United States Environmental

Protection Agency

Office of Water

Washington, DC 20460

EPA-822-R-02-022

March 2002

Methods for evaluating wetland condition

#12 Using Amphibians in

Bioassessments of Wetlands
United States Environmental

Protection Agency

Office of Water

Washington, DC 20460

EPA-822-R-02-022

March 2002

Methods for evaluating wetland condition

#12 Using Amphibians in

Bioassessments of Wetlands

Major Contributors

USGS Patuxent Wildlife Research Center

Donald W. Sparling

King County Department of Natural Resources, Seattle, WA

Klaus O. Richter

Department of Applied Biology, University of Maine

Aram Calhoun

Ohio EPA, Division of Surface Water

Mick Micacchion

Prepared jointly by:

The U.S. Environmental Protection Agency

Health and Ecological Criteria Division (Office of Science and Technology)

and

Wetlands Division (Office of Wetlands, Oceans, and Watersheds)
United States Environmental

Protection Agency

Office of Water

Washington, DC 20460

EPA-822-R-02-022

March 2002

Notice

The material in this document has been subjected to U.S. Environmental Protection Agency (EPA)

technical review and has been approved for publication as an EPA document. The information

contained herein is offered to the reader as a review of the “state of the science” concerning wetland

bioassessment and nutrient enrichment and is not intended to be prescriptive guidance or firm advice.

Mention of trade names, products or services does not convey, and should not be interpreted as

conveying official EPA approval, endorsement, or recommendation.

Appropriate Citation

U.S. EPA. 2002. Methods for Evaluating Wetland Condition: Using Amphibians in

Bioassessments of Wetlands. Office of Water, U.S. Environmental Protection Agency, Washington,

DC. EPA-822-R-02-022.

Acknowledgments

EPA acknowledges the contributions of the following people in the writing of this module:

Aram Calhoun (Department of Applied Biology, University of Maine), Douglas G. Hoskins (U.S.

Environmental Protection Agency), Mick Micacchion (Ohio Environmental Protection Agency), Klaus

O. Richter (King County Department of Natural Resources, Seattle, WA), Don W. Sparling (United

States Geological Survey), and the King County Department of Natural Resources, Washington State,

for supporting this work.

This entire document can be downloaded from the following U.S. EPA websites:

http://www.epa.gov/ost/standards

http://www.epa.gov/owow/wetlands/bawwg
iii

Contents

Foreword .............................................................................................................. v

List of “Methods for Evaluating Wetland

Condition” Modules...........................................................................................vi

Summary ................................................................................................................. 1

Purpose.................................................................................................................. 1

Introduction ........................................................................................................ 1

What Is the Value of Using Amphibians in Bioassessments? .................. 5

Necessary Refinements To The Basic Study Plan..................................... 7

Amphibian Monitoring Protocols ............................................................... 10

Data Analysis..................................................................................................... 27

Possible Metrics.............................................................................................. 27

Limitations of Current Knowledge and Research Needs ................... 29

Conclusions...................................................................................................... 32

References ........................................................................................................ 33

Appendix A .......................................................................................................... 40

Appendix B ...........................................................................................................41

List of Figures

Figure 1: Natural regions of continental herpetofauna .................. 4

Figure 2: Because amphibians use both uplands and

wetlands, it is important to determine landscape

level features of the environment .......................................12
iv

Figure 3: An economical funnel trap can be constructed

from empty 2 L soda bottles ...................................................13

Figure 4: Side view of a wetland showing the different

zones for surveying: shore zone, shallow water

zone, and waterline ....................................................................17

Figure 5: Top view of a wetland showing different zones

for basic egg surveys ............................................................... 18

Figure 6: Pitfall traps can be placed in various configurations,

with and without drift fences .................................................21

Figure 7: Array configurations of pitfalls are also flexible

and can be adjusted to particular needs ........................... 22

Figure 8: The proper setting of pitfall traps involves

burying the pitfall to its lip, burying the drift

fence at least slightly into the ground to prevent

amphibians from crawling underneath, and careful

placement of a lid over the pitfall to prevent

excessive exposure to the elements .................................... 23
v

Foreword

In 1999, the U.S. Environmental Protection Agency (EPA) began work on this series of reports entitled

Methods for Evaluating Wetland Condition. The purpose of these reports is to help States and

Tribes develop methods to evaluate (1) the overall ecological condition of wetlands using biological

assessments and (2) nutrient enrichment of wetlands, which is one of the primary stressors damaging

wetlands in many parts of the country. This information is intended to serve as a starting point for States

and Tribes to eventually establish biological and nutrient water quality criteria specifically refined for

wetland waterbodies.

This purpose was to be accomplished by providing a series of “state of the science” modules concerning

wetland bioassessment as well as the nutrient enrichment of wetlands. The individual module format

was used instead of one large publication to facilitate the addition of other reports as wetland science

progresses and wetlands are further incorporated into water quality programs. Also, this modular

approach allows EPA to revise reports without having to reprint them all. A list of the inaugural set of

20 modules can be found at the end of this section.

This series of reports is the product of a collaborative effort between EPA’s Health and Ecological

Criteria Division of the Office of Science and Technology (OST) and the Wetlands Division of the

Office of Wetlands, Oceans and Watersheds (OWOW). The reports were initiated with the support

and oversight of Thomas J. Danielson (OWOW), Amanda K. Parker and Susan K. Jackson (OST),

and seen to completion by Douglas G. Hoskins (OWOW) and Ifeyinwa F. Davis (OST). EPA relied

heavily on the input, recommendations, and energy of three panels of experts, which unfortunately have

too many members to list individually:

