ELSEVIER Aquatic Toxicology 57 (2002) II-26 

Review 

AQUATIC 

TOXICOlOGY 

www.elsevier.com/locate/aquatox 

Role of selenium toxicity and oxidative stress in aquatic 

birds 

David J. Hoffman ~ ~ 

* 

USGS Patuxent Wildlife Research Center, 12011 Beech Forest Road, Laurel, lvJD 20708-4041, USA 

Received 19 March 2000; received in revised form 18 July 2000; accepted 4 August 2001 

Abstract 

Adverse effects of selenium (Se) in wild aquatic birds have been documented as a consequence of pollution of the 

aquatic environment by subsurface agricultural drainwater and other sources. These effects include mortality, 

impaired reproduction with teratogenesis, reduced growth, histopathological lesions and alterations in hepatic 

glutathione metabolism. A review is provided, relating adverse biological effects of Se in aquatic birds to altered 

glutathione metabolism and oxidative stress. Laboratory studies, mainly with an organic form of Se, selenomethionine, have revealed oxidative stress in different stages of the mallard (Anas platyrhynchos) life cycle. As dietary and 

tissue concentrations of Se increase, increases in plasma and hepatic GSH peroxidase activities occur, followed by 

dose-dependent increases in the ratio of hepatic oxidized to reduced glutathione (GSSG:GSH) and ultimately hepatic 

lipid peroxidation measured as an increase in thiobarbituric acid reactive substances (TBARS). One or more of these 

oxidative effects were associated with teratogenesis (4.6 ppm wet weight Se in eggs), reduced growth in ducklings (15 

ppm Se in liver), diminished immune function (5 ppm Se in liver) and histopathological lesions (29 ppm Se in liver) 

in adults. Manifestations of Se-related effects on glutathione metabolism were also apparent in field studies in seven 

species of aquatic birds. Reduced growth and possibly immune function but increased liver: body weight and hepatic 

GSSG:GSH ratios were apparent in american avocet (Recurvirostra americana) hatchlings from eggs containing 9 

ppm Se. In black-necked stilts (Himantopus mexicanus ), which contained somewhat lower Se concentrations, a 

decrease in hepatic GSH was apparent with few other effects. In adult American coots (Fulica americana), signs of 

Se toxicosis included emaciation, abnormal feather loss and histopathological lesions. Mean liver concentrations of 28 

ppm Se (ww) in the coots were associated with elevated hepatic GSH peroxidase, depletion of hepatic protein bound 

thiols and total thiols, but a small increase in GSH. Diving ducks in the San Francisco Bay area exhibited a positive 

correlation between hepatic Se concentration and GSH peroxidase activity (r = 0.63, P < 0.05), but a negative 

correlation between hepatic Se and GSH concentration (r = - 0. 740, P < 0.05). In willets ( Catoptrophorus semipalmatus) from the San Diego area, positive correlations occurred between hepatic Se concentration and GSSG (r = 0. 70, 

P < 0.001), GSSG:GSH ratio, and TBARS. In emperor geese (Chen canagica) from western Alaska, blood levels of 

up to 9.4 ppm occurred and were associated with increased plasma GSH peroxidase activity (r = 0.62, P < 0.001), but 

with decreased plasma GSSG reductase activity. When evaluating Se toxicity, interactive nutritional factors, including 

other elements and dietary protein, should also be taken into consideration. Further studies are needed to examine 

*Tel.: + 1-301-497-5712; fax: + 1-301-497-5675. 

E-mail address: david_hoffman@usgs.gov (D.J. Hoffman). 

0166-445Xj02f$ - see front matter. Published by Elsevier Science B.V. 

PII: S0166-445X(01)00263-6 
12 D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 

the relationship between different forms of environmentally occurring selenium, arsenic and mercury on reproduction, 

hepatotoxicity and immune function of aquatic birds. Further selenium nutritional interaction studies may also help 

to illucidate the mechanism of selenium induced teratogenesis, by optimizing GSH and other antioxidant defense 

mechanisms in a manner that would stabilize or raise the cell's threshold for susceptibility to toxic attack from excess 

selenium. It is concluded that Se-related manifestations of oxidative stress may serve as useful bioindicators of Se 

exposure and toxicity in wild aquatic birds. Published by Elsevier Science B.V. 

Keywords: Selenium; Aquatic birds; Mallard (Anas platyrhynchos); Glutathione; Oxidative stress; Hepatic 

1. Introduction 

Agricultural drainwater, sewage sludge, fly ash 

from coal-fired power plants, and mining of phosphates and metal ores contribute to the selenium 

(Se) burden in the aquatic environment (Eisler, 

1985; Heinz, 1996). Historically, plant-derived Se 

in excess of 4 ppm in the diet of chickens was 

found to impair reproductive success and to be 

teratogenic (Poley and Moxon, 1938). In more 

recent years, adverse effects of Se in wild aquatic 

birds have been documented as a consequence of 

pollution of the aquatic environment by subsurface agricultural drainwater and other sources. At 

the Kesterson National Wildlife Refuge, in the 

San Joaquin Valley of California, mean Se concentrations of 73 mg/kg occurred during 1983 in 

submerged rooted aquatic plants that were consumed by ducks, and levels of selenium averaged 

over 100 mg Se/kg in aquatic insects (Ohlendorf, 

1989). Biological and physiological effects observed at Kesterson included avian mortality, impaired reproduction and teratogenesis, and 

histopathological lesions with alterations in hepatic glutathione (Ohlendorf et al., 1986, 1988; 

Hoffman et al., 1988; Ohlendorf and Skorupa, 

1989). Similar problems have been discovered· 

elsewhere in the western United States in at least 

a dozen different locations (Skorupa, 1998) but 

most notably in the Tulare Basin in California 

(Skorupa and Ohlendorf, 1991). 

~elenomethionine is a major form of Se in 

wheat (Olson et al., 1970), soybean protein (Yasumoto et al., 1988), and may be a major form of 

s~ in other plants . eilstein an Whanger, 1987). 

