Nondomestic, Exotic, Wild and Zoo Animals–Brief Communication

Experimentally Induced Selenosis inYellow-Bellied Slider Turtles(Trachemys scripta scripta)

David L. Haskins1,2 , Elizabeth W. Howerth3, and Tracey D. Tuberville1

AbstractSelenosis, or selenium toxicosis, occurs in wildlife and livestock, usually because of excessive intake of selenium via selenium-containing plants. Although it is known that wild slider turtles can accumulate large amounts of selenium, little is known about howselenium exposure may affect these reptiles. In this study, the authors report histopathologic changes in yellow-bellied sliders(Trachemys scripta scripta) caused by experimental exposure to selenomethionine. Microscopic changes in kidney and claw tissuewere most significant and resembled those reported in birds. Turtles in the selenium treatment groups had acute tubulardegeneration and regeneration in the kidney, with hyaline droplets in the high-dose animals, and changes in the claws ranging fromepidermal hyperplasia with disorganization and intercellular edema to ulceration, and accumulation of seroheterophilic exudatebetween the epidermis and cornified layer. Although selenium burdens in this study are comparable with values found in wildslider turtles, more data are needed to determine if similar histopathologic abnormalities arise in wild animals exposed to highlevels of selenium.

Keywordsreptiles, selenomethionine, selenosis, turtles, toxicosis, yellow-bellied slider turtles (Trachemys scripta scripta)

Selenosis, or selenium toxicosis, is usually the result of excess

intake of seleniferous forages but can also occur following

parenteral administration. It is characterized by teratogenesis,

morbidity, and mortality in a variety of vertebrate species.8,12

Previous studies have shown that livestock and aquatic bird

species are sensitive to excess amounts of selenium. When

exposed to toxic levels of selenium, birds had microscopic

changes in the liver, kidneys, and keratinized tissues (eg,

claws).8 Feather loss, liver necrosis, and mild nephrosis were

all common in American coots, pied-billed grebes, and mal-

lards exposed to excess amounts of selenium.8,13 Selenosis in

livestock causes a condition called “alkali disease,” which is

characterized by lack of energy, anemia, sloughing of hooves,

damage to other keratinized tissues, and lameness.15

Although the effects of selenosis have been described in

livestock and waterfowl, the effects of this condition in reptiles

are poorly documented. In fact, aside from an exposure experi-

ment with American alligators (Alligator mississippiensis),

there are no reports of selenosis in reptiles.3 However, previous

research shows that slider turtles in selenium-contaminated

environments can accumulate large amounts of selenium and

other inorganic contaminants.9 Inorganic forms of selenium,

such as selenium salts found in coal ash wastes, can be trans-

formed into organic forms (eg, selenomethionine) by bacteria

and plants, which then can be transferred through the

food web.18 Many species of reptiles are also long-lived

(eg, testudines and crocodilians), which suggests that their

exposure could be much longer relative to other vertebrate

species. Coal combustion residues, which are typically rich in

selenium, cause histologic changes in liver tissue of southern

banded watersnakes (Nerodia fasciata fasciata),6 and these

findings suggest that chronic exposure to coal combustion con-

taminants could lead to deleterious effects (such as negative

impacts on reproduction or survival) in watersnakes.

Yellow-bellied sliders (Trachemys scripta scripta) are geo-

graphically widespread, have well-characterized natural and

life histories, and can represent the greatest biomass of turtles

within aquatic ecosystems in the southeastern United States.2,6

In the summer of 2015, we exposed slider turtles to selenium to

examine its impact on their hematology, immune response, and

1Savannah River Ecology Laboratory, University of Georgia, Aiken, SC, USA2D. B. Warnell School of Forestry and Natural Resources, University of

Georgia, Athens, GA, USA3Department of Pathology, College of Veterinary Medicine, University of

Georgia, Athens, GA, USA

Supplementary material for this article is available online.

Corresponding Author:

David L. Haskins, Savannah River Ecology Laboratory, University of Georgia,

Drawer E, Aiken, SC 29802, USA.