 Biological Assessment of Wetlands Workgroup

 New England Biological Assessment of Wetlands Workgroup

 Wetlands Nutrient Criteria Workgroup

More information about biological and nutrient criteria is available at the following EPA website:

http://www.epa.gov/ost/standards

More information about wetland biological assessments is available at the following EPA website:

http://www.epa.gov/owow/wetlands/bawwg
vi

List of “Methods for Evaluating Wetland

Condition” Modules

1 ................. Introduction to Wetland Biological Assessment

2 ................. Introduction to Wetland Nutrient Assessment

3 ................. The State of Wetland Science

4 ................. Study Design for Monitoring Wetlands

5 ................. Administrative Framework for the Implementation of a

Wetland Bioassessment Program

6 ................. Developing Metrics and Indexes of Biological Integrity

7 ................. Wetlands Classification

8 ................. Volunteers and Wetland Biomonitoring

9 ................. Developing an Invertebrate Index of Biological

Integrity for Wetlands

10............... Using Vegetation to Assess Environmental Conditions

in Wetlands

11 ............... Using Algae to Assess Environmental Conditions in

Wetlands

12 ............... Using amphibians in Bioassessments of Wetlands

13 ............... Biological Assessment Methods for Birds

14 ............... Wetland Bioassessment Case Studies

15 ............... Bioassessment Methods for Fish

16 ............... Vegetation-Based Indicators of Wetland Nutrient

Enrichment

17 ............... Land-Use Characterization for Nutrient and Sediment

Risk Assessment

18............... Biogeochemical Indicators

19 ............... Nutrient Load Estimation

20 .............. Sustainable Nutrient Loading

Module # Module Title
1

Summary

Amphibians are important ecological componentsof both wetlands and dry land. Among vertebrates they are distinctive in many ways. For biological assessments, they are especially promising

because of their capability of linking wetlands with

surrounding landscapes. Surveying amphibians and

obtaining reliable data on community characteristics may require multiple samplings of each wetland

within a year because of differences in breeding and

developmental phenology among species and the

complex life histories of amphibians. Thus, more

work may be necessary to conduct an adequate

monitoring program for this assemblage than for

macrophytes, fish, algae, or aquatic invertebrates.

Published studies using amphibians in developing

indices of biological integrity currently do not exist.

However, such studies are in progress with several

attributes proposed, and more can be developed

from a thorough search of literature dealing with

limiting factors in amphibians. Most of the specific

metrics will have to be developed on a regional basis

owing to differences in continent-wide distributions

of amphibian species. We encourage investigators

to examine amphibian communities as a possible

source of metrics in the development of their indices of biological integrity.

Purpose

T his module is intended to encourage the use ofamphibian metrics in wetland bioassessment.

To facilitate use of this valuable information, we describe briefly the basic ecology of amphibians and

their assemblages, and the best available science

on their responses to physical, chemical, and ecological stress. Monitoring protocols for the assessment of amphibian responses along a gradient of

human disturbance are described at various levels

of sampling effort.

Introduction

Carl Linnaeus, arguably among the most famousbiologists of the 18th century, characterized

reptiles and amphibians as “foul and loathsome animals, abhorrent because of their cold body, pale

color, cartilaginous skeleton, filthy skin, fierce aspect, calculating eye, offensive voice, squalid habitation, and terrible venom” (quoted in Hunter et al.

1992). Three centuries later, amphibians (and their

squalid habitation) have gained stature in human

eyes, as their ecological importance becomes

clearer. For example, amphibians may constitute

the highest biomass among vertebrates in some ecosystems and, depending on scale, may be keystone

species (Burton and Likens 1975, Windmiller 1996,

Wyman 1998, Fauth 1999, Petranka and Murray

2001). Within the last decade, amphibians have

the dubious distinction of being in the global spotlight owing to worldwide declines (Barinaga 1990,

Wyman 1990, Wake 1991, Griffiths and Beebee

1992). Suggested causes of declines include various human-induced processes, including habitat loss

or degradation (Reh and Seitz 1990, Griffiths and

Beebee 1992, deMaynadier and Hunter 2000,

Turtle 2000), acid deposition (Freda 1986, Horne

and Dunson 1994), climate warming (Wyman 1990,

Pounds et al. 1999), increases in UV radiation

(Blaustein et al. 1994), spread of toxic substances

(Sparling et al. 2000, 2001), introduction of predators (Funk and Dunlap 1999, Lawler et al. 1999),

and pathogens (Carey and Bryant 1995, Jancovich

et al. 1997, Morrell 1999, Daszak et al. 2001).

These declines raise the global eyebrow because

amphibians are indicators of ecosystem health

(Wake 1991). A thin, moist, highly permeable skin;

jellied, unshelled eggs; possession of aquatic and

terrestrial life histories; restricted home range; and

limited dispersal abilities of many species make

amphibians effective biomonitors. Dramatic changes

in their populations and increased incidences of diseases and malformations, particularly in seemingly
2

pristine areas, highlight concerns about general environmental deterioration.

Scientists studying amphibians have had difficulties sorting out local versus global and single versus

multiple (e.g., synergistic) causes behind population declines and malformations. The formulation

of a useful Index of Biological Integrity (IBI) depends on identifying biological attributes (or metrics)

that provide reliable signals about the condition of

the resource (Karr and Chu 1999). For amphibians, the poor resolution in identifying relationships

between potential causative factors and effects

complicates metric development.

Amphibians as a group are much more closely

associated with water and wetlands than are most

reptiles, birds, or mammals. Most anurans (i.e.,

frogs and toads) and many caudates (i.e., salamanders and newts) lay their eggs in water, have

aquatic larvae, and inhabit forests or other upland

habitats as adults. Treefrogs (Hyla spp.), toads

(Bufo spp.), and salamanders belonging to the mole

salamander group Ambystoma, for example, exhibit such characteristics. Numerous North American frogs, especially those in the common “true frog”

genera Rana and the chorusfrog genera Pseudacris,

live their entire lives in wetlands. Still others, such

as some mole salamanders including the common

northwestern salamander (Ambystoma gracile)

and the widely distributed tiger salamander (A.

tigrinum), may turn into neotenes (i.e., gilled adults),

a life form that is entirely restricted to water (Plate

1). Species in the genera Plethodon and Ensatina

lay eggs in damp locations on land and spend their

entire life in upland habitats (Plate 2).

Among amphibians breeding in aquatic habitats,

different genera, and even species within genera,

select specific aquatic environments on the basis of

hydroperiod, current velocity, and other wetland

characteristics. Some (e.g., wood frog, R.

sylvatica; long-toed salamander, A. macrodactylum) prefer to breed in standing water of vernal

wetlands or temporary pools. Others (e.g., green

frog, R. clamitans; bullfrogs, R. catesbeiana) select quiescent water of permanent ponds (Plate 3).

Plate 1. Among other species, the northwestern

salamander (Ambystoma gracile) commonly occurs as a

neotene (e.g. gilled adult) and consequently can only

successfully breed in permanently flooded wetlands.

© Klaus O. Richter. Used with permission of the photographer.

Plate 2. The Ensatina (Ensatina eschscholtzii) is a species

of amphibian that lives almost entirely on land.