Th1s form of Se is most likely an important 

/t (exposure source for wild aquatic birds since the 

toxic thresholds of the form in eggs for decreased 

hatching success and teratogenicity from lab studies with mallards are very close to those derived 

from field studies (Heinz, 1996). Furthermore, the 

types and frequency of malformations caused in 

lab studies by selenomethionine are very similar 

to those seen in nature (Heinz et al., 1987, 1989; 

Hoffman and Heinz, 1988). Selenomethionine becomes particularly embryotoxic and teratogenic 

when it exceeds 4 ppm Se in the laboratory diet of 

mallards. At higher concentrations it becomes 

toxic to other stages of the mallard life cycle. 

Mallard ducklings exposed for 6 weeks to dietary 

Se as selenomethionine suffered reduced survival 

and growth, with dose-dependent Se accumulation in the liver (Heinz et al., 1988) and hepatotoxicity characterized by oxidative stress 

(Hoffman et al., 1989). Other studies have 

quantified and described histopathological lesions 

of selenomethionine in adult mallards (Albers et 

al., 1996; Green and Albers, 1997). 

Dickson and Tappel (1969) first proposed that 

Se toxicity is related to changes in intracellular 

concentrations of reduced glutathione (GSH), and 

excess Se has been shown to interact with cellular 

sulfhydryls (Combs and Combs, 1986). Most 

mammalian studies have examined effects of excess dietary Se in the selenite form. Ganther 

(1986) demonstrated that Se at the oxidant state 

of selenite undergoes reductive metabolism, utilizing GSH and NADPH as a source of reducing 

equivalents. LeBoeuf et al. (1985) reported that 

3.0-6.0 ppm Se as sodium selenite in the diet of 

rats for 6 weeks caused a significant dose-dependent increase in hepatic GSH, in oxidized glutathione (GSSG), and in the GSSG:GSH ratio. 

When isolated hepatocytes were incubated with 

selenite (30-100 J.LM) in vitro, a depletion of 

thiols including GSH was observed (Anundi et al., 
D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 13 

1984). Other studies revealed that selenite stimulates lipid peroxidation in vitro and in vivo 

(Dougherty and Hoekstra, 1982; Csallany et al., 

1984). Spallholz (1994) concluded that Se toxicity 

manifests itself when oxidative damage exceeds 

antioxidant defenses or the ability of animals to 

form selenoproteins, selenoethers or elemental Se. 

Studies with mallards have compared Se as selenite and selenomethionine, but with further focus 

on selenomethionine because of its likely biological role in nature. These studies have revealed 

oxidative stress in different stages of the mallard 

life cycle. Manifestations of the oxidative stress · 

caused by selenomethionine included alterations 

in hepatic glutathione metabolism towards a more 

oxidized state, accompanied by lipid peroxidation 

(Hoffman and Heinz, 1988; Hoffman et al., 1989, 

1991a). In these studies, as dietary and subsequent 

tissue concentrations of Se were gradually increased, increases in plasma and hepatic GSH 

peroxidase activities occurred, followed by dosedependent increases in the ratio of hepatic oxidized glutathione (GSSG) to reduced glutathione 

(GSH), and ultimately increased hepatic lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS). 

The purpose of this paper is to provide a review, interpretive discussion, and future direction 

for studies with aquatic birds on biological effects 

of excess Se as related to glutathione metabolism 

and oxidative stress. Studies measuring these end 

points include: (1) laboratory studies, mainly with 

mallards and (2) field studies with seven species of 

aquatic birds. While many of these investigations 

were an integration of laboratory and field efforts, 

with the latter related to aquatic birds mainly in 

California, USA, the responses and mechanisms 

are of interest to aquatic toxicologists in general. 

2. Laboratory studies and selenium-related 

oxidative stress in aquatic birds 

2.1. Reproductive effects in mallards and 

black-crowned night herons 

Findings from reproductive studies have indicated that Se as selenomethionine is considerably 

more teratogenic and generally more embryotoxic 

than sodium selenite, probably due in part to 

much higher accumulation in eggs of Se when 

provided as selenomethionine in the diet than as 

sodium selenite (Table 1). When mallards were 

fed a control diet, diets containing 1, 5, 10, or 25 

ppm Se as sodium selenite, or a diet containing 10 

ppm Seas seleno-D,L-methionine, Seat 10 ppm as 

selenomethionine or 25 ppm as sodium selenite 

caused a 40-44% decrease in the total number of 

eggs that hatched compared with controls (Heinz 

et al., 1987; Hoffman and Heinz, 1988). Ten ppm 

Seas selenomethionine was more teratogenic than 

sodium selenite at 25 ppm. Selenomethionine (1 0 

ppm Se) resulted in an incidence of 13.1% malformations that were often multiple, whereas, 

sodium selenite (10 and 25 ppm Se) resulted in 3.6 

and 4.2% malformations. The teratogenicity of 

selenomethionine was confirmed in a second experiment in which mallards received 1, 2, 4, 8, or 

16 ppm Se as selenomethionine, resulting in 0.9, 

0.5, 1.4, 6.8, and 67.9% malformations, respectively (Heinz et al., 1989). These malformations 

included hydrocephaly, microphthalmia, lower 

bill defects and foot defects with ectrodactyly. 

Both forms of Se increased the incidence of edema 

and stunted embryonic growth. Selenomethionine 

(10 ppm Se) resulted in a significant increase of 

approximately, 40% in plasma glutathione peroxidase activity and a 70% increase in sorbitol dehydrogenase activity (indicative of hepatotoxicity) in 

hatchlings (Hoffman and Heinz, 1988). Sodium 

selenite (25 ppm Se) resulted in fourfold elevation 

in plasma uric acid concentration, indicative of 

renal alteration. 

Hatching success of eggs laid by black -crowned 

night-herons (Nycticorax nycticorax) fed a 10 

ppm Se diet, as seleno-D,L-methionine was not 

different from eggs laid by herons fed an unsupplemented diet (Smith et al., 1988). Developmental malformations commonly associated with Se 

exposure in other birds were not observed in 

heron embryos or hatchlings in the 10 ppm group. 

