This protocol was designed by the USGS Northern Prairie Wildlife Research Center to help you make a successful survey for malformed amphibians.

For any successful survey the most important rule is to PLAN AHEAD!

Also, be sure to read the DAPTF Fieldwork Code of Practice before conducting any surveys.

Choosing sites

Which site(s) you choose depends, in part, on your goals:

• If your goal is to verify a report of a malformed amphibian captured in the vicinity of a particular pond, or if you are surveying for a landowner who wants to know if there are malformed amphibians in a pond on his/her land then the choice is simple.
• If you have a more ambitious goal, such as surveying an area, park, wildlife refuge etc., then there several factors to take into consideration. Breeding sites should be randomly chosen from sites known to have an abundance of frogs (your plans should, therefore be made well in advance and include some quick spring frog calling counts). Random means not biased in any way. Sampling the wetland closest to your home base is not random. Numbering the possible wetlands and pulling numbers from a hat is random.
• If you already have an idea which sites have malformed frogs and which do not but are surveying to determine the rate of malformations or the species affected, you should pick pairs of sites where one has only normal animals and the other malformed for purposes of comparison. Again, sites should be randomly chosen.
• How many ponds you sample also depends on the manpower available. For example, the U.S. Fish and Wildlife Service estimates that a sampling effort at a site requires two to three full days with a work team of three people.

Which species should you sample?

Although Leopard Frogs, Green Frogs and Bull Frogs account for 60% of all the reports in the NARCAM database, malformations have been reported in 54 species of amphibians. It is therefore strongly recommended that you do not focus solely on these three species but rather all abundant species on your sites, if possible.

Froglets, juveniles, adults - which animals should you try to catch?

The focus of the sampling should be on newly metamorphosed frogs ("froglets") or salamanders (recently transformed from a tadpole). The reason for this is simple: malformed frogs are not expected to live for very long. A malformation represents an error that occurred early in the process of development from egg to frog or salamander. Most of this development occurs before the tadpole transforms into a froglet or small salamander. Their handicapped situation makes them easy prey for predators and they are often not able to catch food and starve. Few of these individuals will reach adulthood and sampling only adult frogs will therefore underestimate of the malformation rate. Also sampling only adults will tell you about conditions in the pond when that animal was born a year or more ago, depending on the species (assuming that the frog or salamander in question was even born in that pond). Malformations in newly metamorphosed amphibians are the product of factors acting during that year.

The size of frogs at metamorphosis varies by species. For species in the northeast, Green Frogs, Pickerel Frogs, and Leopard Frogs should be 1.5 inches or 3.8 cm or less (measured snout to vent) at metamorphosis. Wood frogs, American toads, and Spring Peepers are about the size of your fingernail. Bullfrogs remain tadpoles for 2 to 4 years and are thus quite sizable at metamorphosis; as large as 2 3/8 inches or 5.9 cm. Again, consult a good field guide for this information before you begin sampling.

If tadpoles have not yet reached metamorphosis they are still worth looking at so long as they have developed both rear and front legs (the hind legs develop long before the front legs). However, malformations involving the spine might be obscured by the presence of the tail. Also some of the metamorphs may still have tails. Since the malformation list includes a category called "Extension of 'Spine' beyond rump" it is important to understand when it is abnormal for a metamorph to have a tail. Please note that this is an uncommon malformation. It is mentioned here not to draw special attention to it but rather to avoid confusion. If the individual still has an oval-shaped mouth it is technically a tadpole and would be expected to have a full tail. A newly metamorphosed frog will have a wide mouth like an adult frog. However, while in transition the metamorph absorbs its tail for nutrition and does not eat. A newly metamorphosed frog in good condition (i.e. as a tadpole it was well nourished and large) may retain a fleshy remnant of its tail. This is not a malformation of the type "Extension of 'Spine' beyond rump".

When should you sample?

For the reasons explained above, sampling should be conducted at the time that metamorphosis is expected to take place for the most abundant frog species in your area. Another reason is that most amphibian species do not use the breeding pond as a foraging habitat - they look for food elsewhere (though some species will return to the pond during periods of draught). This includes, for example, Leopard Frogs, Treefrogs, Chorus Frogs, Toads and Spadefoots (consult a good field guide for this information). Therefore, normal froglets will leave the pond after metamorphosis., while malformed individuals, which are often handicapped and can not travel far, will tend to remain behind. Therefore, if you wait too long to survey for these species there will be disproportionately high number of malformed frogs left in the breeding pond.

For a given species the timing of metamorphosis this varies considerably with latitude (spring comes earlier in the South), altitude (spring comes later in highland sites) and local climate. For a given site the date of metamorphosis for a given species will also vary from year to year - the warmer the weather the faster amphibian larvae develop. You should therefore spot-check the sites at least once a week or confer with a state herpetologist who is working in the field to get an estimated time of metamorphosis for your area. This is another important reason for planning your surveys well ahead of time. Consult field guides or a state herpetologist on when a species usually breeds and how long it usually takes to reach metamorphosis so that you have a rough idea of what to expect.

How many frogs should you catch?

If you catch 20 frogs and one is malformed, what is the malformation rate for all frogs in that pond? 5%? Maybe.

Presumably there are too many frogs in the pond to be able to catch them all, and you can't really know what the true malformation rate is unless you do. Fortunately, however, you can make a good estimate by catching a small sample of frogs. But how small a sample can you get away with? What does one malformed frog out of a total of 20 tell us when we have a population of thousands?