Email: david.haskins@uga.edu

Veterinary Pathology2018, Vol. 55(3) 473-477ª The Author(s) 2017Article reuse guidelines:sagepub.com/journals-permissionsDOI: 10.1177/0300985817750454journals.sagepub.com/home/vet

metabolism.10 Because we observed signs indicative of seleno-

sis, we collected samples for histologic analysis to determine if

selenium exposure caused changes in turtle tissues. Overall, the

objective of this study was to determine if histologic changes

occur in tissues from yellow-bellied slider turtles associated

with experimentally induced selenosis.

Seventy slider turtles (weight range, 154–320 g; plastron

length range, 84–111 mm) were obtained from Concordia Tur-

tle Farms and randomly placed into 1 of 3 treatment groups.

Sliders were dosed weekly via oral gavage (*300 mL) with 0

mg/kg (n ¼ 24), 15 mg/kg (n ¼ 23), or 30 mg/kg (n ¼ 23)

seleno-L-methionine (SeMet) (Sigma S3875; Sigma-Aldrich)

for a total of 5 weeks and were then euthanized. Water was

used as a diluent to create the SeMet mixtures. The dose of

selenium for each turtle was based on individual body weight

and projected weekly food consumption (dose ¼ turtle mass �0.28 � [SeMet]/5,000 mg/kg stock solution, where [SeMet] is

the treatment dose and 0.28 is projected weekly food consump-

tion).1 Doses of SeMet administered in this experiment were

within the range of selenium concentrations reported for prey

species that slider turtles might be expected to consume in

contaminated areas,17 although the stock solution was very

concentrated.

In addition to tissues being harvested for selenium analysis,

liver, kidney, skeletal muscle, and claws, from a subset of each

treatment group (0 mg/kg, n¼ 6; 15 mg/kg, n¼ 5; 30 mg/kg, n

¼ 7), were collected in 10% buffered formalin for histologic

analysis. Claws were decalcified in a 14% neutral ethylenedia-

minetetraacetic acid solution. Formalin-fixed tissues were pro-

cessed routinely and embedded in paraffin, and 4-mm sections

were stained with hematoxylin and eosin. Additional sections

were stained with a modified Brown and Brenn gram stain and

Grocott’s methenamine silver stain for bacteria and fungus,

respectively. For each tissue, severity of change was subjec-

tively scored from 0 to 3, where 0 ¼ none, 1 ¼ mild, 2 ¼moderate, and 3 ¼ severe change. The “grouped” method of

masking tissues for histopathologic examination was used.7

Samples were coded by group, and the pathologist knew the

study design. A series of Fisher exact tests of independence (a¼ 0.05) were used to compare proportions of individuals exhi-

biting histologic changes in claw, liver, kidney, and muscle

tissues among treatment groups.

In our primary experiment, slider turtles treated with the

highest dose of SeMet exhibited multiple signs of selenosis,

including negative hematologic effects (eg, reduced lympho-

cyte counts, increased heterophil counts, and hemolytic ane-

mia), sloughing of claws, and mortality.10 Selenium-treated

turtles from which tissues were collected for histopathology all

had tissue selenium levels that surpassed “level of concern”

thresholds established for avian species.10,14 Average tissue

selenium concentrations from turtles in the present histology

study were highest in the kidney (0 mg/kg, 4.37 + 0.38 mg/kg;

15 mg/kg, 136.31 + 7.96 mg/kg; 30 mg/kg, 237.71 + 15.38

mg/kg), followed by the liver (0 mg/kg, 1.81 + 0.26 mg/kg; 15

mg/kg, 30.21 + 4.07 mg/kg; 30 mg/kg, 75.29 + 15.10 mg/kg)

and muscle (0 mg/kg, 1.95 + 0.19 mg/kg; 15 mg/kg, 29.28 +1.53 mg/kg; 30 mg/kg, 41.15 + 1.81 mg/kg).10 All 4 tissues

examined had changes that were either not seen in the controls

(claw and kidney) or were more severe than in the controls

(muscle and liver) (Table 1).

