8930

Amphibians (Proposed)

Approved by Standard Methods Committee, 2011. Editorial revisions, 2021. Joint Task Group: 22nd Edition—Timothy M. Rice (chair), Katherine Coady, Douglas J. Fort,
Jackson Gross, James R. Rayburn.

8930  A. Introduction
1. Significance its smaller size, larger clutch size, diploid genome, and shorter
generation time.14–18
Amphibians are important organisms in freshwater ecosystems, Members of the genus Rana have long muscular hind limbs,
and many of these species’ populations have been declining for narrow waists, and large, laterally placed eyes. The most fre-
several years.1–3 Because some of the declines likely result from quently used species in toxicology are the Northern leopard frog
exposure to aquatic toxic materials,1,4,5 more focus on amphibi- (Rana pipiens Schreber), the Southern leopard frog (Rana sphe-
ans in aquatic toxicology and standard experimental protocols is nocephala Cope), and the American bullfrog (Rana catesbeiana
imperative. The following summary outlines protocols for anuran Shaw). Adults are semi-aquatic and use their vision to locate liv-
amphibians (Order Anura: frogs and toads). Anurans are the ing prey. Adult R. pipiens and R. sphenocephala range from 50 to
best avenue for standardizing methods because they have been 90 mm (snout to vent) and are marked with large dorsal spots on
the most extensively studied. Salamanders (Order Caudata) have a green or brown background and a pair of dorso-lateral ridges.
been used less frequently in toxicology studies, so they will not Sexes are difficult to distinguish, although females are usually
be specifically addressed. However, the following methods can be larger. Adult R. catesbeiana range from 90 to 150 mm (snout to
modified for salamanders as necessary. vent) and can be green, brown, or heavily mottled dorsally. Males
have a bright yellow throat and an external tympanic membrane
2. Test Organism Characteristics that is larger in diameter than the eye. All 3 species’ larvae have
a keratinized oral disc, which is used to macerate plant or animal
Two genera of anurans have been studied extensively in aquatic material. Features of the oral disk, coloration, and size are used to
toxicology: Xenopus and Rana. Members of the genus Xenopus identify larvae of a particular species.9–13
(family Pipidae) are not native to North America or other tem-
perate zones, but they have been developed as a model for early- References
life-stage tests because they can be easily cultured and bred in the
laboratory. However, Xenopus species have significantly different 1. Blaustein AR, Romansic JM, Kiesecker JM, Hatch A. Ultraviolet
life histories and larval stages than temperate-zone species. radiation, toxic chemicals and amphibian population declines. Diver-
Members of the genus Rana (family Ranidae) are tested when sity Distrib. 2003;9(2):123–140.
2. Collins JP, Storfer A. Global amphibian declines: sorting the hypoth-
a native species is desired (see Section 10900, Plate 25:B). The
eses. Diversity Distrib. 2003;9(2):89–98.
distribution, life history, and biology of these amphibians can be 3. Simon SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fis-
found in the literature.6–18 chm DL, Waller RW. Status and trends of amphibian declines and
The African clawed frogs Xenopus laevis Daudin and Xeno- extinctions worldwide. Science. 2004;306(5702):1783–1786.
pus (Silurana) tropicalis Gray share generally similar morphol- 4. Sparling DW, Krest SK, Linder G. Chapter 1: Multiple stressors and
ogy and natural history.6–8 Adults are fully aquatic, with long, declining amphibian populations: an integrated analysis of cause–
muscular hindlimbs, dorsally located eyes, and a dorso-ventrally effect to support adaptive resource management. In: Linder GL,
flattened body. The hind toes terminate in keratinized claws. Krest SK, Sparling DW, eds. Amphibian decline: an integrated anal-
The dorsal surface is olive or greenish-grey with darker mottling ysis of multiple stressor effects. Pensacola (FL): Society of Environ-
or spots, while the ventral surface is paler. Females are usually mental Toxicology and Chemistry (SETAC) Press; 2003.
5. Burkhardt JG, Bidwell JR, Fort DJ, Sheffield SR. Chapter 4: Chemi-
larger than males and have papillae around the cloaca. Males in
cal stressors. In: Linder GL, Krest SK, Sparling DW, eds. Amphibian
breeding condition develop dark, sticky pads on the digits and decline: an integrated analysis of multiple stressor effects. Pensacola
forelimbs. They eat any live or dead animal material in water that (FL): Society of Environmental Toxicology and Chemistry (SETAC)
will fit in their mouths, and olfaction is an important sense. Lar- Press; 2003.
vae are transparent and feed on fine particles suspended in the 6. Deuchar EM. Xenopus: The South African clawed frog. New York
water column. (NY): Wiley; 1975.
The 2 species differ in size and karyotype.14 Xenopus laevis 7. Kobel HR, Loumont C, Tinsley RC. Chapter 2: The extant species.
has an oligotetraploid genome and typically ranges from 70 to In: Tinsle RC, Kobel HR, eds. The biology of Xenopus. Oxford
125 mm (snout to vent) when mature. In contrast, X. tropicalis (UK): Oxford University Press; 1996.
ranges from 28 to 40 mm and has a diploid genome. Xenopus 8. Rodel MO. Herpetofauna of west Africa, Vol. 1: amphibians of the
west African savannah. Frankfurt au Main, Germany: Edition Chi-
laevis historically has been more frequently used in toxicolog-
maira; 2000.
ical assays, but X. tropicalis recently gained favor because of

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 8930 AMPHIBIANS (PROPOSED) - B. Culture and Maintenance of Test Organisms