© Klaus O. Richter. Used with permission of the photographer.
3

Interestingly, others still (e.g., tailed frog; Ascaphus

truei; torrentsalamander larvae, Rhyacotriton spp.)

are restricted to fast-moving water of small mountain streams, spring heads, and seepages (Plates 4

and 5).

Those that use both uplands and wetlands for different phases of their life cycles often have unique

physiological and behavioral requirements for both

habitat types. Consequently, some amphibian species may be very useful in biological assessments of

wetland health. However, the constellation of species that inhabits a particular region may be better

in assessing the health of both uplands and wetlands. In this way, amphibians can provide useful

metrics to assess the health or condition of entire

landscapes.

Amphibian community composition may vary dramatically across the country, and even across comparatively narrow regions. It is therefore important

for those involved in assessments to be aware of

regional and local amphibian fauna and to avoid

broad generalizations. Readers are referred to the

following atlases, natural histories, and descriptions

of species distributions for their areas: (a) overall

North America (Behler and King 1979, Stebbins

and Cohen 1995, Wright and Wright 1995, Lannoo

1998, Petranka 1998); (b) Northeastern North

America (DeGraaf and Rudis 1983, Hunter et al.

1999, Hulse et al. 2001); (c) Southeastern North

America (Dundee and Rossman 1996, Bartlett and

Bartlett 1999); (d) Eastern and Central North

America (Pfingsten and Downs 1989, Conant and

Plate 4. Tailed frogs (Ascaphustruei) breed in fast-moving

streams and live and feed along their shorelines.

© Klaus O. Richter. Used with permission of the photographer.

Plate 5. Tailed frog tadpoles possess ventral sucotorial

discs that allow attachment to the substrate in swift

moving currents.

© Klaus O. Richter. Used with permission of the photographer.

Plate 3. The bullfrog (Rana catesbeiana) breeds mostly

in permanent ponds.

© Klaus O. Richter. Used with permission of the photographer.
4

Collins 1998, Mitchell and Reay 1999); (f) Western North America (Stebbins 1985); (g) Northwestern North America (Nussbaum et al. 1983, Corkran

and Thoms 1996, Leonard et al. 1993); and (h)

Southwestern North America (Stebbins 1972).

On a continental scale, McDiarmid and Mitchell

(2000) recognized six natural regions for

herpetofauna (i.e., amphibians and reptiles) (Figure

1). Species richness follows somewhat different

patterns for caudates than for anurans. In North

America there are more species of salamanders and

newts (155) than frogs and toads (91), and salamanders tend to be more terrestrial than frogs. The

highest species richness for salamanders is in the

Appalachian Highlands, Gulf and Atlantic Coastal

Plains, and Pacific Northwest. Several species in

the Appalachian Highlands have very restricted

ranges. Other areas with high species richness of

salamanders include the western Sierra Nevadas,

the Edwards Plateau, and the Interior Highlands of

the Central Plain. Frogs also reach their highest

species richness in the southeastern United States

but are most diverse in the lowlands of the Coastal

Plains. South-central Texas is another area of high

salamander species richness. Within each of these

Figure 1: Natural regions of continental herpetofauna (after

McDiarmid and Mitchell 2000).
5

major natural regions there is considerable local

variation in taxa and abundance of which biologists

must be aware.

What Is the Value of

Using Amphibians in

Bioassessments?

Contrary to Linnaeus’ quote above, amphibians are of particular importance in wetland

ecosystems and can perform a significant function

in landscape assessments. To a large extent, they

have been historically ignored in favor of other vertebrates, but amphibians serve as vital links in food

webs and between wetland and upland habitats. In

addition, recent media attention on declines and malformations in amphibians has made them a popular

and conspicuous element in nature. This attention

is useful in creating incentives for recruiting agency

and volunteer support in developing monitoring programs.

Specific advantages of using amphibians in

bioassessments include:

1. Sensitivity. Because of their unique physiology and habitat requirements, amphibians are often

regarded as more exposed and potentially more

vulnerable to changes in their environment than many

other vertebrates (see Sparling et al. 2000). Amphibians may serve as the proverbial “canaries in

the coal mine” in their response to factors such as

habitat fragmentation, hydrologic modifications, alterations in water chemistry, water and airborne contamination, and large-scale climatic variation. This

is exacerbated by production of embryos in clear,

unprotected jelly egg-masses; thin, highly permeable skin exposed to water and the atmosphere;

and their limited dispersal and home ranges (but

utilization of a wide spectrum of habitats across the

aquatic/terrestrial continuum) (Plate 6).

2. Complex life history. Wetland-breeding amphibians exhibit complex life histories, often undergoing dramatic and irreversible morphological and

physiological change from sedentary eggs, to freeswimming aquatic larvae, to semiaquatic or terrestrial adults. Especially because of their utilization of

wetland/upland transitional areas, members of this

class are appropriate for assessing impacts on these

habitats. For instance, the first effects of hydrologic modifications become apparent to eggs that

are exposed to freezing and to tadpoles and newly

metamorphosed individuals that may face desiccation (Plate 7). Whereas some amphibians move

into the adjacent uplands to live the majority of their

life cycle, others spend most of their time on this

Plate 6. Embryos of wetland-breeding amphibians are

“shell-less” and therefore sensitive to water pollutants

and other environmental influences. Consequently,

development, abnormalities, and mortality can readily be

determined and studied.

© Klaus O. Richter. Used with permission of the photographer.
6

fringe between wetland and upland, and both could

serve as “early warning systems” for pending impacts to their aquatic environments.

3. Easily studied. Anurans are very visible, tangible, and popularly enjoyed animals of wetlands

and nearby environments. Adult males tend to be

vocal during the breeding season and tadpoles generally are easily caught, particularly when they reside in small, isolated, seasonally flooded wetlands

and vernal pools. The attractiveness of amphibians

stimulates volunteer recruitment, as exemplified by

programs such as the North American Amphibian

Monitoring Project (NAAMP) and North American Reporting Center for Amphibian Malformations

(NARCAM) (see also Module #8: Volunteers and

Wetland Biomonitoring).