However, 3-day-old hatchlings from herons fed 10 

ppm Se had shorter radius-ulna and femur lengths 

and higher hepatic TBARS (malondialdehyde) 

concentrations than controls. Se residues in heron 

eggs were lower than those reported in mallards 
Table I 

Laboratory studies on effects of excess selenium on glutathione metabolism and oxidative stress in aquatic birds 

Species, age Form of dietary Se (concentration, Observation Effects R

ppm) period 

Mallard, embryo sodium selenite (10) Through 0.5 ppm Se (ww) in eggs, decreased hatching H

hatching success, caused edema and decreased growth, 

increased plasma GSH peroxidase activity (25) 1.3 ppm Se (ww) in e:ggs, caused edema and 

decreased hatching success and growth, increased 

plasma GSH peroxidase activity and uric acid 

concentration 

Seleno-D,L-methionine 

(10) 4.6 ppm Se (ww) in eggs, caused malformations and 

decreased hatching, increased plasma GSH 

peroxidase and SDH activities 

Black-crowned night Seleno-D,L-methionine (10) Through 3.3 ppm Se (ww) in eggs, decreased growth, S

heron, embryo hatching 

increased hepatic TBARS concentration 

Mallard, 

duckling Sodium selenite (10) Day-old 5.0 ppm Se (ww) in liver, increased hepatic GSSG H

through 6 weeks 

reductase activity and GSH concentration et 

(20) 3.2 ppm Se (ww) in liver, decreased growth, 

increased hepatic GSH and GSSG concentrations, 

decreased hepatic protein concentration 

(40) 

2.8 ppm Se (ww) in liver, decreased survival and 

growth, increased plasma GSSG reductase activity, 

hepatic GSH, GSSG and TBARS concentrations, 

decreased hepatic GSH peroxidase activity, hepatic 

total thiol, and protein concentrations Mallard, duckling Seleno-D,L-methionine 

(10) Day-old 4.8 ppm Se (ww) in liver, increased plasma and H

through 6 weeks 

hepatic GSH peroxidase activities et 

(20) 26 ppm 

Se (ww) in liver, decreased growth, 

increased plasma GSH peroxidase activity, hepatic GSSG:GSH ratio, and TBARS concentration, 

decreased hepatic GSH, total thiol, and protein concentrations 

(40) 68 ppm Se (ww) in liver, decreased survival and 

growth, increased plamna GSSG reductase activity, hepatic GSSG, GSSG:GSH ratio, TBARS 

concentration, decreased hepatic GSH, total thiol 

and protein concentrations Mallard, duckling Seleno-D,L-methionine (15) 4 weeks 10.8, 11.6, 14.9 ppm Se (ww) in liver, decreased H

growth, increased hepatic and plasma GSH 19

peroxidase activities 
Table I (Continued) 

Species, age 

Mallard, adult 

Mallard, adult 

Form of dietary Se (concentration, Observation 

ppm) period 

(60) 

Sodium selenite in drinking water 12 weeks 

(3.5 mg/1) 

Selenomethionine in drinking water 12 weeks 

(2.2 mg/1) 

Seleno-D,L-methionine (2) 14 weeks 

(4) 

(8) 

(16) 

(32) 

Effects 

49, 56, 72 ppm Se (ww) in liver, decreased survival 

and growth, increased histopathological lesions, 

plasma and hepatic GSH peroxidase activities, 

GSSG:GSH ratio, TBARS, and plasma AST, 

decreased plasma alkaline phosphatase activity, 

hepatic GSH and total thiol concentrations 

Refe

Increased serum AL T Fair

5 ppm (ww) Se in liver, impaired immune function, 

increased serum AL T, and plasma GSH peroxidase 

activities 

2 ppm Se (ww) in liver, increased plasma GSH Hof

peroxidase activity 

4 ppm Se (ww) in liver, increased plasma GSH 

peroxidase activity 

12 ppm Se (ww) in liver, increased plasma GSH 

peroxidase activity, hepatic GSSG concentration and 

GSSG:GSH ratio 

21 ppm Se (ww) in liver, decreased hemoglobin 

concentration, increased plasma GSH peroxidase 

activity, hepatic GSH and TBARS concentrations 

29 ppm Se (ww) in liver, decreased survival, body 

weight, hematocrit, and hemoglobin concentration, 

increased hepatic lesions, plasma GSH peroxidase and 

alkaline phosphatase activities, total protein 

concentrations, hepatic GSH and TBARS 

concentrations, decreased DNA:RNA ratio 
16 D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 

fed the same amount of Se. Results of this study 

suggested that herons may be less sensitive to the 

toxic effects of Se than mallards. 

2.2. Survival and growth in mallard ducklings 

Se as selenomethionine resulted in generally 

greater oxidative stress than did Se as selenite, as 

reflected by effects on hepatic glutathione 

metabolism and lipid peroxidation in mallard 

ducklings (Hoffman et al., 1989). These findings 

may reflect the much greater accumulation of Se 

in the liver and other organs as dietary selenomethionine than as selenite. However, both forms of 

Se reduced duckling growth and survival (Heinz 

et al., 1988). When day-old ducklings were fed 

0.1, 10, 20, or 40 ppm Seas seleno-D,L-methionine 

or sodium selenite for 6 weeks, Se from selenomethionine accumulated in a dose-dependent manner 

in the liver. Hepatic and plasma GSH peroxidase 

activity was initially elevated at 10 ppm Se as 

selenomethionine, whereas, GSSG reductase activity was elevated at higher dietary concentrations of Se. A decrease in the concentration of 

hepatic-reduced glutathione (GSH) and total hepatic thiols (SH) at 20 ppm Se in the diet was 

accompanied by an increase in the ratio of oxidized glutathione (GSSG) to GSH, and an increase in TBARS concentration as evidence of 

lipid peroxidation (Hoffman et al., 1989). 