9. Conant R, Collins JT. A field guide to the reptiles and amphibians 15. Song MO, Fort DJ, Mclaughlin DL, Rogers RL, Thomas JH,
of eastern and central North America. 3rd ed. (expanded). Boston Buzzard BO, Noll AM, Myers NK. Evaluation of Xenopus tropicalis
(MA): Houghton Mifflin Co.; 1998. as an alternative test organism for frog embryo teratogenesis assay–
10. Minton SA. Amphibians & reptiles of Indiana. 2nd ed. (revised). Xenopus (FETAX). Drug Chem Toxicol. 2003;26(3):177–189.
Indianapolis (IN): Indiana Academy of Science; 2001. 16. Fort DJ, Rogers RL, Thomas JH, Buzzard BO, Noll AM, Spauld-
11. Hinshaw S. Northern leopard frog. In: Hunter ML Jr, Calhoun AJK, ing CD. Comparative sensitivity of Xenopus tropicalis and Xen-
McCullough M, eds. Maine amphibians and reptiles. Orono (ME): opus laevis as test species for the FETAX model. J Appl Toxicol.
University of Maine Press; 1999. 2004;24(6):443–457.
12. Albright J. Bullfrog. In: Hunter ML Jr, Calhoun AJK, McCullough 17. Fort DJ, Rogers RL. Chapter 3: Enhanced frog embryo teratogenesis
M, eds. Maine amphibians and reptiles. Orono (ME): University of assay Xenopus model using Xenopus tropicalis. In: Ostrander GK,
Maine Press; 1999. ed. Techniques in aquatic toxicology, Vol. 2, Section 1: techniques
13. Bury RB, Whelan JA. Ecology and management of the bullfrog; U.S. for assessment of toxicity in whole organisms. Boca Raton (FL):
Department of Interior Fish and Wildlife Service. Resource Publica- CRC Press; 2005.
tion No.: 155. Washington DC: U.S. Department of Interior; 1984. 18. Berg C, Gyllenhammar I, Kvarnryd M. Xenopus tropicalis as a test
14. Hirsch N, Zimmerman LB, Grainger RM. Xenopus, the next genera- system for developmental and reproductive toxicity. J Toxicol Envi-
tion: X. tropicalis genetics and genomics. Dev Dyn. 2002;225(4):422– ron Health. 2009;72(3–4):219–225.
433.

8930  B. Culture and Maintenance of Test Organisms

1. Obtaining Test Organisms life stages of Rana spp. can be cultured easily, but maintaining a
continuous viable breeding facility for successive generations can
Xenopus laevis and X. tropicalis of various ages can be obtained be difficult for several reasons.
for toxicity testing from commercial breeders, biological supply a. Water supply and culture system: Natural water is preferred
houses, or an in-house culture facility. An in-house breeding facil- for maintaining adult Xenopus species. Supplies from wells or
ity is recommended and may be required because most assays springs usually are more uniform in quality than those from surface
involve amphibians in early life stages. Ensure that specimens— waters. Dechlorinated tap water can be used if residual chlorine
particularly those from outside sources—are certified as to is monitored and at low levels (dechlorination can be incomplete;
their identity, age, and freedom from disease. Examine animals preferably dechlorinate with sodium bisulfite). The salt solution
on arrival for skin lesions or red patches on ventral surfaces. used for the Frog Embryo Teratogenesis Assay-Xenopus (FETAX)
Quarantine new arrivals from the resident population for at test and for breeding adults and culturing early stages also can
least 2 weeks.1–4 be used for maintenance, but is only suitable for small colonies
Rana specimens of various ages can be obtained from biological because of cost and formulation time. Maintain water tempera-
supply houses, hatcheries, and bait shops. Such specimens proba- ture at 20 ± 2 °C for X. laevis (26 ± 0.5 °C for X. tropicalis) before
bly were field-collected, so in general, avoid them due to inaccu- adding adults. Provide aeration, if needed, to maintain dissolved
rate species identity, the potential for disease (from shipping), and oxygen (DO) levels. Take quarterly measurements of pH, total
lack of locality data. Preferably obtain specimens of Rana spp. by dissolved solids (TDS), total organic carbon (TOC), organophos-
collecting them from habitats with known toxicant exposure his- phate pesticides, organic chlorine [or organochlorine pesticides
tory. Such collections are subject to breeding-season constraints, plus polychlorinated biphenyls (PCBs)], chlorinated phenoxy
but during this time a variety of life stages and sizes, as needed, herbicides, perchlorate, chlorate, ammonia, bromide, beryllium,
can be gathered easily and inexpensively. Quarantine specimens cadmium, chlorine, chloramine, chromium, copper, fluoride,
before use in experiments. In-house breeding cultures are difficult iodide, iron, lead, manganese, mercury, nickel, selenium, silver,
to establish for Rana spp. because adults require live food and and zinc. The water’s physical and chemical limits include pH 6.5
eggs must be artificially fertilized.5 to 8.5, TOC ˂10 mg/L, and alkalinity and hardness between 16
If laboratory gloves are worn while handling specimens and 400 mg/L as CaCO3.1–4,8,9 Water requirements for Rana spp.
or cleaning containers, be cautious when using latex gloves. are similar to those for X. laevis.
Amphibian larvae are extremely sensitive to direct and indirect The FETAX assay (8930 C.1) requires the following salt solu-
contact with latex. Vinyl and nitrile gloves are less toxic, but spec- tion for all experiments with X. laevis:
imens should be monitored for increased mortality not associated Sodium chloride (NaCl) 625 mg
with chemical exposure.6–9 Sodium bicarbonate (NaHCO3) 96 mg
Potassium chloride (KCl) 30 mg
2. Culturing and Care of Test Organisms Calcium chloride (CaCl2) 15 mg
Calcium sulfate (CaSO4 ⋅ 2H2O) 60 mg
All stages of Xenopus spp.—from egg to adult—may be cul- Magnesium sulfate (MgSO4) 75 mg
tured successfully in the laboratory through several generations. Reagent water 1L
Basic information on establishing and maintaining a culture The final solution’s pH must be between 7.6 and 7.9. Use this
facility has been developed for X. laevis and is presented below.1 solution for breeding, static or renewal assays, and flowthrough
Requirements are similar for X. tropicalis, with adjustments for experiments whenever possible. Other water sources must allow
their smaller size and higher preferred temperatures.2–4 Separate for embryonic growth at the same rate as this solution with