4. Selecting amphibians for specific purposes.

Researchers can utilize amphibians’ multiple life

stages, varied species-specific habitat preferences,

and broad and diverse landscape utilization to assess a variety of wetland classes and habitats over

a relatively long annual cycle. There are regional

differences in habitat preferences, such as for permanent wetlands, seasonally flooded wetlands, and

vernal pools; but similarities can be drawn upon as

well to facilitate comparisons among amphibians

(Plates 8–10). For example, Plethodons are terrestrial, ambystomids use water and land, and ranids

Plate 7. The health of amphibian eggs can provide

valuable information on wetland hydrologic conditions.

This totally frozen northwestern salamander egg mass was

attributable to dropping water levels between spawning

and survey date.

© Klaus O. Richter. Used with permission of the photographer.

Plate 8. Permanent wetlands are the preferred breeding

habitats for bullfrogs, green frogs, northwestern

salamanders, and a select few other species of

amphibians.

© Klaus O. Richter. Used with permission of the photographer.

Plate 9. Vernal pools are the preferred breeding habitats for

many species of ranids, chorus frogs, and treefrogs.

© Klaus O. Richter. Used with permission of the photographer.
7

tend to prefer stable bodies of water to breed and

live near aquatic environments for more of their life

cycle than do bufonids (toads).

5. Existing information. Although there are obvious gaps in our knowledge about amphibian ecology, there is also enough scientific literature on the

effects of environmental stressors on amphibians to

assist in understanding their needs and limitations

(see below).

6. Further study will lead to better understanding. The additional information that would be gathered during amphibian biological assessments should

fill in some of these data gaps and contribute to a

better understanding of amphibian relationships to

gradients of human disturbance and the reasons for

their population declines.

Necessary

Refinements To The

Basic Study Plan

Modules #4: Study Design for Monitoring

Wetlands and 6: Developing Metrics and

Indexes of Biological Integrity provide the basics

for bioassessment of wetlands and construction of

an index of biological integrity, and we will not repeat that information here in detail. Each group of

organisms—macrophytes, algae, invertebrates, fish,

and amphibians—requires special procedures or

modifications of that basic protocol. In this section

we discuss some of the considerations necessary in

using amphibians in bioassessments.

1. Uplands and wetlands are important. Compared with other assemblages such as macrophytes

or aquatic invertebrates, one difference in using

amphibians in assessing wetlands is that many species are restricted to water for only part of their life

cycle whereas others are totally aquatic. Most

amphibians breed and lay eggs in water (or moist

environments); their larval stages depend on standing water but the metamorphosed juveniles and

adults outside of the breeding season are terrestrial

or arboreal. For example, many adult toads,

treefrogs, and metamorphosed ambystomid salamanders spend more of their time on land than in

water. Therefore, inclusion of upland areas adjacent to wetlands may be essential in developing a

disturbance gradient and understanding local amphibian distributions. This is especially true for forested wetlands and other sites where uplands are

integral components of a wetland/upland complex.

2. Amphibian populations fluctuate widely.

Reliance on the presence or absence of amphibian

species from limited studies may not be a good indicator of overall wetland health. Short-term studies especially may be misleading because of substantial annual variation in numbers at a given body

of water (Pechmann et al. 1989, Pechmann 1991)

and because many amphibian species exist as

metapopulations, using several nearby wetlands interchangeably (Gill 1978, Dodd and Cade 1998,

Semlitsch 2000). Numbers of breeding amphibians at any wetland can vary widely for several reasons. Specifically, in some species only a small

percentage of the adult population breeds in any

given year, and the reasons for not breeding are

unknown in most instances. In others, annual variation in weather can have a large effect on the numPlate 10. After rains, the Great Basin spadefoot toads

(Spea intermontana) often breed and lay their eggs in

puddles, roadside ditches, and other shallow ephemeral

waters. Their eggs hatch in a few days and they

metamorph within a few weeks.

© Klaus O. Richter. Used with permission of the photographer.
8

bers of species and individuals migrating or breeding (Packer 1960, 1963, Hurlbert 1969, Semlitsch

1985).

3. Repeated visits may be necessary. In most

regions it is impractical or even a mistake to monitor amphibian species by making only one visit to a

wetland. The sampling windows for some amphibians, especially adults, are short in duration, noncontinuous among species, and vary from year to

year within a species. Following breeding there is

typically a larval period lasting from a few weeks to

several months and then a comparatively short period of metamorphosis. Individual species may have

discrete breeding seasons, although larval periods

may overlap. Investigators need to know the life

histories, approximate breeding phenologies, and

habitat preferences of the amphibians likely to be

encountered in their areas. All other factors being

equal, if only a single trip can be afforded, it should

be timed to encounter the greatest species richness

of larvae.

4. Temporal variation may differ across regions. The variability in breeding can become magnified in the southern United States, where seasons

can last much longer than farther north. For example, in Florida it is not unusual for amphibians,

especially anurans, to exhibit breeding behavior for

more than half the year (Conant and Collins 1998).

In contrast, some amphibians at the northern extent

of their ranges or in arid regions of North America

breed for a period of a week or less (Stebbins and

Cohen 1995). To further complicate the situation,

some species such as green frogs may metamorphose in the year that they were laid when breeding

seasons are sufficiently long, but delay metamorphosis if laid late in the season or where breeding

seasons are shorter (Conant and Collins 1998). It

is often difficult or impossible to have monitoring

schedules coincide with periods of activity in all

species. Most adult ambystomatid salamanders in

the Midwest and the Pacific Northwest are latewinter, early-spring breeders (Petranka 1998), and

monitoring periods for adults of these species need

to coincide with their life cycles. Many frogs in the

Midwest, however, breed slightly later, and earlyto mid-spring are the best times to encounter these

adults. Thus, it is best to design a study that will

allow monitoring of wetlands on two or more occasions during the breeding season.

5. Multiple life stages should be considered. A

study design that monitors several amphibian life

stages will provide valuable information for developing metrics and IBIs. Call counts of adult anurans

provide early indication that frogs and, by extension, salamanders inhabit a wetland. Because of

species specificity, calling males can be used to assess relative species abundance and richness.

Counts of egg masses can also provide an index to

breeding efforts. Frog and toad tadpoles and salamander and newt larvae may indicate a level of successful breeding or may be used in other ways in

bioassessments. Metamorphosing juveniles provide a higher level of evidence for the suitability of a

wetland than egg and larval censuses alone, and the

frequency of malformations among juveniles may

additionally be a useful metric.