Se from selenite. accumulated in the liver to an 

apparent maximum at 10 ppm in the diet, resulting in an increase in hepatic GSH and GSSG, 

accompanied by a small decrease in hepatic total 

SH. Sodium selenite resulted in increased hepatic 

GSSG reductase activity at 10 ppm and in plasma 

GSSG reductase activity at 40 ppm. A small 

increase in lipid peroxidation occurred at 

40 ppm. 

Further studies with Se in ducklings confirmed 

the previous findings for selenomethionine, including dose-dependent oxidative stress (Hoffman 

et al., 199lb, 1992a,b). In these studies, decreased 

growth with increased plasma and hepatic GSH 

peroxidase activities occurred at 15 ppm Se in the 

diet. At 60 ppm Se in the diet, decreased survival 

and growth with increased histopathological lesions occurred. Other effects included, decreased 

plasma alkaline phosphatase act1v1ty related to 

decreased bone growth, and increased plasma aspartate aminotransferase (AST) activity related to 

hepatotoxicity. Pronounced oxidative stress included decreased hepatic GSH and total thiol 

concentrations with increased ratio of 

GSSG:GSH and TBARS concentration. 

Recent studies have suggested that seleno-L-methionine may be more toxic to ducklings than 

seleno-D,L-methionine under certain dietary conditions; effects on survival and growth revealed 

that Se as seleno-L-methionine in a wheat-based 

diet resulted in higher mortality at 30 ppm Se 

than did either seleno-D,L-methionine or other Se 

groups that were studied, including selenized yeast 

and wheat (Heinz et al., 1996; Hoffman et al., 

1996). In contrast, when a different and commercially-based basal diet was provided, survival of 

ducklings was not affected by 30 ppm Se. Many 

of the previous avian toxicity studies with selenomethionine have used the mixture of 

stereoisomers, seleno-D,L-methionine, that was 

commercially available. Therefore, it is possible 

that under certain dietary conditions, the potential toxicity of Se as naturally occurring seleno-Lmethionine may have been underestimated. Se as 

selenized yeast was found to be less toxic than 

seleno-D,L-methionine, seleno-L-methionine, or 

selenized wheat, as reflected by fewer effects on 

growth, plasma alkaline phosphatase activity, and 

hepatic oxidative stress. Here, greater oxidative 

stress for DL and L diets was manifested by significant decreases in hepatic GSH and total SH. This 

may be related in part to the nearly double tissue 

accumulation of Se from selenomethionine diets 

than from selenized yeast. 

2.3. Subchronic effects on immune function and 

hepatotoxicity in adult mallards 

The subchronic effects of selenomethionine and 

sodium selenite on several immunologic, hematologic, and serologic parameters in adult male mallards were measured, using concentrations in 

drinking water of 0, 0.5, or 3.5 mgjl Se (Se) as 

sodium selenite; or 2.2 mg/1 (Se) as selenomethionine for 12 weeks (Fairbrother and Fowles, 

1990). A battery of in vivo and in vitro immuno-
D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 17 

logic assays was performed on each bird throughout the study. The selenomethionine-treated birds 

displayed an impaired delayed-type hypersensitive 

(DTH) response to tuberculin (M. bovis), as measured by the number of positive reactions present 

24 h post purified protein derivative (PPD) challenge. This group also exhibited increased serum 

alanine aminotransferase (ALT) and plasma GSH 

peroxidase activities. Se concentration in the liver 

and breast muscle was significantly elevated 4-

and 14-fold, respectively, over controls. Sodium 

selenite-treated birds did not display any detectable differences in immune function or Se 

accumulation in tissues above that of controls. 

Serum ALT activity was increased in the 3.5 mg/1 

group, although to a lesser extent than in selenomethionine-treated birds. 

Concentrations of Se as sodium selenite did not 

affect the immune system, whereas, low concentrations of selenomethionine (2.2 mgjl Se) appeared to suppress certain aspects of the mallard 

immune response. 

When adult male mallards received a control 

diet (0.2 ppm Se) or diets containing 1, 2, 4, 8, 16, 

or 32 ppm Se as selenomethionine for 14 weeks, 

Se accumulated readily in the liver in a dose-dependent manner, reaching a mean concentration 

of 29 ppm (ww) in the 32 ppm group (Hoffman et 

al., 199la). Dietary Se of 2 ppm or greater increased plasma glutathione peroxidase activity. 

Mortality (10%) and histopathological effects, including bile duct hyperplasia and hemosiderin 

pigmentation of the liver and spleen, occurred in 

the 32 ppm group. These histopathological effects 

were accompanied by lower hemoglobin concentrations (16 and 32 ppm groups) and hematocrit 

(32 ppm group), and elevated plasma alkaline 

phosphatase activity (32 ppm group), indicative of 

cholestatic liver injury. Manifestations of hepatic 

oxidative stress included significant linear dose-response increases in GSSG concentration and ratio 

of GSSG:GSH with increasing Se; both of these 

measurements were significantly greater than controls for groups receiving 8-32 ppm Se. Mean 

hepatic GSH and TBARS concentrations were 

significantly elevated in the 16 and 32 ppm 

groups. 

3. Field studies and selenium-related oxidative 

stress in aquatic birds 

3.1. Effects on growth and immune function in 

avocet and stilt chicks 

Excess Se in the aquatic food chain is embryotoxic and teratogenic to avocets, stilts, and other 

waterbirds. Normal growth and immune function 

may also be compromised (Table 2). Avocets 

(Recurvirostra americana) hatched from eggs collected from the south Central Valley of California 

were studied to determine the impact of elevated 

concentrations of Se, , arsenic, and boron (B) on 

the immune system and growth to maturity (Fairbrother et al., 1994). The reference site for this 

study had low Se (1.2 ppb), As (29 ppb) and (B 

not measured) in the \.Vater~ The study site with 

the highest concentrations of Se ( 450 ppb ), also 

had elevated As (127 ppb) and B (109 ppm). Size 

at hatch and growth rate of chicks was significantly reduced at the highest Se site. Chicks from 

that site exposed solely through in ovo transfer of 

these elements had significantly increased heterophil:lymphocyte ratios. The phagocytic activity 

of macrophages was also significantly reduced. 