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 8930 AMPHIBIANS (PROPOSED) - B. Culture and Maintenance of Test Organisms

acceptable mortality and malformation rates.1 Some types of in adult Rana spp. can only be induced in animals collected
test solutions will require modifying the above formulation by during breeding season. Collect pairs that are in amplexus and
decreasing or increasing certain salts, or using a more concen- place them in an undisturbed holding tank with water; fertilized
trated or more dilute solution.10 eggs can be collected in 24 h. Alternatively, artificially induce
Maintain adult specimens of Xenopus spp. in glass aquari- ovulation in gravid females collected from the field. The standard
ums or fiberglass or stainless steel raceways at densities of 4 to induction method involves intraperitoneal injection of pituitaries
6/1800 cm3 and containing water 7 to 14 cm deep. Use opaque from several adults into a gravid female. Extrude ova into a petri
tanks or shield tank sides (e.g., with black plastic) so specimens dish 24 to 48 h after injection by applying pressure to the female.
are not disturbed by outside activity. Use a photoperiod of 12 h Inseminate the ova by mincing testes (previously removed from
day:12 h night.1–4 For adult Rana spp., adjust tanks to provide a a male) in buffer and applying the sperm suspension over the
dry area because these species are not completely aquatic. This extruded ova.5,12,13 Sperm and ova might also be collected via
dry area can be created by tipping the tanks at one end so water injections of synthetic human luteinizing hormone-releasing hor-
pools at the opposite side, or by adding platforms above the water mone (LHRH) in a method similar to those described for Xenopus
level so specimens can climb out of the water. Keep adult Xeno- and chorionic gonadotropin (¶ b above). These techniques do not
pus spp. and Rana spp. in same-size groups to decrease cannaba- require destroying animals but are not frequently used.12–14
lism and competition for food.1–5 c. Collection and maintenance of embryos: If necessary for
b. Establish breeding colonies: To establish an X. laevis breed- certain experiments, begin dejellying anuran embryos immedi-
ing colony, use males that are at least 2 years old and 75 to 100 cm ately after egg laying. Gently swirl embryos for 1 to 3 min in a
long (snout to vent), and females that are at least 2 years old 2% (w/v) l-cysteine solution prepared in FETAX solution (¶ a
and 100 to 125 cm long.1 To establish an X. tropicalis breeding above). Adjust the pH of the cysteine solution to 8.1 with 1 N
colony, use adults that are at least 4 months old.2–4 Breed males NaOH, and continue dejellying until all jelly is removed. Do not
and female Xenopus spp. as single pairs. Females of both species treat embryos too long because survival will be reduced. Place
should have visible cloacal papillae and swollen sides indicative embryos in 60-mm glass or plastic petri dishes (25 embryos and
of full ovaries (sides of the abdomen appear swollen from dorsal 10 mL FETAX solution) and keep these in a constant-­temperature
view). Males should have darkened nuptial pads on the palms of room or a suitable incubator. Renew the solution every 24 to
their forelimbs and light mating strips on the underside of their 48 h.1–4 Periodically identify the developmental stage of embryos
arms.1–4 according to standard guides.15–17
Breeding can be conducted in any container with a mesh false d. Rearing larvae and metamorphs: Transfer free-swimming
bottom. Fit a 20- or 40-L (5- or 10-gal) glass aquarium with a larvae of Xenopus spp. or Rana spp. to glass aquariums. Loading
1-cm mesh (nylon or plastic only) suspended about 3 cm from depends on the size of larvae and the type of tank (static versus
the bottom of the aquarium so deposited eggs can lie undisturbed flowthrough). Determine developmental stages using standard
on the bottom. Shield sides of the breeding aquarium (e.g., with guides.15–17 Transfer metamorphs and small adults of Xenopus
black paper) and add an aerator if desired. Cover the top of the spp. to tanks similar to those of adults. Transfer Rana larvae to
aquarium with an opaque porous material (e.g., a fiberglass fur- adult tanks with dry areas when forelimbs emerge and tail reab-
nace filter).1 sorption begins.18–20
Plastic containers can be used by stacking 2 dish pans on top e. Food and feeding: Feed Xenopus spp. larvae a finely ground
of one another. Perforate the uppermost pan with enough holes animal chow (e.g., Salmon Starter pellets or Sera Micron). Sug-
to create the mesh false bottom.1–4 A useful alternative to glass gested rations are 30 to 80 mg/animal/d for X. laevis8–9 (6 to
aquaria or plastic dish pans is a sifting cat litter box. These boxes 10 mg/animal/d for X. tropicalis21), depending on the develop-
come equipped with an outer base and an inner perforated sifting mental stage. Rana larvae can be fed a variety of foods, including
grate, which functions as the mesh false bottom. The boxes are trout chow, boiled lettuce and spinach, and fish flakes. A con-
opaque and have a base area of 1260 cm2.11 venient diet for Rana larvae is a combination of ground rabbit
Breed adult Xenopus spp. in the same dilution water in which chow (Harlan Teklad), agar, and gelatin, which is heated and then
the test will be conducted. Hold water temperature at 20 ± 2 °C cooled until congealed.22–24 Feed Rana an equivalent Xenopus diet
for X. laevis (26 ± 0.5 °C for X. tropicalis). To induce breeding, if studies require comparison between the 2 genera. If larvae will
inject a male with 250 to 500 IU of human chorionic gonadotro- be used for experiments, feed early-stage larvae (actively feeding
pin into the dorsal lymph sac, and a female with 500 to 1000 IU. for <20 d) until the test begins. Withhold food from later-stage
The specific amount injected depends on the time of year and larvae for 48 h before test begins.25,26
the condition of adults. The hormone concentration should be Feed postmetamorphic and adult Xenopus spp. raw beef liver
1000 IU/mL in sterile 0.9% NaCl. Use a 1-mL tuberculin syringe 3 times a week. Xenopus tropicalis can be fed Salmon Starter
fitted with a 1.3-cm (0.5-in.) long, 26-gauge needle. Amplexus or equivalent at a rate of 40 to 50 mg/female and 25 to 30 mg/
normally occurs within 2 to 6 h and egg deposition about 9 to 12 h male.2–4 This food should also be adequate for X. laevis, but
after injection. The fertility rate should be >75%. Do not use eggs amounts should be increased due to this species’ larger size. Sup-
laid in “strings” or those that are not perfectly round, because they plement food with liquid multiple vitamins and screen it for mate-
develop abnormally.1–4 rials that will be tested.1–4 Adult Rana spp. will not eat nonmoving
Eggs of Rana spp. and other North American anurans can only food. Feed them appropriate-sized crickets and other insects or
be collected during the breeding season, either directly from the earthworms. Dust food with a vitamin powder. If food animals—
field or from reproductively active adults. Obtain proper permits particularly crickets—will be maintained for extended periods,
to collect eggs or adults. Collect fertilized egg masses during feed diets fortified with calcium and vitamins. Anurans will absorb
breeding season and treat as in paragraph c below. Reproduction these supplements. Large R. catesbeiana eat fish, crawfish, and