6. A variety of sampling methods may be necessary. Investigators should be familiar with the

various types of amphibian monitoring techniques

that are available (Heyer et al. 1994, Bonin and

Bachand 1997, Enge 1997, Olson et al. 1997,

Crouch and Paton 2000, Mitchell 2000). Specific

methods are appropriate for particular species and

life stages but not for others. For example, adult

treefrogs may be censused by inserting a 1 m long,

5 cm diameter PVC pipe partially into the ground

whereas their larvae can only be censused with dipnets, seines, or aquatic funnel traps. Combinations

of methodologies increase the likelihood of getting

an accurate representation of the amphibians

present. The type of monitoring protocols used must

be weighed against the time and money available to

conduct the study.

7. Animal care and use concerns. Although

care should be taken with any living organism, invertebrates, macrophytes, and algae generally have

sufficiently high numbers or reproductive potential
9

to tolerate moderate collection. However, amphibians are vertebrates with lower fecundity or total

numbers than these “lower” groups, and extensive

collecting may impact populations. Also, various

States restrict the collecting of amphibians and may

require permits. Animal care and use guidelines

dictate that certain procedures (e.g., keeping animals in comfortable conditions, euthanasia through

approved means) are followed to ensure humane

treatment of amphibians. Every effort should be

made to develop monitoring techniques that are

noninvasive and nonlethal. Individuals to be released should be held for as short a period of time

as possible. Researchers need to be sensitive to

keeping the skin of amphibians moist but not allowing metamorphs and adults to drown in water. When

taking vouchers or preserving individuals for later

laboratory use, it is best that larvae rather than adults

be sacrificed because removal of larvae will have

less impact on populations. All of these precautions become more important when dealing with

species that are federally, State, or even locally

listed. Encounters with listed species should be well

documented and reported to the appropriate Federal or State agency as soon as possible.

8. Avoid spreading disease. Whatever protocols are selected for monitoring, researchers need

to ensure that they incorporate proper procedures

to guard against spreading contamination and pathogens from one wetland study site to another. Monitoring equipment will need to be site-specific or

treated between uses at different sites to ensure that

the transmittal of any diseases, parasites, or other

nuisance organisms is held to a minimum. Investigators should be thorough in their decontamination

procedures and be aware that boots, waders, hands,

and gloves are potential carriers of contamination.

Detailed field practices to guard against contamination when working with amphibians can be found

on the North American Reporting Center for Amphibian Malformations (NARCAM) website, http:/

/www.npwrc.usgs.gov/narcam/techinfo/daptf.htm.

9. Abnormalities in frogs (and other amphibians) are of critical interest. Amphibian malformations are closely monitored and studied throughout the country. Any incidence of malformed amphibians greater than 2% or 3% of metamorphosing or juvenile frogs, or any incidence of bizarre

malformations, should be reported to NARCAM

(Plates 11–13). It is important that this information

be relayed to the reporting center quickly to allow

for any necessary followup studies. See the home

page of NARCAM, http://www.npwrc.usgs.gov/

narcam.html, for complete information and reporting procedures.

Plate 12. Mouth malformation in the Pacific treefrog

(Pseudacris regilla) is another example of a deformity.

© Klaus O. Richter. Used with permission of the photographer.

Plate 11. Blistering and edema in the heart region of a

northwestern salamander larva.
10

Amphibian Monitoring

Protocols

General Field Methods

Obligate wetland-breeding amphibians typically include four life stages: eggs, aquatic larvae, a rapidly

developing metamorph stage, and terrestrial or semiterrestrial adults. There are some exceptions. For

example, some salamanders may be neotenic and

become sexually mature without metamorphosing

(Dent 1968) or, like the mudpuppy, Necturus

maculosus, never exhibit a terrestrial phase at all

(Plate 14). Collectively, amphibians exploit a diverse variety of wetland and upland habitats. These

differences must be considered in monitoring if one

wishes to inventory sites, determine relative abundance, and establish population health. For example, anurans may be highly vocal during the breeding season, whereas most caudates are silent.

Therefore, well-designed calling surveys can be

used to survey breeding anurans but are inappropriate for caudates. Conversely, cover board surveys may be ideal for surveying salamanders and

newts but are generally deficient in establishing the

distribution and abundance of frogs and toads.

Some methods, such as aquatic funnel traps, and

pitfall traps may capture both anurans and caudates,

but all methods have some bias.

Overall Recommendations

Numerous methods are available for censusing

amphibians (reviewed in Heyer et al. 1994, Fellers

and Freel 1995, Halliday 1996, Olson et al. 1997).

These include visual encounters, egg surveys, calling censuses, terrestrial cover boards, dipnets,

seines, aquatic funnel traps, and terrestrial pitfall

traps.

Regardless of monitoring method, several factors

need to be considered to obtain reliable, unbiased

data. These include:

1. Regional modification and calibration.

Sampling designs need to be as specific as possible

to region, local species, and habitats.

2. Sample size. The number of samples per wetland should be based on the total size of the wetland and on the diversity of vegetation within the

wetland. Very large wetlands (> 5 hectares) can

be divided into two or more units at constriction

points. It is a good idea to sample vegetation communities in proportion to their approximate dominance within the wetland. Wetlands with diverse

patches of vegetation and cover may require more

sampling than those with simple communities and

structure. When resources are very limited and only

Plate 14. The mudpuppy (Necturus maculosus) inhabits a

wide variety of permanent aquatic habitats, with clear as

well as muddy waters that include large streams, lakes,

ponds, and wetlands.

© Klaus O. Richter. Used with permission of the photographer.

Plate 13. Extra set of front digits in the rough-skinned newt

(Taricha granulosa) is an example of deformities called

fluctuating asymmetry.

© Klaus Richter. Used with permission of the photographer.
11

a single sample of a wetland can be taken, do so

when most anurans are calling or when most species have larvae present. Of these two, the period

of peak larvae presence will probably record the

most complete representation of species. However, identification of larvae and especially frog tadpoles may be problematic. Censuses conducted

biweekly during spring and summer (March to July)

will yield the greatest species richness and most

accurate estimates of relative abundance.

3. Timing of surveys. Each of the methods described below has an optimal sampling window.

Adult call counts produce their greatest species richness following rain or during a light rain that does

not impair hearing. Cover board censuses and drift

fence/trapline captures should take place during

periods of annual migrations (e.g., late winter, early

spring), under moist soil and cool temperature conditions. Aquatic funnel traps, seining, and dipnetting

for larvae will be effective throughout the late spring/

summer in most locations. Ultimately, the number

and timing of surveys will depend on: (a) the goals

of the survey (e.g., collect data on one life stage of

a single species or several life stages of one or more

species); (b) local and regional wetland conditions;

(c) altitude of study site; and (d) wetland orientation (wetlands fully exposed to sunlight may be

warmer and more advanced than those in deep

shade at a given time of spring).