In kidney (Figs. 1–4), there was tubular degeneration, necro-

sis, and regeneration in both selenium-dosed groups. The his-

tologic scores were higher in the selenium-dosed group relative

to the control group (P ¼ .0008). Abnormalities were more

severe in the high-dose group: although both low-dose and

high-dose scores ranged from 1 to 3, more high-dose turtle

kidneys were scored 3 (Suppl. Table S1). Histologic scores in

controls were all 0. Hyaline droplets were seen in the tubular

epithelium in 5 of 7 of the high-dose turtles with scores of 2 or

3, but not in the control or low-dose groups (P ¼ .003).

Claws from control group turtles had no changes (Figs. 5

and 6), while claws from turtles of both selenium treatment

groups had similar changes (P ¼ 8.08 � 10–5), but these were

more severe in the high-dose group (low-dose score range, 1–3;

high-dose scores were all 3). Changes in the epidermis were

most severe over the claw tip and along the palmar/plantar

surface and included epidermal hyperplasia with disorganiza-

tion, vacuolar degeneration, intercellular edema, necrosis, and

ulceration (Figs. 7–9). Inflammatory cells and proteinaceous

material separated the epidermis or dermis from the stratum

corneum. The dermis was mildly edematous and thickened by

fibrosis with a mild lymphocytic infiltration. Infectious agents

were not identified on gram and Grocott’s methenamine silver

stains.

Table 1. Histologic Lesions in Yellow-Bellied Slider Turtles After Administration of 0, 15, or 30 mg/kg Seleno-L-Methionine.a

Treatment Group Claw Lesionb Kidney Degenerationc Kidney Hyaline Dropletsd Muscle Degeneratione Liver Lipidosis

0 mg/kg 0 (0/6) 0.17 + 0.4 (1/6) 0 (0/6) 0.8 + 0.4 (5/6) 1.7 + 0.5 (6/6)15 mg/kg 1.5 + 0.6 (4/4) 1.2 + 0.5 (5/5) 0 (0/5) 1.6 + 1.3 (4/5) 3 (5/5)30 mg/kg 3 (7/7) 2.1 + 1.2 (7/7) 0.71 + 0.5 (5/7) 1.7 + 1.3 (6/7) 2 + 1.2 (6/7)

aData are expressed as mean + SD for histologic scores (number of turtles with lesions/number of turtles evaluated). Changes in tissues (used for means) werescored 0 ¼ none, 1 ¼ mild, 2 ¼ moderate, and 3 ¼ severe.bClaw lesion characterized by epidermal hyperplasia, intercellular edema, and disorganization to ulceration and serocellular exudate at the cornified layer junction.cKidney degeneration characterized by tubular degeneration, necrosis, and regeneration.dKidney change characterized by tubular hyaline droplets.eMuscle degeneration characterized by acute myofiber swelling with hypercontraction bands.

474 Veterinary Pathology 55(3)

Hepatic lipidosis was present in all turtles except 1 in the

high-dose group (P ¼ 1.00 for all comparisons). In this individ-

ual, hepatocytes were small with glassy cytoplasm consistent

with atrophy, probably indicating emaciation. Lipidosis was

most severe in the low- and high-dose groups (control score

range, 1–2; low-dose score range, 2–3; high-dose score range,

1–3, excluding the turtle that did not have hepatic lipidosis).

In the skeletal (pectoralis) muscle of all groups, most turtles

had rare to many myofibers with acute degenerative changes

characterized by hyalinization and hypercontraction bands

(P ¼ 1.00 for all comparisons). This was most apparent in the

high-dose group (score range, 1–3), followed by the low-dose

group (score range, 0–3) and control group (score range, 0–1).

Similar to these turtles, changes in keratinized structures

such as hooves, claws, and beaks are a major sign of selenosis

in many species of mammals and birds.15,16 In humans, in

whom there has been an increase in cases of selenosis due to

misformulated dietary supplements, nail loss (onycholysis and

onychoptosis) is also a common finding.13 The microscopic

lesions in these turtles were similar to the more severe lesions

described in experimental selenosis of mallard ducks, in which

there was epidermal necrosis and accumulation of cellular and

proteinaceous material that separated the cornified layer from

the dermis.16 Those birds also had similar changes in the max-

illary beak. It is also possible these turtles had beak lesions, but

unfortunately the beaks were not examined.