In another study, hatchling morphology and 

oxidative stress were examined in american avocets and black-necked stilts (Himantopus mexicanus). Eggs were collected from three sites in the 

Tulare Lake Basin of California and hatched in 

the laboratory (Hoffman et al., 2002). These sites 

included the Tulare Lake Drainage District-north 

(TLDD-N, water 2.5 ppb Se), TLDD-south 

(TLDD-S, water 8.6 ppb Se) and Westfarmers 

(WF, water 190 ppb Se). Highest egg Se concentrations occurred at WF (geometric mean 9 ppm, 

ww for avocets and 6 ppm for stilts), mean egg Se 

concentrations were 2 ppm for avocets and 2.5 for 

stilts at TLDD-S, and 1 ppm for avocets and 0.7 

for stilts at TLDD-N. Hatching success, incidence 

of malformations, body and organ weights and 

bone lengths did not differ significantly among 

locations. However, ratio of liver to body weight 

was greater for avocets from WF than TLDD-N. 

With increasing Se concentration, hepatic oxidative stress was most apparent in avocet hatchlings 

from WF in which GSH peroxidase activity in-
Table 2 

Field studies related to excess selenium and oxidative stress in aquatic birds 

Species, age Location Tissue Se Effects 

(concentration, 

ppm) 

American avocet (R. americana), hatchling Evaporation ponds in the 4.8 ppm (ww) in Malformations, decreased size at hatch, 

through adult Tulare Basin, CA eggs possible impaired immune function 

American avocet (R. americana), embryo, Evaporation ponds in the 9 [4-17] ppm (ww) Embryo growth, increased liver:body 

hatchling Tulare Basin, CA in eggs weight ratio, increased oxidative stress 

(increased hepatic GSH peroxidase, ratio 

GSSG:GSH, decreased G6PDH activity) 

Black-necked stilt (H. mexicanus), Evaporation ponds in the 6 [1.5-15] ppm Decreased hepatic GSH 

embryo, hatchling Tulare Basin, CA (ww) in eggs 

America 

coot (Fulica americana), adult Kesterson Reservoir, CA. 28 ppm (ww) in Emaciation, abnormal feather loss, 

liver histopathological lesions, decreased hepat

glycogen and protein, increased oxidative 

stress (hepatic GSH peroxidase, total thio

PBSH, GSH) 

Surf scoter (Melanitta perspicillata), adult San Francisco Bay, CA 36 [17-60] ppm Positive correlation between hepatic GSH 

(ww) in liver peroxidase and hepatic Se concentration 

(r = 0.63, P<0.05) 

Ruddy duck ( Oxyura jamaicensis ), adult San Francisco Bay, CA 12 [2-21] ppm Decreased hepatic GSH with increased S

(ww) in liver (r = -0.740, P<0.05) 

Emperor geese (C. canagica), adult Alaska 2.9 [0.1-9.4] ppm Positive correlation between plasma GSH 

(ww) in blood peroxidase and blood Se concentration 

(r = 0.62, P<O.OOl), negative correlation 

between plasma GSSG reductase and 

blood Se concentration (r = -0.44, 

P<0.01) 

Willet (C. semipalmatus ), adult San Diego, CA 1.3 [0.2--4.5] ppm Positive correlation between hepatic Se 

(ww) in liver concentration and the following: GSSG 

(r=0.70, P<0.001), GSSG:GSH (r=0.5

P<0.01), and TBARS (r = 0.55, P<0.05
D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 19 

creased, glucose-6-phosphate dehydrogenase (G6-PDH) decreased, and GSSG concentration increased, as well as, the ratio of GSSG to GSH. 

In stilts, hepatic GSH was lower in WF hatchlings, with few other effects. 

3.2. Selenium toxicosis in adult american coots 

Severe gross and microscopic lesions and 

other changes found in american coot embryos 

and adults from Kesterson Reservoir, Merced 

County, CA were attributed to excess Se, readily 

identified in the water, food items, and tissues 

of the birds (Ohlendorf et al., 1988). Adult birds 

were emaciated, had subacute to extensive 

chronic hepatic lesions and excess fluid and 

fibrin in the peritoneal cavity. Biochemical 

changes in their livers included elevated glutathione peroxidase activity, glycogen and GSH 

concentrations, but lower protein, total 

sulfhydryl, and protein-bound sulfhydryl concentrations. Congenital malformations observed 

grossly in embryos were often multiple and included anophthalmia, micropthalmia, abnormal 

beaks, amelia, micromelia, ectrodactyly, and hydrocephaly. Mean concentrations of Se in livers 

(28 ppm, ww) and kidneys (29 ppm) of adult 

coots collected at the Kesterson ponds were 

about 10 times those found at a nearby control 

area (2.5 and 3. 7 ppm). 

3.3. Selenium and glutathione metabolism in 

diving ducks 

Adult male greater scaup (Aythya marila) 

(GS), surf scoters (Melanitta perspicillata) (SS), 

and ruddy ducks ( Oxyura jamaicensis) (RD) 

were collected from Suisun Bay and coastal 

Tomales Bay in the greater San Francisco Bay 

area to assess exposure to inorganic contaminants. Hepatic Se concentrations were highest in 

GS (geometric mean= 67 ppm, dw) and SS (119 

ppm) in Suisun Bay, whereas, hepatic mercury 

(Hg) was highest (19 ppm) in GS and SS from 

Tomales Bay. Hepatic Se and Hg were lower in 

RD and did not differ between locations. Hepatic GSH-peroxidase activity was higher in SS 

and RD, and G-6-PDH higher in GS and SS 

from Suisun Bay than Tomales Bay. GSSG-reductase was higher in SS from Suisun Bay. The 

ratio of GSSG:GSH was greater in all species 

from Tomales Bay. The following significant relationships were found in one or more species 

with increasing hepatic Hg concentration: body, 

liver and heart weights, hepatic GSH concentration, G-6-PDH and GSH-peroxidase activities 

decreased whereas the ratio of GSSG to GSH, 

and GSSG-reductase increased. With increasing 

hepatic Se concentration, GSH-peroxidase mcreased but GSH decreased. 