4. Sampling location. See Module #4: Study

Design for Monitoring Wetlands of this series for

descriptions of targeted and random sampling designs. A targeted design program selects habitats

or other aspects of a wetland and assigns a sampling regimen to maximize the representation of these

aspects. As the name implies, random designs develop from a sampling strategy that maximizes the

representation of the entire wetland, regardless of

habitat structure. Both types of designs are applicable to amphibians, depending on species and

objectives. If an estimate of relative abundance or

density is desired, random sampling designs are

probably more appropriate. If the objective is to

identify all the species inhabiting a site without regard to abundance, targeted designs may be more

efficient and representative.

 5. Wetland maps. Good maps such as United

States Geological Survey (USGS) topographic

maps; aerial photographs; National Wetland Inventory maps and other available State, county, or local wetland maps; or detailed sketches drawn to

scale are invaluable (Figure 2). They help in identifying existing conditions such as wetland and habitat classifications, perimeter and area of wetlands,

deep and permanently flooded locations, and plant

communities. The location of amphibian surveys

and other conditions through time can then be directly transferred to such maps and sketches to mark

changes in these factors. The maps should be sufficiently detailed to show location of aquatic plant

communities, the width and nature of immediate

buffers around the wetland, and the adjacent upland cover and habitats beyond buffers. Because

amphibians integrate wetland and upland features,

details of land use within a species’ range of dispersal, and hence gene flow to adjacent wetlands

and amphibian populations (Dodd 1996, Richter

1997, Semlitsch 2000), should also be included.

Specific Monitoring Methods

Of the many methods available for sampling amphibians,some are specific for aquatic stages such

as frog tadpoles or salamander larvae and, to some

extent, adults that live in the water or at the shoreline. Other methods are more specific for adults,

either in the water, moving between wetland and

upland habitats, or in the uplands proper.

Aquatic surveys

Several techniques are available for sampling

aquatic life stages of amphibians. These methods

include funnel trapping, egg mass searches, call surveys, dipnetting, and seining. Each has advantages

and disadvantages, and their use would depend on

the objectives of the survey and available human

and monetary resources.
12

Funnel trapping

If there were only one method of sampling anurans

to recommend, it would be funnel trapping. This

method requires an additional visit to a wetland

compared with some other techniques such as dipnetting or seining, yet it provides a reasonably economic method of sampling. This method captures

most larvae, including cryptic forms that might go

unnoticed with other techniques. When done correctly, it also has less potential for bias and a greater

potential for obtaining information on the entire

amphibian breeding assemblage than other aquatic

methods (Adams et al. 1997). It is highly recommended for these advantages (Mushet and Euliss

1997). Traditional aquatic funnel traps are selective for larval amphibians (Griffiths 1985) as well as

neotenes of some salamanders (Richter and Kerr

2001). Modifications for floating on the surface

have additionally proven funnel traps effective for

adult frog captures (Casazza et al. 2000). Aquatic

funnel trap captures confirm successful breeding,

egg development, and hatching. Clearly, when adults

are captured, the traps are also valuable in directly

determining their arrival times. Finally, with an appropriate study design, funnel trapping may also be

used for determining abundances and population

trends (Richter and Adams 1997).

Some funnel traps are easy to construct from inexpensive materials (Figure 3), making the method

cost-effective (Griffiths 1985, Richter 1995). Even

commercially manufactured nylon traps are relatively inexpensive (~$7.00). A great advantage of

aquatic funnel trapping is that, like another passive

Figure 2: Because amphibians use both uplands and wetlands, it is

important to determine landscape level features of the environment.
13

method, pitfall trapping, but unlike the more active

methods such as dipnetting, seining, or direct observation, the skill and experience of the surveyor

have little impact on reliability of data obtained (Plate

15). Thus, trapline sampling can be consistently

repeated over time, among wetlands, and with different crews. It is simple to use, requires minimal

training of technicians or volunteers, and can be used

for surveying and monitoring most aquatic amphibians (Adams et al. 1997).

One drawback of any sampling method that emphasizes larval forms is the difficulty of identifying

Figure 3: An economical funnel trap can be constructed from

empty 2 L soda bottles.
14

captures by species. Taxonomic keys such as Altig

et al. (1999), Petranka (1998), or several excellent

regional keys (e.g. Pfingsten and Downs 1989,

Hunter et al. 1999, Mitchell and Reay 1999) can

be quite helpful in identification. With similar-looking species, culturing larvae through metamorphosis may be required. In other cases, voucher specimens should be preserved to ascertain identifications in the future. A second potential drawback is

the interpretation of capture data. For example,

what is the role of mutual exclusion/attraction among

and between species (Wilson and Pearman 2000)

and the impact of predation by conspecifics and

other taxa (i.e., neotenes eat smaller captured larvae, Ditiscid beetle larvae destroy tadpoles, Richter pers. obs.)? Answers to these questions should

be considered in the interpretation of capture data.

Because of the potential for mortality if animals

are trapped too long, and species-specific differences in activity periods, funnel trapping normally

should be conducted over a 24-hour period. Times

shorter than this may be selected for some species.

However, much longer periods may result in elevated

mortality. Funnel trapping will always require at

least two site visits within the sampling time span.

Trapping within a variety of habitats and plant communities during breeding and larval development

increases the probability of identifying all species

using a wetland. Some statistical analytical methods such as power analysis (Gibbs 1993) can be

used to estimate the number of trap-nights needed

to provide a statistically valid sample of the

population.

The number of animals caught per unit time can

sometimes be used as a rough index of relative

abundance, but more precise estimates of abundance require solid study designs to be sure that all

assumptions required for population estimates are

valid. Consequently, reliable population estimates

(as contrasted to relative abundances) may be impractical. Most IBI analyses do not require the

statistical precision or rigor of experimental hypothesis testing (see Module #6: Developing Metrics

and Indexes of Biological Integrity). Besides, many

amphibian populations exist as metapopulations and

therefore show substantial variation in numbers from

year to year, and presence and absence (i.e. failure

to detect) data may be more reliable in reflecting

changes in amphibian numbers over a broad area

than absolute numbers (Fellers and Drost 1994).