Renal lesions, although mild, were the other prominent find-

ing of selenosis in these turtles. High levels of orally adminis-

tered selenium were associated with tubular damage whose

severity varied in a dose-dependent manner. There were also

hyaline droplets in the tubular epithelium of the high-dose

group, suggesting proteinuria.4 Lesions similar to those

Figure 1. Normal kidney; yellow-bellied slider turtle (control group). Figures 2–4. Experimentally induced selenosis, kidney; yellow-belliedslider turtles fed 30 mg/kg seleno-L-methionine weekly for 5 weeks. Figure 2. Histologic score ¼ 2, characterized by necrosis of renal tubuleswith loss of nuclei and hyaline droplets (arrow), and regenerative tubules with decreased diameter and attenuation of tubular epithelium. Inset:Higher magnification of cytoplasmic hyaline droplets. Figure 3. Histologic score ¼ 3. Renal tubules are necrotic with loss of nuclei, orregenerative with decreased tubular diameter, attenuated tubular epithelium, and crowding of nuclei. Figure 4. Histologic score ¼ 3. Renaltubules are necrotic with loss of nuclei and hyaline droplets (arrow), or regenerative and lined by flattened tubular epithelium.

Haskins et al 475

described in these turtles have been reported in mallards with

experimental selenosis,8 and renal calcinosis has been attrib-

uted to selenium toxicosis in trout.11

Acute degenerative changes in skeletal muscle were present

in both control and treated groups, so they were probably asso-

ciated with stress and handling at the time of euthanasia. How-

ever, changes were more pronounced in the treated groups,

suggesting that exposure to high levels of selenium might make

muscles more prone to exertional rhabdomyolysis.

Factors other than high selenium levels, such as a high-

energy diet or an inadequate photoperiod, were likely the pri-

mary cause of hepatic lipidosis in these turtles, as it was

observed in both control and treated groups. However, lipidosis

was more severe in the treated groups, suggesting that high

levels of selenium may also have had an adverse effect on the

liver or on energy metabolism. Watersnakes fed a contami-

nated diet that contained selenium as well as other trace ele-

ments had hepatic changes.5 Although changes were described

as fibrosis, this could have been due to the extended length of

time the snakes received the contaminated diet. Mallards with

experimentally induced selenosis also had liver lesions,

although different in character to the lipidosis described here.8

Selenosis in slider turtles was associated with changes in

tissues that are similar to those reported in avian species. The

most notable changes in this study were found in kidney and

claw tissues, with the high-dose group exhibiting the most

severe changes. Selenium blood and liver content in our sliders

from this study are comparable with levels found in wild sliders

in coal-contaminated environments.10 However, more research

is needed to determine if exposure in the wild results in similar

histopathologic abnormalities in slider turtles.

Acknowledgments

We would like to thank Caitlin Kupar, Matt Hamilton, Katrina Woods,

Naya Eady, Jarad Cochran, Amanda Jones, and Sam Dean for their

help with animal husbandry, selenium administration, turtle dissec-

tions, and tissue preparation. This research was funded by the US

Department of Energy under award number DE-FC09-07SR22506

to the University of Georgia Research Foundation and by Savannah

River Nuclear Solutions – Area Completions Project.

Figures 5 and 6. Figure 6. Normal distal digit and claw; yellow-bellied slider turtle (control group). Figure 5. Normal digital bone and thindermis and epidermis. Figure 6. Normal dermis and epidermis near tip of the toe. Figures 7–9. Experimentally induced selenosis, distal digitand claw; yellow-bellied slider fed 30 mg/kg seleno-L-methionine weekly for 5 weeks; histologic score ¼ 3. Figure 7. Along the palmar surface,there is abrupt transition to hyperplastic epidermis (black arrow) that extends over the tip of the toe (T). The dermis (D) is thickened by fibrosis.Figure 8. Hyperplastic, disorganized epidermis with epithelial swelling and vacuolation. A small amount of serocellular exudate (asterisk) ispresent between the epidermis and stratum corneum. Figure 9. Necrosis of the palmar epidermis of the claw, which is extensively hyperplasticand disorganized with intercellular edema. The epidermis is separated from the stratum corneum by a thick layer of serocellular exudate(asterisk).