3.4. Elevated blood selenium and plasma enzymes 

of emperor geese 

As high as 10 ppm Se (ww) was found in 

blood samples collected from incubating female 

emperor geese (Chen canagica) on their breeding 

grounds of the Yukon-Kuskokwim Delta in 

western Alaska (Franson et al., 1999). Incubating adult females captured in late May through 

mid-June 1997 had higher concentrations of Se 

in their blood (mean= 5.60 ppm) than adult females captured during wing molt in late-July 

1996 (mean= 2.78 ppm). Females that nested 

early or were in good body condition had 

higher concentrations of Se in their blood than 

did other nesting females. Blood samples 

from 4 of 29 goslings had detectable levels 

of Se (mean= 0.14 ppm). Findings suggest that 

emperor geese are exposed to more Se 

in the marine environment of their wintering 

and staging areas on the Alaska Peninsula than 

on the breeding grounds. Mercury levels were 

generally low since the highest concentration of 

Hg found in the blood of emperor geese was 

0.24 ppm in an adult female during wing 

molt, but the mean concentration of Hg was 

0.05 ppm. Hg was not correlated with Se. During the subsequent year, as high as, 11.3 

ppm Se was found in the blood of incubating 

females (mean= 5.0 ppm) and a positive correlation between blood Se concentration and 

plasma GSH peroxidase activity was found but 

a negative correlation was observed with plasma 

GSSG reductase activity (Hoffman, unpublished 

data). 
20 D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 

3.5. Selenium and glutathione metabolism in 

willets 

Willets (Catoptrophorus semipalmatus) that 

were overwintering in southern California near 

San Diego were found through analysis of ingesta 

to be feeding primarily on mollusks and crustacea 

(Hui and Beyer, 1998). Liver Se concentrations of 

the willets were found to contain up to 4.5 pm Se 

(ww), and positive correlations were found between hepatic Se concentration and ratio of hepatic GSSG:GSH, and and TBARS concentration 

(Hoffman and Hui, unpublished data). 

4. Discussion 

Antioxidants are a major resource of most living organisms for protection against diverse free 

radical and other oxidative stressors, including 

excess selenium, to which they invariably become 

exposed (Cross et al., 1987; Griffith, 1999). Glutathione is an important antioxidant and cofactor 

for antioxidant enzymes; high electron-donating 

capacity combined with high intracellular concentration endows GSH with great reducing power, 

which is used to regulate a complex thiol-exchange system (-SH~-s-S-). This functions at 

all levels of cell activity, from the relatively simple 

(circulating cysteinej-SH thiols, ascorbate, other 

small molecules) to the most complex (cellular 

-SH proteins) (Kidd, 1997). Changes in GSH 

status including depletion are thought to constitute an important component of injury to liver 

and other tissues. As cellular GSH is depleted, 

individual cells first die in local areas most affected, followed by zones of tissue damage often 

occurring where content of polyunsaturated lipids 

are highest and/or antioxidant defenses are most 

meager. Therefore, repletion of glutathione appears to be central to intrinsic adaptive strategies 

for meeting the challenge of sustained (or acute) 

oxidative stress (Levine and Kidd, 1985; Kidd, 

1993). 

In keeping with the above, many of the avian 

studies described within this review have reported 

hepatic oxidative stress at different stages of the 

life cycle due to excess dietary Se, mainly in the 

organic form (seleno-D,L-methionine, seleno-Lmethionine or Se incorporated into wheat or 

yeast). Manifestations of the oxidative stress included alterations in hepatic glutathione 

metabolism towards a more oxidized state, accompanied by lipid peroxidation. As dietary and 

subsequent tissue concentrations of Se increased, 

plasma and hepatic GSH peroxidase activities increased, followed by dose-dependent increases in 

the ratio of hepatic oxidized to reduced glutathione (GSSG:GSH), and ultimately increased 

hepatic lipid peroxidation. These dose-related effects were apparent in the four studies with mallard ducklings (Hoffman et al., 1989, 1992a,b, 

1996) and two studies with adult mallards (Fairbrother and Fowles, 1990; Hoffman et al., 1991a). 

Elevated plasma GSH-peroxidase activity is an 

indicator of Se exposure and the onset of oxidative stress in birds (Hoffman et al., 1989, 1991a,b) 

and mammals (Combs and Combs, 1986; 

Whanger, 1986; Levander, 1986). In mallard studies, two factors accounted for the dose-dependent 

increase in ratio of GSSG to GSH: (1) a dose-dependent increase of oxidized glutathione by, as 

much as, 240%; and (2) a dose-dependent depletion of reduced glutathione (GSH) with increasing 

concentrations of hepatic Se, sometimes falling to 

less than 25% of corresponding control values. A 

depletion of total hepatic thio1s often appeared to 

parallel the depletion of GSH but was, as much 

as, twice that of the GSH depletion in terms of 

micromoles of GSH equivalents. Related hepatic 

lesions occurred and included hepatic cell necrosis 

and bile duct hyperplasia. 

Similar findings have been reported for Se as 

selenite under in vitro conditions with isolated rat 

hepatocytes where partial to total depletion of 

thiols occurred with a concomitant increase in 

GSSG and 0 2 consumption (Anundi et al., 1984). 