Presence data that are compared with expected

species presence based on reliable regional guides

might be an attribute for evaluating wetland condition.

There are several steps to a funnel trapping design, as outlined below. For more details see Adams

et al. (1997), Fronzuto and Verrell (2000), or

Mitchell (2000). Here we present enough detail to

form a general understanding of the processes involved.

Plate 15. Aquatic funnel trapping using recycled plastic

pop bottles is one method of cheaply monitoring

amphibians.

© Klaus O. Richter. Used with permission of the photographer.
15

1. Identify and map aquatic habitat types.

Identify and map areas characterized by water regimes, habitat classes/subclasses, and community

types that occur between mean shoreline and open

or deep (>1.5 m) water. Tadpoles and adult frogs

seldom venture into deep or open water, where risks

from predation by fishes may be greater. This mapping can be part of the overall assessment of a wetland and may be adapted to other assemblages in

addition to amphibians. Habitat types vary by wetland classification (e.g., Cowardin et al. 1979, also

Module #7: Wetlands Classification) and desired

level of detail and may include:

i. Hydrology, especially permanently and

semipermanently flooded areas

ii. Vegetation by major types of plant communities and including open areas

iii. Type of bottom material (gravel, silt, sand)

iv. Ecotones, the 1 m edge area where two different communities meet (see Richter and

Adams 1997).

2. Determine number, size, and location of

sampling points. If a priori methods to determine

adequate sample size cannot be utilized, we recommend at least 10 randomly selected sampling

points within each major habitat for funnel trapping.

i. Map to scale all habitat types, being certain to

include 1 m wide ecotones at boundaries between habitat types.

ii. Use a planimeter or overlay scale to calculate

the area (in m2

) of each habitat type, including

ecotones.

iii. Divide total m2 area of each unique habitat

unit and ecotone by 10 to obtain a sample

overlay grid size. Apportion these sample

grids according to habitat block size.

iv. Identify sampling points within each habitat unit

by sequentially sketching and numbering the

total number of m2

 plots in each unit and use

some method such as a random number table

or random number generator to select sample

points; repeat random selection until 15 sampling points are identified; the last five sampling points chosen serve as alternative contingency points. Set funnel traps at the crossing of diagonals on the map.

v. Randomly select new surveying locations for

each monitoring activity every year to avoid

trapping biases and to take into consideration

yearly changes in hydrology and plant communities.

For wetlands with very simple structural complexity, sampling points can be randomly selected without regard to cover types. Start at a set point along

the wetland’s edge. Use a random numbers table

or generator to obtain a fixed unit of distance in

meters to space the funnel traps. A second random number may be used to determine whether

the first trap is set to the left (e.g. even number) or

right (odd number) of the set point.

3. Sample the population. Surveys should take

place during comparable times and conditions of

animal presence and development. For example,

choose similar conditions of water depth, temperature, and initial set time (morning or afternoon) to

standardize efforts. A two-person crew (one for

placement, the other for carrying funnel traps and

assisting) is essential for trapping; three persons are

better. More than 100 traps generally require a 4-

person crew. In hot weather, reduce trapping time

to 18 hours to reduce mortality; set traps in the afternoon and remove them the next morning. Funnel traps should be set so that there is an air space

at the top for breathing. They may be marked with

brightly colored flagging and set about 2 m in a specific direction (e.g., north) of each trap to reduce

the risk of predation. Lay traps on the substrate or

diagonally attach to dowels with openings near the

substrate. The sample data sheet for funnel traps

(Appendix A) provides some ideas for data collection and recording.

4. Egg surveys. Counts of egg masses have

served as another source of information on species
16

breeding in wetlands and their relative abundance

(e.g., Richter and Ostergaard 2001, Crouch and

Paton 2000). Unlike call counts that identify species using wetlands but do not provide data on

breeding effort (see below), egg searches confirm

breeding of amphibians and, with some training,

provide a repeatable index to relative breeding effort (Richter and Ostergaard 2001). The abundance and health of eggs also may serve as indices

of wetland and population health, in that a discrepancy between numbers of adults and eggs or an

abundance of parasitized or diseased masses could

denote less than optimal conditions (Plate 16).

Although there may be some problems in identifying the species of egg masses where several species of the same genus are breeding concurrently,

most egg masses can be identified by their shape,

size, oviposition depth, substrate, and breeding

chronology. Egg masses may be single small groups,

grapelike clusters, or rafts; they may be free or attached to vegetation; or they may take on unusual

forms (Stebbins and Cohen 1995). For example,

bullfrog eggs and masses are larger than those of

green frogs. Northwestern salamanders breeding

in wetlands with natural conditions almost exclusively attach their orange-sized, clear, dense jelly

egg masses to 3-6 mm diameter, thin-stemmed

emergent plants and woody stems, whereas the

long-toed salamanders attach their eggs in small

clumps or long clusters. Consequently, they can be

readily distinguished from each other (Richter pers.

obs.). Breeding phenology may also separate species. In the Mid-Atlantic, southern leopard frogs

(Rana sphenocephala or R. utricularia) breed

earlier than either green frogs or bullfrogs. In the

Puget Sound Lowlands of the Pacific Northwest,

long-toed salamanders are the first to breed in late

winter or early spring. Nevertheless, where species richness is particularly high, identification of frog

egg masses or the separation of some frog egg

masses from salamander egg masses may be problematic. Although some local field guides include

keys to egg masses (e.g., Corkran and Thoms

1996), we are not aware of technically strong, peerreviewed regional keys to identifying egg masses.

To get a total count, egg mass searches should be

conducted soon after breeding has ceased for those

species with relatively short breeding periods. For

species with prolonged breeding (e.g., cricket frogs,

Pseudacris spp., or bullfrogs), multiple searches

may have to be conducted. Historical records of

breeding may help to decide when to start searching, although annual weather conditions are important in initiating egg laying. In particular, rainfall often initiates breeding activity, and searches may be

most productive a few days after a good thunderstorm.

There are three main zones for an egg mass survey: the waterline, shallow water areas, and the

shore zone (Figure 4). The waterline is the physical line at which water and land meet at the time of

the survey. This line will change seasonally and annually, and in some cases daily, through natural or

manipulated fluctuations in water level. The shallow water zone includes all wadable water, from

the waterline to a depth that can be safely walked

Plate 16. Abnormally high percentages of dead eggs

within originally healthy egg clusters indicate potential

problems with wetland conditions.

