TOE-CLIPPING OF FROGS AND TOADS

STANDARD OPERATING PROCEDURE
ARMI SOP No. 110
Revised, 16 February 2001

1. PURPOSE. This SOP outlines a safe, simple, sanitary and humane method to remove toe tips from frogs and toads (toe clipping), and the proper method to preserve the clipped toe tips for genetic analyses, histologic examinations for infectious diseases, or to determine (estimate) the age of the amphibian.


2. SCOPE: The described procedure is limited to fully metamorphosed anurans (frogs and toads), but is NOT recommended for tadpoles, larvae and salamanders.


3. EQUIPMENT & SUPPLIES.
   1. 2-3 pairs of scissors, stainless steel, surgical-type, 4-6 inches long Note: Finger-nail and toe-nail clippers are NOT recommended because they cannot be easily disinfected between animals.
   2. Squeeze bottle of clean, potable water
   3. Ethanol, 85-95%
   4. Bactine® spray
   ®
   5. Vials, screw cap, 1-5 ml size (or 1 to 4 dram size), filled with 70-75% ethanol
   6. Labeling tape or paper; pencil or indelible ink pen


4. BACKGROUND. Toe-clipping to mark wild animals has been practiced on amphibians, waterfowl and small rodents for decades. Although the procedure is considered less than humane by some groups, this SOP will not debate anthropomorphic issues. Most published reports of toe-clipping in amphibians document few adverse effects, but there are exceptions (Donnelly et al., 1994; Clarke, 1972; Reaser and Dexter, 1996; Lemckert, 1996; Golay and Currer, 1994). In those publications that document adverse effects, the precise methods in which the procedure was done are not reported. Hence, it is difficult to assess the skill and training of the toe-clipper, the cleanliness of the technique, wound treatments, and environmental microbiology. This SOP will present, from the veterinary medical and surgical view-point, a safe, simple, sanitary and humane method to remove toe tips from frogs and toads; this procedure should eliminate or greatly reduce any adverse effects of toe clipping. This SOP will not present patterns and numbering systems for unique identification of animals (See, Donnelly et al., 1994).
   
   Amphibians have delicate skin that is quite different from the skin of reptiles, birds and mammals. Hence, amphibian skin cannot be prepared for surgery as would the skin of reptiles and endotherms. Alcohols, phenolics, and iodine-based skin antiseptics (and disinfectants) cannot be used on amphibians, because these chemicals will removed essential mucus and waxy layers in the epidermis that protect amphibians from dehydration, viruses, bacteria and fungi. Likewise, ointments and sprays that are oil-based or alcohol-based are contra-indicated in amphibians. Furthermore, amphibians are capable of absorbing into the body a wide variety of chemicals that are applied to the skin. Hence, alcoholic, iodine and phenolic based antiseptics and disinfectants could be toxic to amphibians when absorbed through the skin.
   
   Skin Antiseptics. At present, there is only one commercially available, over-the-counter antiseptic and antibacterial preparation that does not contain oil, grease or alcohol. This product is Bactine® spray. If other preparations become available that also lack oil, grease and alcohol, these may be recommended for use in amphibians. Hence, at present, Bactine® is the only preparation that is recommended for application to the wound following toe-clipping; the product also may be used to prepare the skin for toe-clipping or biopsy.
   
   Initially, toe-clipping was done simply to mark and identify individual small animals for recapture studies when tags, collars, brands, paints, etc, were impractical. Currently, toe-clipping has three additional major functions (only if the clipped toes are saved): the toes can be used 1) in genetic/DNA studies (population genetics, microbial disease detection, etc), 2) for histological examinations for infectious diseases (most notably chytrid fungi and ranavirus infections), and 3) for determining the age of amphibians by counting the growth rings in the phalanges.
   