Excess GSSG can react with protein sulfhydryls, 

contributing to the total thiol and protein bound 

thiol depletions, observed in mallard studies, by 

the formation of mixed glutathione: protein 

disulfides. Formation of mixed disulfi.des may be 

part of a significant mechanism in regulating 

metabolic activity as well as integrity of cell membranes in response to oxidative stress (Keeling et 

al., 1982; Ondarza, 1989; Kidd, 1997). In mallard 
D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 21 

duckling studies, excess dietary Se as selenomethionine had a more pronounced effect than 

sodium selenite on hepatic glutathione 

metabolism and lipid peroxidation as reflected by 

greater GSH and total SH depletions. Differences 

in response between the two forms may be related 

in part to the much higher hepatic accumulation 

of Se as selenomethionine. When excess dietary Se 

was provided as sodium selenite to ducklings, an 

increase in hepatic GSH occurred, accompanied 

by an increase in GSSG similar to that reported in 

laboratory rodents (LeBoeuf et al., 1985). However, the GSSG to GSH ratio did not significantly 

increase, presumably due to an adaptive increase 

in GSH, which most likely represented an attempt 

to maintain a nearly normal ratio of GSSG to 

GSH (LeBoeuf and Hoekstra, 1983). Elevated 

GSSG reductase activity was consistent with this 

observation. Furthermore, selenite had less of a 

stimulatory effect than selenomethionine on lipid 

peroxidation in ducklings, which would appear to 

be a further reflection of the adaptive increase in 

GSH. Such an increase in reducing equivalents 

would combat lipid peroxidation, in contrast to 

the large depletion of GSH and total SH caused 

by selenomethionine. A similar hepatic Se accumulation pattern for selenite has been reported in 

young chickens in which a maximum accumulation occurred at 10 ppm dietary Se with a slight 

decrease at higher dietary levels (Jensen, 1975). 

Manifestations of Se-related oxidative effects 

on glutathione metabolism were also apparent in 

field studies in seven species of aquatic birds at 

different stages of the life cycle including embryonic development. Exposure during embryonic 

development to a variety of environmental contaminants is known to cause in ovo and in utero 

death of embryos and fetuses, and to often initiate 

birth defects (teratogenesis) in the surviving offspring. Yet, limited understanding exists on the 

exact biochemical mechanisms, or the determinants of teratological susceptibility. Recent in 

vitro and in vivo studies primarily based on rodent models suggest that many potential embryotoxic xenobiotics are actually proteratogens that 

must be bioactivated by enzymes including cytochromes P450 or by peroxidases such as 

prostaglandin H synthase to produce reactive intermediary metabolites that are teratogenic (Wells 

and Winn, 1996). These reactive intermediates 

generally are electrophiles or free radicals that 

bind covalently (irreversibly) to, and either directly or indirectly oxidize embryonic cellular 

macromolecules, such as, DNA, protein, and 

lipid, irreversibly altering cellular function. Target 

oxidation, known as oxidative stress, often appears to be mediated by reactive oxygen species 

(ROS), such as, hydroxyl radicals. Largely due to 

immature or otherwise compromised embryonic 

pathways for detoxification, cytoprotection and 

repair, the embryo is relatively susceptible to reactive intermediates, and teratogenesis via this 

mechanism can occur even from exposure to therapeutic concentrations of drugs or supposedly 

safe concentrations of environmental chemicals, 

including Se. In keeping with the above, studies 

with cultured rat embryos that examine the effects 

of depletion of reduced glutathione (GSH) on 

selenium (Se) embryotoxicity concluded that embryonic GSH status plays a role in the embryotoxicity of selenite and selenate (Usami et al., 

1999). There are several mechanisms in which 

selenium may express its embryotoxicity and teratogenicity to the avian embryo. One possible 

mechanism would involve tissue injury as a result 

of oxidative stress, possibly with the formation of 

CH3Se which either enters a redox cycle and 

generates superoxide and oxidative stress, or 

forms free radicals that bind to and inhibit important enzymes and proteins (Spallholz and Hoffman, 2002). Largely due to immature or otherwise 

compromised embryonic pathways for detoxification, cytoprotection and repair, the embryo is 

relatively susceptible to reactive intermediates and 

teratogenesis via this mechanism. In order to test 

this hypothesis further, feeding studies or egg 

injection studies could be done with Se as selenomethionine under conditions of restricted antioxidants or possibly containing an inhibitor of 

GSH synthesis such as buthionine sulfoximine 

(BSO). Comparisons could be conducted with 

baseline controls, as well as other groups containing highly supplemented antioxidants including 

glutathione and/or its precursors, as well as vitamins, such as C and E. Another possible mechanism for selenium teratogenesis in birds may be 
22 D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 

related to the presence of excess selenium analogs 

of sulfur-containing enzymes and structural 

proteins as a consequence of high levels of selenomethionine in the diet. This hypothesis could 

perhaps be further tested by examining the effects 

of other methionine analogues on development 

for potential teratogenesis including type and frequency of malformation and for percent impairment of hatching success by means of egg 

injection and ultimately feeding studies with 

mallards. 

In addition to hepatotoxicity and teratogenesis 

other potentially toxic and biologically relevant 

effects of excess selenium have been reported in 

avian laboratory and field studies. One of these 

effects includes reduced immune function which 

occurred in conjunction with oxidative stress 

(Fairbrother and Fowles, 1990; Fairbrother et al., 

1994) In mammals, the proliferation, growth and 

differentiation of immune cells is dependent on 

GSH and is most likely so in birds as well. Both 

T and B lymphocytes require adequate levels of 

intracellular GSH to differentiate, and with relatively low lymphocyte GSH concentrations were 

found to have significantly lower CD4 counts 

(Kinscherf et al., 1994). Intracellular GSH is also 

required for the T -cell proliferative response to 

mitogenic stimulation, for the activation of cytotoxic T 'killer' cells, (Droge et al., 1994) and for 

many specific T -cell functions, including DNA 

synthesis for cell replication, as well as for the 

metabolism of interleukin-2 which is important 

for the mitogenic response (Wu et al., 1994). 

Experimental depletion of GSH inhibits immune 

cell functions, sometimes markedly and in a number of different experimental systems the intracellular GSH of lymphocytes was shown to 

determine the magnitude of immunological capacity (Fidelus and Tsan, 1987; Droge et al., 1994). 

These and other findings indicate that intracellular GSH status plays a central role in the functioning of immune cells, and that disturbances in 

GSH status by selenium could be potentially disruptive. Further studies are needed to examine the 

relationship between different forms of environmentally occurring selenium as well as interaction 

with other elements including arsenic and mercury 

on immune function. 

