and anorexic appearance (Table 2; Figure 3), no gross deformities were observed in fish fed 57.6 mg of Se kg-1 diet at 5- and 9-month (Table 2). Abnormal swimming behaviors characterized as swimming belly up or lying motionless on the bottom of the tank were only observed in fish fed 26.0 and 57.6 mg of Se kg-1 diet at the end of 5- and 9-month. No dose-response relationship in liver histopathology was observed at 5- and 9-month (Table 2). However, several types of histological changes in liver were associated with Se exposure including cytoplasmic glycogen depletion, fatty vacuolar degeneration, eosinophilic protein droplets, and macrophage aggregate (cf. Figure 6 vs Figures 7 and 8). Reduction in liver glycogen was associated with an apparent increase in cytoplasmic basophilia of these hepatocytes VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Liver section of juvenile splittail fed 0.4 mg of Se kg-1 diet shows normal morphology of glycogen-rich hepatocytes (arrows) at the end of 9-month exposure. Hematoxylin and eosin stain. Bar ) 20 µm.

FIGURE 7. Liver section of juvenile splittail fed 6.6 mg of Se kg-1 diet shows severe glycogen depletion (basophilic cytoplasm) and moderate fatty vacuolar degeneration (arrows) at the end of 9-month exposure. Arrowheads point to pyknotic cells with eosinophilic cytoplasm. MA ) macrophage aggregate. Hematoxylin and eosin stain. Bar ) 20 µm. (Figures 7-8). Fish exposed to 26.0 mg of Se kg-1 diet had higher mean lesion scores for hepatic fatty vacuolar degeneration [prevalence of lesion (PL) ) 3 of 15 fish] and cytoplasmic protein droplets (PL) 6) while fish exposed to 57.6 mg of Se kg-1 diet had higher mean lesion scores for macrophage aggregates (PL ) 7) and glycogen depletion (PL ) 5) at 5-month (Table 2). Higher mean lesion scores were observed for (i) hepatic fatty vacuolar degeneration (PL ) 5) in fish exposed to 6.6 mg of Se kg-1 diet and (ii) 6090

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macrophage aggregates (PL ) 10), glycogen depletion (PL ) 8), single cell necrosis (PL ) 3), and cytoplasmic protein droplets (PL ) 7) in fish exposed to 57.6 mg of Se kg-1 diet at 9-month (Table 2).

Discussion Selenium is a nutritionally essential trace mineral but can become toxic to fish at concentrations not much higher than the nutritional requirement, that is, at 3-10 mg of Se kg-1

FIGURE 8. Moderate eosinophilic protein droplets (arrowheads) and mild fatty vacuolation (arrows) and glycogen depletion in liver of juvenile splittail exposed to 26.04 mg of Se kg-1 diet for 5 months. Hematoxylin and eosin stain. Bar ) 20 µm. in the diet (13, 21, 32). In this study, the dietary Se doses were positively correlated with reduced growth and survival at the end of 5 and 9 months of feeding. However, no apparent dose-response relationships were evident for gross deformity at 5- and 9-month dietary Se exposure. Liver histopathology was dose-dependent at 5-month but not at the end of 9-month dietary Se exposure. Overall, the results indicate that chronic Se toxicity is a complex interplay between dose and duration of exposure. In addition, we conclude that gross deformity and histopathology were more sensitive indicators of toxicity than growth, as commonly recognized. Namely, growth and mortality were only significant at 26.0 and 57.6 mg of Se kg-1 diet while deformities and histopathology became significant at 6.6 mg of Se kg-1 diet in juvenile splittail. The latter corresponded to threshold tissue burdens of 26.8 ( 4.0 (liver) and 15.1 ( 0.4 (muscle) mg of Se kg-1 DW for juvenile splittail, which are approximately two times higher than those proposed by Lemly (32). These results suggest that benthivore juvenile splittail is probably more tolerant to Se than higher trophic level fish species such as bluegill sunfish (Lepomis macrochirus), chinook salmon (Oncorhynchus tshawytscha), fathead minnow (Pimephales promelas), and striped bass (Morone saxatilis) (13, 14, 21, 24, 25, 32, 33) where growth and survival were adversely affected in juvenile fish fed diets in the range of 6.5-9.6 mg of Se kg-1 (32). Therefore, from the food chain transfer standpoint, the insensitivity of splittail to Se may pose a significant threat to higher trophic level predator fish such as salmon and striped bass in the San Francisco Estuary. Se bioaccumulation from diet into the liver of juvenile splittail seemed to reach equilibrium by 5-month exposure as the Se tissue burden patterns were similar between 5- and 9-month exposure (cf. Figure 2A). Although the highest BCF found was in liver with a factor of 54, Se burden of muscle (maximal BCF ) 18) was more responsive to dietary Se concentrations than that of liver (Figure 2B). These results indicate that muscle may be better suited for evaluating Se bioaccumulation in splittail than liver.