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 8930 AMPHIBIANS (PROPOSED) - B. Culture and Maintenance of Test Organisms

small laboratory mice. Offer food on the tank’s dry area, but items 12. Easley KA, Culley DD Jr, Horsemen ND, Penkala JE. Environmen-
that float or swim will be eaten from the water.5,27–29 tal influences on hormonally induced spermiation of the bullfrog,
f. Parasite and disease control: Observe animals in quarantine Rana catesbeiana. J Exp Zool. 1979;207(3):407–416.
daily for signs of disease. Treat diseased frogs appropriately. Look 13. Waggener WL, Carroll EJ, Jr. A method for hormonal induction of
sperm release in anurans (eight species) and in vitro fertilization in
for dull or dry skin, wounds or infection, poor posture, decreased
Lepidobatrachus species. Dev Growth Differ. 1998;40(1):19–25.
body condition, and lethargy.30,31 Withhold sick frogs from breed- 14. Cope RB, Miller CA, Post M, Mateus-Pinnilla NE, Murphy JE, Bea-
ing for 4 to 6 weeks after treatment. Remove dead individuals sley VR. Use of synthetic human luteinizing hormone releasing hor-
and waste, and use separate cleaning and transfer equipment for mone for induction of breeding in the cricket frog, Acris crepitans. J
each tank. Herpetel Med Surg. 2000;10(1):7–8.
15. Gosner KL. A simplified table for staging anuran embryos and larvae
3. Acclimating and Holding Test Organisms with notes on identification. Herpetologica. 1960;16(3):183–190.
16. Nieuwkoop PD, Faber J. Normal table of Xenopus laevis (Daudin).
New York (NY): Garland Publishing; 1994.
Use embryos immediately for embryo–larval stage tests.1–4
17. Bantle JA, Dumont JN, Finch RA, Linder G. Atlas of abnormalities:
Acclimate later-stage larvae before use by changing water from a guide for the performance of FETAX. Stillwater (OK): Oklahoma
100% holding water to 100% dilution water over 48 h. Hold sub- State Publications Department; 1991.
jects at 100% test water for 48 h.25,26 18. Chen TH, Gross JA, Karasov WH. Sublethal effects of lead on
Northern leopard frog (Rana pipiens) tadpoles. Environ Toxicol
References Chem. 2006;25(5):1383–1389.
19. Chen TH, Gross JA, Karasov WH. Adverse effects of chronic copper
1. Standard guide for conducting the frog embryo teratogenesis assay– exposure in larval Northern leopard frogs (Rana pipiens). Environ
Xenopus (FETAX) (ASTM E1439-98(2004)). In: Annual book of Toxicol Chem. 2007;26(7):1470–1475.
ASTM standards, Vol. 11.05. West Conshohocken (PA): ASTM 20. Gross JA, Chen TH, Karasov WH. Lethal and sublethal effects of
International; 2004. chronic cadmium exposure on Northern leopard frog (Rana pipiens)
2. Fort DJ, Rogers RL. Chapter 3: Enhanced frog embryo teratogenesis tadpoles. Environ Toxicol Chem. 2007;26(6):1192–1197.
assay: Xenopus model using Xenopus tropicalis. In: Ostrander GK, 21. Mitsui N, Fujii T, Miyahara M, Oka T, Kashiwagi A, Kashiwagi
ed. Techniques in aquatic toxicology, Vol. 2, Section 1: techniques K, Hanada H, Urushitani H, Santo N, Tooi O, et al. Development
for assessment of toxicity in whole organisms. Boca Raton (FL): of metamorphosis assay using Silurana tropicalis for the detection
CRC Press; 2005. of thyroid system-disrupting chemicals. Ecotoxicol Environ Saf.
3. Song MO, Fort DJ, McLaughlin DL, Rogers RL, Thomas JH, 2006;64(3):281–287.
Buzzard BO, Noll AM, Myers NK. Evaluation of Xenopus tropi- 22. Hirschfield WJ, Richards CM, Nace GW. Growth of larval and juve-
calis as an alternative test organism for frog embryo teratogenesis nile Rana pipiens on four laboratory diets. Am Zool. 1970;10(3):315–
assay-Xenopus (FETAX). Drug Chem Toxicol. 2003;26(3):177–189. 316.
4. Fort DJ, Rogers RL, Thomas JH, Buzzard BO, Noll AM, Spauld- 23. Gross JA, Johnson PTJ, Prahl LK, Karasov WH. Critical period of
ing CD. Comparative sensitivity of Xenopus tropicalis and Xen- sensitivity for effects of cadmium on frog growth and development.
opus laevis as test species for the FETAX model. J Appl Toxicol. Environ Toxicol Chem. 2009;28(6):1227–1232.
2004;24(6):443–457. 24. Cary Coyle TL, Karasov WH. Chronic, dietary polybrominated
5. National Academy of Sciences. Amphibians: guidelines for the diphenyl ether exposure affects survival, growth, and development of
breeding, care, and management of laboratory animals. Washington Rana pipiens tadpoles. Environ Toxicol Chem. 2010;29(1):133–141.
DC: National Academy Press; 1974. 25. Standard guide for conducting acute toxicity tests on test materi-
6. Gutleb AC, Bronkhorst M, van den Berg JHJ, Murk AJ. Latex als with fishes, macroinvertebrates, and amphibians (ASTM E729-
laboratory-gloves: an unexpected pitfall in amphibian toxicity assays 96(2002)). In: Annual book of ASTM standards, Vol. 11.05. West
with tadpoles. Environ Toxicol Pharmacol. 2001;10(3):119–121. Conshohocken (PA): ASTM International; 2004.
7. Cashins SD, Alford RA, Skerratt LF. Lethal effect of latex, nitrile, 26. Standard guide for conducting acute toxicity test on aqueous ambient
and vinyl gloves on tadpoles. Herpetol Review. 2008;39(3):298–301. samples and effluents with fishes, macro invertebrates, and amphibi-
8. Organisation of Economic Cooperation and Development. OECD ans (ASTM E1192-97(2003)). In: Annual book of ASTM standards,
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phosis assay. Paris (France): OECD; 2009. 27. Minton SA. Amphibians & reptiles of Indiana. 2nd ed. (revised).
9. Endocrine disruptor screening program test guidelines, OPPTS Indianapolis (IN): Indiana Academy of Science; 2001.
890.1100: amphibian metamorphosis (Frog) (EPA 740-C-09-002). 28. Hunter ML Jr, Calhoun AJK, McCullough M. Maine amphibians and
Washington DC: U.S. Environmental Protection Agency; 2009. reptiles. Orono (ME): University of Maine Press; 1999.
10. Tietge JE, Ankley GT, Defoe DL, Holcombe GW, Jensen KM. 29. Bury RB, Whelan JA. Ecology and management of the bullfrog; U.S.
Effects of water quality on development of Xenopus laevis: a frog Department of Interior Fish and Wildlife Service. Resource Publica-
embryo teratogenesis assay—Xenopus assessment of surface water tion No.: 155. Washington DC: U.S. Department of Interior; 1984.
associated with malformations in native anurans. Environ Toxicol 30. Mader DR. Reptile medicine and surgery. Philadelphia (PA): W.B.
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11. McCallum ML, Rayburn JR. A simple method for housing Xenopus 31. Fowler ME, Miller RE. Zoo and wildlife medicine: current therapies.
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8930 C. Toxicity Test Procedures