First we shall assume your sample was truly random and that malformed and normal froglets had equal chances of being captured. Such a sample means, then, that there is a 95% chance that the malformation rate is anywhere from just above 0% to 27%, a range of 27 percentage points! This "range of uncertainty" (the "95% Confidence Interval") will decrease as sample size increases (though it is also dependent upon the number of malformed individuals you catch), as can be seen in the table:

The table suggests that more is better, but if you are observant you will notice that gains in precision decrease with increasing sample size, i.e. each new frog that you catch will increase the precision of your estimate but not by as much as the last one. More is better but we don't want to waste our time violating the law of diminishing returns, so what is optimal? Once you have caught somewhere around 50-60 animals, the addition of one more frog to the sample will decrease the size of the 95% Confidence Interval (Maximum minus Minimum) by less than 1% (the exact number depends on the number of malformed individuals in the sample). Therefore we will make these arbitrary recommendations:

• Collect at least 60 individuals if your you are finding that approx. 5% or more are malformed, but...
• If you have the time/manpower to collect more, do so.
• You will need to collect more frogs in order to detect malformation rates that are lower than 5% with a reasonable degree of precision.
• If you're having trouble catching enough frogs, remember that a small sample is sill better than no sample

Equipment Needed

• Hip boots
• Bait nets (2 sizes recommended: 12 inch frame with 4 foot handle, and 6 inch frame with 1 foot handle - the deeper the net the better)
• Small aquarium net
• Five gallon buckets with lids and/or old pillow cases
• Data sheets with malformation check lists (like the Standard Field Forms available on this website). If you will be working in foul weather consider printing the forms on water proof paper.
• Pens and pencils
• Camera and slide film (to help document the malformations seen)
• Cooler
• Ice/Blue Ice

Other useful items would be a GPS unit if are surveying ponds in remote areas and electrofishing equipment if you have the experience and are conducting large scale surveys.

Making the Catch and Recording Data

Froglets tend to congregate in groups along the edge of the wetland. Once you find a rich spot fish (frog?) it out thoroughly before moving on.

Some have found that different nets work best for different sites, and having a large net and a small net in hand can be useful for herding frogs. Inexpensive nets are often available at bait shops (a potential source of inexpensive nets is provided below*). Some sites have such dense vegetation that capturing frogs with nets is nearly impossible. A group surveying at Mason Neck Refuge in Virginia encountered such a problem so they tried electrofishing. This method proved effective and caused no apparent mortality. Seining with a fine mesh net can also be effective.

Holding the frogs prevents recapture bias. As froglets are captured they can be placed in buckets that have a little water from the site in them, or in a moistened pillow case tied to your belt. On hot days, froglets in the pillowcase and/or buckets can be periodically transferred to a cooler that has a little water from the site and some ice or blue ice (preferably) to await processing. Keep the water in the buckets and/or cooler shallow - some newly metamorphosed frogs can actually drown if the water is over their head and they are not able to pull themselves out or climb onto to some object (in the pond this is never a problem).

Once you have collected your approx. 60 froglets, they should be processed. Study each frog carefully for signs of malformations and if you believe a froglet is malformed set it aside. Normal froglets should be released as soon as possible. Please catalog each species separately, and catalog adults separately from metamorphs.

Reexamine the malformed individuals more carefully and fill out the survey data sheets. Excellant data collection field forms for malformation surveys are available on this Web site. Try to record such things as left and right, fore and hind, etc. While it may not be likely that malformations are more likely to occur in one limb over another, this can not be ruled out if we do not have the data. Also, note whether the malformation is bilaterally symmetric. As pointed out in the introduction, some researchers feel that since chemical agents in solution are more likely to affect the entire individual they would only give rise to malformations that affect both sides of the body in the same way. Other researchers dispute the notion that chemical agents would never give rise to asymmetrical malformations. Most agree that parasites are more likely to create asymmetrical malformations.

If possible take some photos of representative malformations. (Suggestion: to avoid strong shadows, take the photos in a shaded area outdoors without using a flash. Use a gray or white background). Pictures of malformed frogs are available on the page What do malformations look like? and can be printed out and taken into the field. If the abnormality is obviously trauma related (blood apparent, or leg broken during capture) record the frog as normal. All frogs should be released back to the site once recorded.

* Brunken Manufacturing Company Inc. , 4205 W. Jackson St., Pensacola, FL 32505, (850)438-2478(The 6 inch net is model BN-15A)

What do I do with the information?

After recording this information in the field, the data will need to be sent to NARCAM so that it can be entered into our database (the basis of the maps found under Where Have Malformations Been Reported?). This can be done online using the reporting form on the NARCAM site.

Entering your data into these forms should be fairly self-explanatory. However, if this survey was conducted on a federal reserve, please enter "NWR-" or "WMD-" under "Name" before your name to alert the NARCAM that this survey is coming from a National Wildlife Refuge or Wetland Management District. Under "Location of Survey," please enter the name of your NWR or WMD in the space provided for "Town/City".

For more information on National Wildlife Refuges or Wetland Management Districts consult the U.S. fish and Wildlife Service's List of Offices By Region.

This surveying protocol was developed from an older U.S. Fish and Wildlife Service protocol for surveying national wildlife refuges.