   In order to assure the greatest possible use of the toe(s) that are clipped, 70-75% ethanol is the recommended preservative. Normally, formalin is recommended for histology, but formalin has a small hazardous potential and formalin-fixed tissue is nearly unusable for recovery of DNA. Ethanol is the preferred fixative for clipped toes for several reasons: 1) it has none of the hazards associated with formalin (formaldehyde), 2) ethanol-fixed tissues retained excellent cell morphology for histologic examinations, 3) ethanol will be needed for other field procedures, so it makes sense to carry slightly more ethanol than to need two different chemicals, and 4) ethanol-fixed tissues can be used for molecular tests, including recovery of DNA for genetic analyses.
   
   


5. ANATOMY OF THE FOOT. All normal endemic adult frogs and toads in the USA have 5 toes (digits) on each hindlimb (leg, foot). In some toads, a tubercle near the first toe can be fairly prominent and could be mistaken for a toe. If in doubt, count toes from the lateral (outside) region inwards. Toes (digits) usually are assigned Roman numerals I through V, with the first, shortest or medial digit being digit I; digit IV is the longest. Each toe consists of 2 types of bones: one metatarsal and multiple phalanges. Digits I and II have two phalanges each, digits III and V have three phalanges each, and digit IV has four phalanges.


6. METHOD.
   1. Anesthesia of the amphibian is recommended prior to toe clipping, but is optional.
   
   
   2. Digit (toe) III or V are recommended for clipping if, and only if, a toe is to be clipped solely as a biopsy for various analyses and not as a method of individual identification of the amphibian. If "fingers" are to be clipped, many herpetologists recommend that digit I ("thumb") not be clipped from male frogs and toads, because this digit swells in some species during breeding season and may be needed by the male for clasping the female during amplexus.
   
   
   3. Clean the foot gently by spraying a stream of clean water over the skin and toes in order to remove mud and debris. The stream of water should be at least 5 continuous seconds each on both the dorsal and ventral surfaces of the toes. The foot may then be sprayed briefly with Bactine® (optional).
   
   
   4. Position of amphibian for toe-clipping. Hold up the foot and toe(s) in a position that is higher than (above) the rest of the foot and vent; this will prevent dirty water, feces and urine from running down the leg and contaminating the toe skin. This may require holding the animal vertically with the nose pointed to the ground and the toes pointed sky-ward.
   
   
   5. Clipping. The blades of the disinfected scissors are placed on each side of the selected toe. It is recommended that the portion of the scissors near the hinge (rather than near the tip) be used for clipping the toe. It is recommended that the selected toe be severed next to the web, however, if toe webs extend to the toe tip, then a portion of web may have to be removed. It is recommended that 1.5 to 2 full toes bones (phalanges) be clipped off. Cutting through a joint (inter-phalangeal joint) between toe bones (phalanges) is recommended, but is optional.
   
   
   6. If two people are present for the procedure, it is recommended that the second person gently hold the tip of the toe while it is being clipped (with a pair of forceps or "tweezers") to prevent flinging of the toe tip and its loss in soil, grass, stream, etc. Please do not crush the amputated toe in the grasp of forceps, as this could destroy the usefulness of the toe for histological examination and skeletochronology.
   
   
   7. Bleeding from the wound usually is minimal. Three to five drops of blood at the stump are to be expected. If more than 5 drops of blood flow from the wound, then the wound should be sprayed with Bactine® and a sterile cloth, tissue, cotton ball or Nobuto filter paper strip may be applied to the stump to stem bleeding and enhance clotting.
   
   
   8. Disinfection of the wound. Bleeding usually is minimal and usually lasts for only 15-30 seconds. When bleeding has ceased, the wound should be sprayed with Bactine® just prior to release of the animal. The Bactine® spray should be allowed to dry for 1-2 minutes before the amphibian is released.
   
   
   9. Release of the amphibian should be onto land rather than water, because water will immediately wash off the Bactine® and some ponds contain high levels of bacteria and water molds that could potentially infect the wound. However, if the amphibian is an aquatic stage (larva or neotene), then it should be released into water.
   