1. Frog Embryo Teratogenesis Assay–Xenopus laevis (FETAX) 2) Test initiation—Induce breeding in 3 mating pairs and har-
vest clutches separately. Conduct 3 definitive tests using embryos
FETAX is used to evaluate how toxicants affect animals’ devel- from only one mating pair in each test. Keep clutches separate
opmental processes. It is designed not just for amphibians but because embryos from one mating pair that initially appear
also other species (e.g., mammals) and, therefore, is useful for acceptable might develop poorly. If the embryos were mixed
screening substances that are potential teratogens in humans.1 (from different mating pairs), then all embryos would have to be
The FETAX assay can be modified to accommodate species other discarded.
than X. laevis (e.g., X. tropicalis2–4) with different developmental For each test concentration, use 2 dishes each containing 25
times or maintenance requirements. Details on FETAX assays are embryos and 10 mL test solution. For each control, use 4 dishes
presented in other sources.1–4 The general procedure and modifi- of 25 embryos each. Maintain a temperature of 20 ± 2 °C for X.
cations to the standard protocol are discussed below. laevis 1 (26 ± 0.5 °C for X. tropicalis2–4) throughout the test to
a. Scope and application: FETAX procedures can be used prevent malformations and allow proper growth of controls. For
to test chemicals either individually or in formulations, as well a positive control or reference toxicant, use 6-aminonicotinamide
as temperature, DO, pH, physical agents, aqueous surface and at 5 mg/L (the 96-h LC50) and 2250 mg/L (the 96-h EC50 for
ground waters, leachates, aqueous extracts of water-insoluble malformations)—4 dishes of each.
materials, and solid-phase samples (e.g., soils and sediments, 3) Solution renewal—Replace test material every 24 h, and
particulate matter, sediment, and whole bulk soils and sediment). measure the pH of the control and the highest test concentration
The assay uses X. laevis embryos, so it requires no feeding, little during renewal. Remove the test solution using a Pasteur pipet
maintenance, and minimal laboratory space. with an orifice that has been enlarged and fire-polished to accom-
b. Range-finding test and selection of concentrations: Conduct modate embryos without damage in case embryos are acciden-
range-finding tests to select proper concentrations for definitive tally picked up. Complete the renewal as quickly as possible to
tests. Use at least 7 concentrations that differ by a factor of 10. minimize embryo desiccation.
Conduct the test and calculate the 96-h lethal concentration (LC) 4) Biological data recording—Measure mortality by count-
(LC5, LC16, LC50, LC84, and LC95) and the effective concentra- ing and removing dead embryos during solution renewal. At 24
tion (EC) (EC5, EC16, EC50, EC84, and EC95). If necessary, use h, death is determined by an embryo’s pale skin pigmentation,
results of the first definitive experiment as another range-finder decomposition, and lack of response to prodding. At 48, 72, and
and readjust test concentrations. 96 h, use the lack of heartbeat as an unambiguous sign of death.
c. Specific test procedures: Record the final number of dead embryos at 96 h of exposure and
1) Facilities and equipment—Conduct tests in an incubator (see the time of first hind-limb buds in controls. Fix dead embryos
8930 B.2c). Ensure that all equipment and facilities that contact and remaining live embryos in 3% formalin. Record the devel-
stock solutions, test solutions, or water in which embryos will be opmental stage and malformations at the end of 96 h according
placed do not contain leachable or dissolvable substances at lev- to standardized guides.7–9 Compare exposed embryos to control
els that would adversely affect embryo growth or development. embryos. Measure head-to-tail length at the end of each test after
For new test facilities, conduct a nontoxicant test in which all test embryos are fixed in 3% formalin.
chambers contain FETAX solution with no added test material. 5) Exogenous metabolic activation system (MAS)—Use an
The embryos must grow, develop, and survive in numbers con- exogenous MAS when using FETAX to evaluate developmen-
sistent with an acceptable test. To count and evaluate abnormal tal toxicity for human-health hazard assessment, because early
embryos, use a binocular dissection microscope (magnifications X. laevis embryos have limited xenobiotic metabolic capabilities,
up to 303×), a darkroom enlarger (to enlarge embryo images), particularly cytochrome P450. Make the MAS from rat liver mic-
and a map measurer or an ocular micrometer. Alternatively, count rosomes and nicotinamide adenine dinucleotide (the generator),
embryos using a digitizer interfaced to a microcomputer. and Aroclor 1254 (the cytochrome P450 inducing agent). Use
For toxicity tests of fluids, use 60-mm glass or plastic petri MAS-only dishes as the negative control, and 4 mg/mL cyclo-
dishes as the exposure containers and modify them as necessary.1–4 phosphamide with and without MAS as the positive control.
For aquatic sediment testing, use a 250-mL (9-oz) specimen bot- Make a P450 control (to demonstrate that the cytochrome P450
tle with a glass tube/PTFE mesh insert. Add 35 g test soil to the system is responsible for observed bioactivation) by adding di­
bottle, followed by the insert and 140 mL FETAX solution. Place thionite directly to the microsomes and bubbling carbon monox-
embryos on top of the insert.5 For in situ field exposures, partic- ide through for 3 min to inactivate P450. To prepare 20 mL test
ularly with native species, construct an exposure chamber from a solution, place an appropriate volume of FETAX solution into
2.5-cm-wide, 7.0-cm-diam circlet of plastic polyethylene mesh a 50-mL flask, add MAS components and an appropriate vol-
(1-mm mesh). Use a central nylon bolt or wing nut to hold the ume of test material stock to give the desired concentration, then
chamber together, and line the chamber with PTFE mesh to retain adjust the final volume to 20 mL with FETAX solution. Divide
embryos. Anchor the chamber in water with a stainless steel this test mixture between replicate petri dishes to which embryos
stake, and mark the location with a fishing bobber. Equilibrate are added.
embryos by placing a laboratory shipping container directly into 6) Duration of test—For X. laevis, run the FETAX assay for
the sample location until temperatures are within 2 °C. Use 4 to 96 h, at which point the control embryos should have hind-limb
6 exposure cages per onsite sample unit, with 10 to 25 embryos buds (Nieuwkoop and Faber Stage 468). If 90% of controls do not
in each cage.6 show hind limb buds by 96 h, then extend the test another 3 h. The