Although splittail fed 26.0 and 57.6 mg of Se kg-1 diets grew significantly slower, had higher mortality, and accumulated higher Se burden in both liver and muscle (p < 0.05), the reason is not clear for the lower prevalence of gross deformity in fish fed these Se concentrations. However, it is conceivable that sublethal Se effects in liver may have played a significant role in lowering the incidence of deformity for the highest Se diets. First, 26.0 and 57.6 mg of Se kg-1 diets were not lethal to the juvenile splittail but caused sublethal effects including abnormal feeding (i.e., anorexia; Figure 3C) and swimming behavior (i.e., lying at the bottom of the tank). Although feeding activity was not measured, higher frequency of uneaten food left in fish exposed to 26.0 and 57.6 mg of Se kg-1 diets was observed. In addition, swimming mobility of those fish was apparently reduced (personal observations). However, significantly higher liver and muscle Se concentrations were observed in fish exposed to 26.0 and 57.6 mg of Se kg-1 diets (Figure 2A,B), which suggest that food intake was not completely avoided but likely significantly reduced. The low food intake, slow mobility, weight loss, and muscle wasting (Figure 3) were typical signs of cachexia-anorexia syndrome resulting from chronic malnutrition and Se toxicity. When livers from these fish were examined histologically, sublethal Se toxicity was evidenced by the presence of higher mean lesion scores for macrophage aggregates, glycogen depletion, and single cell necrosis (Table 2). Macrophage aggregates have been proposed and used as indicators of contaminant exposure and, more often, as a generalized nonspecific response to stresses such as starvation, heat stress, bacterial infections, parasitic infestation, and aging (34-36). Thus, it is reasonable to assume that macrophage aggregates signify prior cell necrosis resulting from Seassociated starvation. Consistent with the above assumption is the increase in single cell necrosis in livers exposed to the two highest Se diets. Single cell necrosis is an irreversible cellular injury as a result of progressive failure to synthesize new structural and metabolic components of the cell to repair Se-induced damages, leading to cell death. Single cell necrosis and VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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macrophage aggregates may be linked to glycogen depletion, which could result from increased glycogenolysis in liver due to starvation. This effect may reflect the excessive energy and precursor demand for tissue repairs. On the basis of these observations, we believe that starvation and liver incapacitation may be the major contributing factors to decreased growth and increased mortality in fish fed 57.6 mg of Se kg-1 diet. In addition, the observation of cytoplasmic protein droplets and fatty vacuolar degenerations in Se-exposed hepatocytes (Figures 6-8) could reflect altered structural protein turnover and abnormal lipid metabolism (e.g., lipid peroxidation), which could in turn be related to failure of the liver to metabolize and excrete biochemicals. Therefore, it is possible that the lack of gross deformities in fish fed 57.6 mg of Se kg-1 diet in this study is due to (i) a failure of the liver to metabolically transform Se to form(s) that can exert the sublethal deformities effects and/or (ii) the inability of the liver to excrete the active form(s) to the blood stream for transport to the target organs. In comparison, at lower Se doses (most notably 6.6 mg of Se kg-1 diet for 9-month exposure), higher prevalence (53.3%) of deformity and hepatic fatty vacuolar degeneration was prominent while glycogen depletion and protein droplets were less evident in juvenile splittail. These morphological effects could reflect excessive lipid peroxidation (37-39), but maintenance of metabolic activity so that generation of toxic Se form(s) and transport to target organs may occur. As discussed above, deformity effects (Table 2) are both doseand time-dependent. In this context, lower toxic doses of Se could maintain appropriate liver functions over a longer period of time than excessively high Se doses to cause morphological changes. Because of the high variability in environmental factors and varieties of natural and anthropogenic stressors, it is likely that juvenile splittail with irreversible spinal deformities are less likely to survive to adult stage in the wild. In summary, the dietary threshold for chronic toxic effects of selenium is difficult to define and appeared to be both time- and dose-dependent. These findings indicate the importance of evaluating the chronic effect of Se using multiple end points over a longer period of exposure. Results also indicate that diet concentrations above 26.0 mg of Se kg-1 directly affect growth and survival of juvenile splittail in the lab while diet of 2.71-12.57 mg of Se kg-1 may depend on ecological dietary factors. Thus, the effects observed in this study will need to be taken into consideration together with comprehensive food web pathways of Se (40) in order to gain an understanding of the long-term effect on the health of the splittail population in the San Francisco Estuary. This is the first-ever study addressed the chronic effects of dietary selenium on a California native fish species Sacramento splittail (P. macrolepidotus). Chronic exposure to 6.6 mg of Se kg-1 diet induced deleterious health effects (e.g., deformity and histopathology) that can potentially impact survival of juvenile splittail in the wild.

Acknowledgments This study was funded by CALFED 99-N07, California State Water Resources Control Board (STF1153 & STF1168), and UC Toxic Substances Research and Teaching Program. Facility was provided by the Center for Aquatic Biology and Aquaculture at University of CaliforniasDavis.

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Received for review March 24, 2004. Revised manuscript received August 17, 2004. Accepted September 2, 2004. ES049545+

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