When evaluating different aspects of Se toxicity, including biological effects and oxidative 

stress, interactive nutritional factors appear to 

play an important role. Given the great seasonal 

and species variability in diets of wild birds, including aquatic birds, there is good reason to 

believe that different forms of Se may interact 

with nutritional variables in the field to affect the 

health of birds. Important interactive nutritional 

factors studied in mallards have included: concentrations of dietary protein, methionine, and certain other elements, such as As, B and Hg 

(Hoffman et al., 199lb, 1992a,b; Heinz, 1996; 

Heinz and Hoffman, 1998; Stanley et al., 1996). 

Adequate dietary protein, supplemental methionine, As and Hg appear to act antagonistically with 

Se in mallards, ameliorating adverse effects including: mortality, impaired gro\vth, histopathological lesions, and manifestations of oxidative 

stress. Arsenate alone reduces hatching success 

and hatchling survival, but in combination with 

Se ameliorates Se-related reproductive impairment 

and teratogenesis (Stanley et al., 1994). When the 

interaction of arsenic and selenium at the subcellular level in rat kidney cells was studied using 

electronic probe microanalysis that permits detection of elements in the intracellular organelles, 

arsenic and selenium were concentrated and precipitated in the lysosomes of the renal cells in the 

form of insoluble selenide (As2Se) (Berry and 

Galle, 1994). In the long term, the lysosomes and 

their precipitate are eliminated in the urine. This 

intralysosomal concentration and precipitation 

may serve as a model for the mechanism of 

interaction of selenium in other tissues and species. Diving ducks in the San Francisco Bay area 

exhibited positive correlations between hepatic Se 

concentration and GSH peroxidase activity, but a 

negative correlation between hepatic Se and GSH 

concentration (Hoffman et al., 1998). However, 

high concentrations of Hg were a confounding 

factor in this study. Interestingly, in willets in the 

San Diego area where little Hg was apparent, a 

positive correlation between hepatic Se concentration and oxidative stress occurred with only modestly elevated hepatic Se concentrations (Hui et 

al., unpublished data). Similarly in emperor geese, 

there was little apparent blood Hg present, but 
D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 23 

elevated blood Se concentration, resulting in elevated plasma GSH peroxidase activity and decreased plasma GSSG reductase (Hoffman, 

unpublished data). In adult mallards combinations of methylmercury and selenomethionine 

were antagonistic, but appeared additive or synergistic in toxcity to mallard embryos in terms 

of embryo survival and teratogenisis (Hoffman 

and Heinz, 1998). In mammalian studies, when 

selenium compounds were coadministered with 

mercuric mercury, a remarkable depression was 

noted in the acute toxicity of mercury through 

reduction of mercury accumulation in the kidneys, prolonged retention of mercury and selenium as inert high molecular weight complexes 

in the blood, particularly in the erythrocytes, 

and the formation of stable and nondiffusible 

complexes of mercury and selenium in various 

organs such as the liver and kidneys (Imura and 

Naganuma, 1991). Further investigation in birds 

is warranted to compare the interactions of various forms of mercury and selenium particularly 

with respect to effects on reproduction. Other 

selenium and nutritional interaction studies are 

called for that would optimize GSH and other 

antioxidant defenses in a manner that would 

stabilize or raise the cell's threshold for susceptibility to toxic attack from excess selenium. Possible dietary means for attaining this include 

increasing dietary concentration of one or more 

of the following: GSH, L-methionine, 'activated' 

methionine metabolite known as SAM (Sadenosyl methionine), L-cysteine, N-acetyl cysteine (NAC), as well as glycine, L-glutamine, 

and L-taurine. Optimizing antioxidant capacity 

through other sources including vitamins E and 

C could also prove to be beneficial. 

5. Conclusion 

Laboratory studies, mainly with Se as organic 

selenomethionine, have revealed oxidative stress 

in different stages of the mallard (Anas 

platyrhynchos) life cycle. As dietary and tissue 

concentrations of Se increase, increases in 

plasma and hepatic GSH peroxidase activities 

occur, followed by dose-dependent increases in 

the ratio of hepatic GSSG:GSH, and ultimately 

hepatic lipid peroxidation. One or more of these 

oxidative effects were associated with teratogenesis (4.6 ppm wet weight Se in eggs), reduced 

growth in ducklings (15 ppm Se in liver), diminished immune function in adults (5 ppm Se in 

liver) and histopathological lesions (29 ppm Se 

in liver). 

Manifestations of Se-related effects on glutathione metabolism have also been documented 

in field studies in seven species of aquatic birds, 

including american avocets, black-necked stilts, 

american coots, several species of diving ducks, 

emperor geese and willets. Accompanying biological effects in these species ranged from reduced growth and immune function to 

emaciation, histopathological lesions, and mortality. There are several mechanisms in vlhich 

selenium may express its embryotoxicity and teratogenicity to the avian embryo that require further investigation, including: (1) tissue injury as 

a result of oxidative stress; and (2) the action of 

selenium analogs of sulfur-containing enzymes 

and structural proteins as a consequence of high 

levels of selenomethionine in the diet. Intracellular GSH status plays a central role in the functioning of immune cells and disturbances in 

GSH status by selenium could be potentially 

disruptive. Therefore, fur!Jler studies are ne~ 

to examine the relations~rent 

£- s o environmentally occurring selenium as 

well as interaction with other elements incl 

rsemc an mercury on immune function. Further investigation in birds is warranted to compare the interactions of various forms of 

mercury and selenium particularly with respect 

to effects on reproduction. Other selenium and 

nutritional interaction studies are called for that 

would optimize GSH and other antioxidant defenses in a manner that would stabilize or raise 

the cell's threshold for susceptibility to toxic attack from excess selenium. 

In conclusion, Se-related manifestations of oxidative stress may provide an insight into the 

nature of different aspects of selenium related 

toxicosis in birds and serve as useful bioindicators of Se exposure and toxicity in wild aquatic 

birds. 
24 D.J. Hoffman/ Aquatic Toxicology 57 (2002) 11-26 

Acknowledgements 

E.C. Colket and C.A. Erwin provided valuable 

assistance in the preparation of this review. 

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