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 8930 AMPHIBIANS (PROPOSED) - C. Toxicity Test Procedures

FETAX assay can be conducted in 48 h for X. tropicalis.2–4 For tests, maintain a DO level between 60% and 100% of saturation
Rana spp. or other species, the test duration may need to be modi­ in each test chamber at all times.
fied to accommodate differences in developmental time. Report d. Specific test procedures:
deviations from standard FETAX conditions. 1) Test initiation—Distribute test organisms randomly among
7) Mixture-interaction studies—Interaction studies can be con- the test chambers. For static and renewal tests, place the test
ducted to evaluate synergistic or antagonistic developmental toxi­ organisms in chambers within 30 min after the test material is
city among 2 or more combined substances. Two methods have added. For flow-through tests, place the test organisms in cham-
been used successfully with the FETAX assay.10–12 In toxic unit bers after the test solutions have been flowing through chambers
analysis, binary mixtures are made in specific ratios (1:3, 1:1, long enough for their concentrations to have reached steady state.
and 3:1), and LC50s are plotted on an isobole diagram to deter- 2) Feeding—Do not feed larvae 24 to 48 h before or during an
mine additive, synergistic, or antagonistic effects. In the second acute toxicity test. Food can add another uptake route, and fecal
method, greater or less than expected responses from a particular matter and uneaten food decrease the DO concentration and bio-
concentration of chemicals with known effects are tested. This logical activity of some test materials.
method requires less time but does not yield the quality of infor- 3) Physical and chemical data recording—For static and renewal
mation of toxic unit analysis. tests, measure water quality parameters at the beginning, the end,
and at least every 48 h during the test in the controls and the high,
2. Other Short-Term Acute Tests for Embryos and Later medium, and low test concentrations as long as live organisms
Stage Larvae remain. Measure maximum and minimum temperatures daily in
at least one test chamber. For flow-through tests, measure water
Procedures for testing toxicants on postembryonic, free swim- quality parameters (e.g., hardness, alkalinity, conductivity, DO,
ming amphibian larvae (tadpoles) in an acute exposure can be and pH) at the beginning of the test and measure temperature
designed similar to tests for fish and macroinvertebrates. Standard daily in at least one test chamber. To monitor test-solution con-
methods for acute exposures have been described in detail13,14 and centrations, take water samples from a point midway between the
are discussed in general below. top, bottom, and sides of the test chamber. In static and renewal
a. General procedure: Larvae are maintained for 2 to 8 d in tests, measure the concentration of test material in at least the
each of 2 or more treatments in one or more test chambers. In controls and the high, medium, and low concentrations of test
each of the one or more control treatments, larvae are maintained material at the beginning of the test, at the end of renewal periods,
in dilution water to which no test solution has been added. This and at the end of the test. For flow-through tests, measure concen-
procedure measures the test’s acceptability based on, for exam- tration of test material in the chambers as often as practical during
ple, the quality of test subjects, dilution water, test conditions, and test. In each treatment, the highest concentration measured during
handling procedures. It also provides a basis for interpreting data the test should be less than 1.5 times the lowest concentration.
obtained from the other treatments. In each treatment, larvae are 4) Biological data recording—Measure the number of dead
maintained in one of several concentrations of test solution. Data and affected organisms (lack of movement or response to gentle
on biological effects in each test chamber usually are obtained prodding, loss of equilibrium) in each test chamber every 24 h
periodically during the test and analyzed to determine LC50s or after test begins. Remove dead organisms at least once every 24 h
EC50s for various exposure periods. if it can be done without stressing live organisms. Measure the
b. Types of experimental design: Two types of experimental weights of test organisms after exposure is complete.
design are appropriate in most cases. For an acute test intended 5) Test termination—Conduct the test for at least 96 h or long
to allow calculation of an LC50, EC50, or inhibitory concentra- enough to ensure that a time-independent toxicity level can be
tion (IC) (IC50), use one or more control treatments and a geo- determined or estimated mathematically. If organisms will not
metric series of at least 5 concentrations of test material. Except be substantially affected by starvation for at least 8 d, conduct
for the controls and the highest concentration, each concentration the test for at least 8 d to determine whether more organisms are
should equal at least 60% of the next higher one. Alternatively, if affected or killed after 96 h. As an optional test, place remaining
it is only necessary to determine whether a specific concentration test organisms in dilution water that does not contain any added
is acutely toxic to larvae or whether the LC50, EC50, or IC50 is test material for 2 to 8 days and feed them to determine whether
above or below a specific concentration, then only that concen- delayed effects occur.
tration and the controls are necessary. Use 2 more concentrations
at about one-half and twice the specific concentration to increase 3. Amphibian Metamorphosis Assay (AMA)
confidence in the results. For both test designs, ideally, at least
one concentration of test material kills or affects a percentage of A variety of environmental chemicals adversely affect endo-
larvae (other than 0% or 100%) near the percentage for which the crine systems. The thyroid system is one of the most vital
LC, EC, or IC is to be calculated. endocrine systems in vertebrates; it is particularly important in
c. Test setup: In static and renewal tests, load 0.5 to 0.8 g/L of regulating development and metamorphosis in amphibians. The
organisms in each test chamber, adding fewer animals at higher Amphibian Metamorphosis Assay (AMA) is a simple, standard-
temperatures. In flow-through tests, load no more than 1 g/L ized screen for substances that affect the hypothalamic–pituitary–
organism in each test chamber. For consistency, use temperatures thyroid (HPT) axis.15–18 The assay uses Nieuwkoop–Faber (NF)
similar to those in other assays (e.g., FETAX). For static tests, Stage 51 X. laevis,8 and consists of a 21-d exposure to the sus-
maintain a DO level between 60% and 100% of saturation in each pected chemical. Periodic observations during exposure include
test chamber during the first 48 h of the test, and between 40% mortality, hind-limb length, snout-to-vent length, developmental
and 100% of saturation afterward. For renewal and flow through stage, weight, and thyroid histology. The AMA is a test guideline

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 8930 AMPHIBIANS (PROPOSED) - C. Toxicity Test Procedures