476 Veterinary Pathology 55(3)

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to

the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, author-

ship, and/or publication of this article.

ORCID iD

David L. Haskins https://orcid.org/0000-0002-6692-3225

References

1. Canadian Council of Animal Care. CCAC species-specific recommendations

on: amphibians and reptiles. Available at: https://www.ccac.ca/Documents/Stan

dards/Guidelines/Add_PDFs/Wildlife_Amphibians_Reptiles.pdf. Accessed

December 13, 2017.

2. Congdon JD, Greene JL, Gibbons JW. Biomass of freshwater turtles: a geo-

graphic comparison. Am Mid Nat. 1986;115(1):165–173.

3. Finger JW Jr, Hamilton MT, Glenn TC, et al. Dietary selenomethionine admin-

istration in the American alligator (Alligator mississippiensis): hepatic and renal

Se accumulation and its effects on growth and body condition. Arch Environ

Contam Toxicol. 2017;72(3):439–448.

4. Fisher ER. Hyaline droplets of renal tubular and glomerular epithelium: observa-

tions concerning their nature and derivation. Exp Mol Pathol. 1964;3:304–319.

5. Ganser LR, Hopkins WA, O’Neil L, et al. Liver histopathology of the southern

watersnake, Nerodia fasciata fasciata, following chronic exposure to trace

element-contaminated prey from a coal ash disposal site. J Herpetol. 2003;

37(1):219–226.

6. Gibbons JW. The slider turtle. In: Sheffield R, ed. Life History and Ecology

of the Slider Turtle. Washington, DC: Smithsonian Institution Press; 1990:

1–18.

7. Gibson-Corley KN, Oliver AK, Meverholz DK. Principles for valid histopatho-

logic scoring in research. Vet Path. 2013;50(6):1007–1015.

8. Green DE, Albers PH. Diagnostic criteria for selenium toxicosis in aquatic

birds: histologic lesions. J Wildl Dis. 1997;33(3):385–404.

9. Haskins DL, Hamilton MT, Jones AL, et al. Accumulation of coal combustion

residues and their immunological effects in the yellow-bellied slider (Tra-

chemys scripta scripta). Environ Pollut. 2017;224:810–819.

10. Haskins DL, Hamilton MT, Stacy NI, et al. Effects of selenium exposure on the

hematology, innate immunity, and metabolic rate of yellow-bellied sliders (Tra-

chemys scripta scripta). Ecotoxicology. 2017;26(8):1134–1146.

11. Hilton JW, Hodson PV. Effect of increased dietary carbohydrate on selenium

metabolism and toxicity in rainbow trout (Salmo gairdneri). J Nutr. 1983;

113(6):1241–1248.

12. Koller LD, Exon JH. The two faces of selenium-deficiency and toxicity—are

similar in animals and man. Can J Vet Res. 1986;50(3):297–306.

13. Morris JS, Crane SB. Selenium toxicity from a misformulated dietary supple-

ment, adverse health effects, and the temporal response in the nail biologic

monitor. Nutrients. 2013;5(4):1024–1057.

14. Ohlendorf HM, Heinz GH. Selenium in birds. In: Beyer WN, Meador JP,

eds. Environmental Contaminants in Biota. Boca Raton, FL: CRC; 2011:

668–701.

15. O’Toole D, Raisbeck MF. Pathology of experimentally induced chronic

selenosis (alkali disease) in yearling cattle. J Vet Diagn Invest. 1995;7(3):

364–373.

16. O’Toole D, Raisbeck MF. Experimentally induced selenosis of adult Mallard

ducks: clinical signs, lesions, and toxicology. Vet Pathol. 1997;34(4):

330–340.

17. Unrine JM, Hopkins WA, Romanek CS, et al. Bioaccumulation of trace ele-

ments in omnivorous amphibian larvae: implications for amphibian health and

contaminant transport. Environ Pollut. 2007;149(2):182–192.

18. Young T, Finley K, Adams W, et al. What you need to know about selenium. In:

Chapman PM, Adams WJ, Brooks ML, et al, eds. Ecological Assessment of

Selenium in the Aquatic Environment. Pensacola, FL: SETAC Press; 2010:7–46.

Haskins et al 477