   
   10. Optional blood collection from the toe stump. For larger amphibians (>25g), it may be desirable to have one fresh Nobuto filter paper strip handy to collect any blood that drips from the wound. Applying the paper strip to the wound often will promote or hasten clotting. If blood is collected from the toe wound, it should be so labeled to distinguish it from blood collected from the heart or a vein; it also is desirable to record whether Bactine® was applied to the toe before it was clipped (to know whether Bactine could be present on the filter paper mixed with the blood).
   
   
   11. The amputated toe is promptly placed in a vial of 70-75% ethanol. Volume of ethanol should be at least 10 times the volume of the toe(s). The vial is labeled (either internal paper label, or external label on tape). One vial per amphibian is recommended; multiple toe tips from one amphibian may be placed in the same vial, but toes from multiple animals should not be placed in one vial.
   
   
   12. Disinfection of scissors between animals. Scissors must be disinfected after use on each animal to prevent spread of diseases and build up of contaminants on the scissors. Numerous methods of disinfection are available, as well as several liquid disinfectants. To speed field operations, it would be desirable to have 2 or 3 scissors. For field work, there are three main choices for disinfection of scissors:
       1. Soak scissors for 15 minutes in 70% ethanol. To prevent the ethanol from becoming heavily contaminated with blood, tissues and debris, the scissor blades should be wiped off prior to being placed in the ethanol. The entire blades, hinge and a portion of the handles need to be placed into the disinfecting solution. The scissors should be allowed to air dry for about 60 seconds before being used to clip another toe.
       2. Flame the scissors by wetting the blades in 90% ethanol and then immediately putting the blades into a small flame (candle or cigarette lighter) and allow the alcohol to burn off completely. The blades may need to cool for 15-60 seconds before being used on the next animal. SAFETY TIPS: A) Keep ethanol and open flame well separated, and do not hold flaming scissors over the open container of ethanol. B) Hold flaming scissors perfectly level or with the blades slightly pointed down to prevent flaming alcohol from flowing sown the handles onto fingers and hands.
       3. Soak scissors in a bleach solution for 5 minutes. The solution should be 1 ounce of full strength bleach per liter (or quart) of clean clear water. Again, blood and debris on the scissors should be wiped off with a towel or cloth before being placed in the disinfectant solution. Prior to being used to clip another toe, the scissors should be rinsed in fresh water to prevent any bleach solution on the scissors from causing a caustic skin burn on the amphibian.
       4. Sites. Disinfect scissors and other instruments (forceps, nets, boots, etc) before arriving at the next site.


7. CITED LITERATURE:
   1. CLARKE, R. D. 1972. The effect of toe clipping on survival in Fowler's toad (Bufo woodhousei fowleri). Copeia 1972:182-185.
   2. DONNELLY, M. A., C. GUYER, J. E. JUTERBOCK, and R. A. ALFORD.1994. Techniques for marking amphibians. In W. R. Heyer, M. A. Donnelly, R. W. McDiarmid, L. C. Hayek, and M. S. Foster (eds.), Measuring and Monitoring Biological Diversity, Standard Methods for Amphibians, pp. 277-284. Smithsonian Inst. Press, Washington, D.C.
   3. GOLAY, N., and H. DURRER. 1994. Inflammation due to toe-clipping in natterjack toads (Bufo calamita). Amphibia-Reptilia 15:81-83.
   4. LEMCKERT, F. 1996. Effects of toe-clipping on the survival and behavior of the Australian frog Crinia signifera. Amphibia-Reptilia 17:287-290.
   5. MARTIN, D., and H. HONG. 1991. The use of Bactine® in the treatment of open wounds and other lesions in captive anurans. Herpetol Rev 22: 21.
   6. REASER, J. K., and R. E. DEXTER. 1996. Rana pretiosa (Spotted Frog). Toe clipping effects. Herpetol. Rev. 27:195-196.

D. Earl Green, DVM, DACVP
U.S.G.S., National Wildlife Health Center
6006 Schroeder Road
Madison WI 53711-6223
Tel: 608-270-2482
E-mail: david_green@usgs.gov