suggested by the Organisation for Economic Cooperation and the control treatment group. Choose 5 stage-matched larvae from
Development (OECD)15 and is also incorporated into Tier 1 of each replicate tank for histopathological observations. Consult a
the U.S. Environmental Protection Agency’s Endocrine Disrup- pathologist for determination of histological effects caused by the
tor Screening Program.16 This procedure has been adapted for X. test chemical, because basic statistical analysis is not useful for
tropicalis.19 The general procedures are described below. these endpoints.
a. Obtaining test subjects: Breed 3 to 5 pairs of adult X. laevis d. Data analysis: Tests considered to be negative for thyroid
(see 8930 B.2b). Retain the best individual spawns and do not activity are accepted as valid if
mix. Discard all obvious dead or abnormal eggs via pipet. Trans- (a) mortality in any treatment does not exceed 10%;
fer healthy embryos from each spawn into hatching tanks for 4 d, (b) at least 2 treatment levels, with all 4 uncompromised repli-
and transfer the best spawn to a rearing tank (see 8930 B.2c). cates, are available; and
Through the first week, remove dead larvae and replace daily. (c) at least 2 treatments with no obvious toxicity are available.
Clean tanks daily by siphoning, and feed larvae twice each day Tests considered to be positive for thyroid activity are accepted
(see 8930 B.2e). During pre-exposure, use the same culture water, as valid if mortality in the control group does not exceed 2 tad-
light, and feeding regimen used during the exposure phase. Do poles per replicate. Accelerated development, asynchronous
not exceed rearing densities of 4 larvae per liter (static exposure) development, or marked histopathology of the thyroid gland rela-
or 10 larvae per liter (flow-through exposure). Larvae should tive to the control specimens indicates the test material’s potential
develop from NF Stage 45/46 to 51 in 12 d. effects on the thyroid. Advanced development is determined via
b. Test vessels and solutions: Conduct tests in glass or stainless statistical analyses of
steel aquaria (22.5 × 14 × 16.5 cm) with 4 to 10 L test solution • hind-limb length (normalized by snout-to-vent length) on
(10 to 15 cm minimum water). Flow-through test systems are pre- Study Day 7;
ferred over static renewal systems. Ensure that iodide concentra- • hind-limb length (normalized by snout-to-vent length) on
tions in the test water are between 0.5 and 10 μg/L because iodide Day 21;
needs to be available for proper thyroid gland function. When • developmental stage on Day 7; and
determining concentrations of test material, set the high test con- • developmental stage on Day 21.
centration by either the test substance’s solubility limit, the max- Consider the test positive for accelerated development if a sta-
imum tolerated concentration (MTC), or 100 mg/L, whichever tistically significant effect is detected in any of the 4 endpoints
is lowest. If solvent carriers for the test substance are required, above.20
then include a solvent control among the test solutions. Use at
least 3 test concentrations and a clean water control. Separate the 4. Chronic Toxicity Tests
highest and lowest concentrations by a minimum of one order of
magnitude. Few toxicology protocols have been developed to expose
c. Test initiation and conduct: Initiate the exposure on Day 0 amphibians from embryo through the larval period to adulthood.
when enough larvae have reached NF Stage 518 and are ≤17 d Investigators have rarely attempted to expose amphibian lar-
postfertilization. Hind-limb morphology is the most prominent vae for the entire metamorphic period because this lasts several
and preferred landmark for morphological staging. To select test months for many species, and because larvae are extremely sensi-
animals, pool normal-looking larvae into one vessel containing tive and difficult to keep alive at metamorphic climax. Examining
dilution water. To determine developmental stage, remove larvae toxicant effects during amphibian metamorphosis is particularly
individually and transfer them to a transparent chamber (e.g., valuable because certain critical stages of development might be
100-mm petri dish) containing dilution water. Avoid anesthesia; sensitive to even low levels of toxicant. The following methods
however, if necessary for proper staging, anesthetize larvae with are useful in examining later-stage larvae under exposure times
100 mg/L buffered tricaine methanesulfonate (MS-222). Distrib- longer than a few days and as a complement to FETAX assays.
ute larvae randomly into treatment tanks, 20 per tank (5 per L), a. 10- and 30-d embryo–larval exposures: Transfer 4-d-old
with 4 replicate tanks per treatment. Replace unhealthy larvae embryos (e.g., after FETAX assay) to glass aquariums and con-
from the pooling tanks. Continue monitoring mortality every tinue exposure through 10 d. Monitor the same endpoints as in
day and remove dead larvae. Record any abnormal behavior or the FETAX assay (change exposure water on Day 7).21 If desired,
excessive visible malformations throughout the test. Sample test extend the exposure for 30 d.22–26 Feed subjects as needed, depend-
solutions from each replicate tank for chemical analysis on Day 0 ing on tank size, and renew exposure water every 48 h. After 30 d,
and once every week thereafter. measure length and weight and identify hind-limb abnormalities.
On Day 7, remove 5 randomly chosen larvae from each test Fix representative specimens in 3% formalin at pH 7.0.
tank, euthanize in 150 to 200 mg/L tricaine methanesulfonate, b. Exposures through metamorphosis: The following procedures
rinse, and blot dry. Measure body weight (nearest mg), hind- have been used successfully to expose R. pipiens from embryo
limb length, snout-to-vent length, and developmental stage. On through tail reabsorption27–29 and may be adaptable to other species
Day 21, terminate exposure by removing and euthanizing all (with necessary adjustments for larval size and development dura-
remaining larvae. Rinse, dry, and determine body weight, snout- tion). Collect embryos as above (8930 B.1). Maintain in 150-mL
to-vent length, and developmental stage for each larva. For any petri dishes, 30 to 40 embryos per dish, with 100 mL of appro-
histological assessments, place all larvae or head tissue samples priate water or test solution. Renew solutions daily. When larvae
containing the lower jaw in Davidson’s fixative for 48 to 72 h. reach Gosner Stage 25,7 transfer them from their respective petri
Segregate larvae in each test tank according to stage. Evaluate the dishes to 18.9-L, low-density polyethylene (LDPE) plastic tanks
distribution of stages across the test by comparing larvae from all (I-CHEM certified cubitainers or equivalent) containing 15.14 L
treatments to the median developmental stage of all larvae from

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 8930 AMPHIBIANS (PROPOSED) - C. Toxicity Test Procedures

test solution. Feed larvae ground rabbit chow daily, ad libitum. c. Respiratory behavior in larvae: Place larvae in individual
Change the full volume of water every 48 h. Check periodically tanks, or uniquely mark individuals in group tanks.43,44 Monitor
for dead larvae and record length, weight, and malformations on larvae for 5 d. For each individual, count the number of move-
a subset of larvae; the frequency of data collection depends on the ments longer than one body length (activity), the number of trips
experiment’s objectives. As larvae reach Gosner Stage 42 (fore- to the surface for 15 min, and the number of times the buccal floor
limb emergence7), record the time to metamorphosis, size, and is elevated for 1 min. Larvae with compromised respiratory abili­
mass. Transfer individual Stage 42 larvae into plastic boxes with ties will become less active, head to the surface more frequently
divided compartments (115 × 51 × 45 mm) (e.g., a plastic tackle to gulp aerial oxygen, and increase their buccal pump rate.45
box). Add enough test solution to keep each larva hydrated, and
change the solution daily. Slant the boxes so larvae can emerge 6. Statistical Analysis
from the water. Do not feed larvae; nutrition comes from reab-
sorbing the tail. Maintain larvae until complete tail reabsorption Assemble, analyze, evaluate, and report data as described in
(Gosner Stage 46).7 Euthanize larvae in 0.5 g/L tricaine methane- Section 8010 G.
sulfonate (MS-222) and freeze for tissue analysis.
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Published Online: August 27, 2018