© Klaus O. Richter. Used with permission of the photographer.
17

(usually less than 1 m deep). The shore zone is the

land bordering the pond to within about 3 m of the

waterline. The use of polarized glasses or an umbrella that casts shade over the immediate search

area may help to see and identify eggs under water.

Figure 5 shows one possible search method.

Recommended survey methods are described

below for each zone; however, procedures may

warrant adjusting based on site conditions. For

example, a small vegetatively homogeneous wetland or flooded meadow may not readily be divisible into distinct survey zones and can be surveyed

as a whole. Moreover, shoreline topography or

vegetation affects accesses to or survey effectiveness of the near-shore zone, making it difficult or

dangerous to survey at such locations. In some lentic habitats, deeper water (more than 1 m) can be

sampled by boat or float-tube.

Search the waterline first. If the shallow water

zone is narrow (less than 1 m), one person walks

along the waterline scanning both the waterline and

the shore zone while a second person walks in the

shallow water zone about 2 m from the first. This

allows both surveyors to search a 2 m wide strip.

Where the shallow water zone is extensive, both

members of the team can wade parallel to the waterline, one about 2 m from shore, the other about

2 m further out. We recommend that the pair stop

every 5 m to more thoroughly search nearby areas

and to scan ahead (see Figure 5).

Total egg counts can be accomplished in small

wetlands for certain species; partial counts can be

made for others. Sightings per unit area in large

uniform habitats will provide a partial quantification

of density. In situations where only a partial count

can occur, a time-constrained sample can provide

estimates on relative abundance. Search for a fixed

period of time and count all egg masses identified

during that time. Alternatively, the number of masses

per linear distance of shoreline may be used. In

wetlands with complex cover or a diverse habitat

structure, counts should be in proportion to the

major cover types. The method employed must be

consistent across all wetlands sampled. At a minimum, identify, clearly mark, and map areas of

Figure 4: Side view of a wetland showing the different zones for

surveying: shore zone (dry land to 3 m from water’s edge), shallow

water zone (to 1 m deep), and waterline (edge of water).

Shore zone Shallow water zone

Waterline

Wadable

Area

1 m
18

greater concentrations. Egg masses can be marked

by placing flagged dowels or poles at 2 m distance

with the number of egg masses and date written on

the flagging. Sometimes egg masses of different

females can be laid in close proximity or even overlap each other. An estimate of the number of separate egg masses in such clusters can be made by

careful inspection of the masses or by dividing the

volume of the cluster by the typical volume of an

individual mass.

5. Dipnet sweeps. Dipnetting is a fast, easy

method of capturing and identifying slow-swimming

amphibians and larvae in open water or along shallow shorelines within soft-stemmed emergent vegetation (Plate 17). Amphibians readily netted include most Rana, Hyla, Bufo, and Pseudacris species. It does not seem to be as effective on salamander adults, neotenes, or larvae as it is on

anurans, although some species of Ambystoma,

Taricha, and Notopthalmus can be collected by

this method. It is inappropriate in wetlands or habitats where dense vegetation or woody debris limit

visibility and inhibit sweeps.

Adult and larvae captures can provide information on the presence and health of some species.

For some purposes, such as collecting metamorphs

for examination of malformations, dipnetting may

be a preferred method because of its efficiency.

Dipnetting efforts can be standardized by consistently using the same net size (i.e., mesh and opening dimension) as well as pull length (usually 1 m),

by using time-constrained sampling, and by standardizing for time of day, water depth, and microhabitat. Consequently, one can roughly determine

volume of water sampled and make some judgment of density.

Reference stake(s) 

Reference stake(s) 

1 meter 

2 meters 

Transects 

(2 m apart) 

Figure 5: Top view of a wetland showing different zones

for basic egg surveys.
19

6. Calling surveys. Males of many anuran species vocalize in wetlands. Sexually mature male frogs

and toads call to attract mates and establish territories. Immatures may use warning and distress calls

when disturbed. Consequently, vocal species can

be identified by unique calls, and an approximation

of their relative abundance can be determined by

the number of calls heard. This information can be

used to develop population trends based on the

number of calls heard over repeated censuses. Such

surveys can provide information on which species

are breeding and a rough estimate of their abundance, but do not provide any information on the

success of breeding. It is possible that adults may

be attracted to wetlands that produce little recruitment because of contamination, high predator abundance, or other detrimental impacts. Call surveys

are not good for salamanders, which generally are

mute. Several good audio sources of frog and toad

calls are available (e.g., Davidson 1995, Hunter et

al. 1999) and should be consulted for species recognition. Moreover, standard procedures for call

surveys have been developed by the NAAMP. We

advise anyone starting a call count survey to obtain

a copy of the NAAMP protocol available through

their website: http://www.mp1-pwrc.usgs.gov/

amphibs.html.

Typical surveys include 3 to 4 nights over the

course of 4 to 6 months with a total of 1 to 2 hours

per survey listening for calling frogs or toads. A

typical call route has 10 stops. At each stop, several minutes are spent listening and taking notes on

species heard and frequency of calling using the

scoring criteria: 0 = no frogs and toads can be heard

calling; 1 = individual calls not overlapping; 2 = calls

are overlapping, but individuals are still distinguishable; and 3 = numerous frogs or toads can be heard,

chorus is constant and overlapping. Surveys must

be standardized for season, air temperature, wind

speed, and occurrence of rain. If temperatures are

too cool, winds too high, or rain too heavy, frog or

toad calls may not be audible.

 Terrestrial surveys

1. Pitfall trapping. Several studies have investigated ways of increasing the efficiency of pitfall

trapping (e.g., Greenberg et al. 1994, Corn 1994,

Dodd and Scott 1994, Enge 1997, Christiansen

and Vandewalle 2000). The method is generally

most appropriate for adult or juvenile amphibians.

Pitfall trapping, in addition to providing a species

inventory, can be combined with individual marking

of amphibians to determine relative population densities, comparative estimates among wetlands, and

population changes within wetlands. If an objective of a study is to obtain reliable estimates of these

measures, surveys must be site-dependent and need

specific multiple recapture methods and analysis

(Donnelly and Guyer 1994). Consequently, they

may require considerable planning and statistical

expertise beyond the scope of this document.

Two trapline methodologies are suggested for

descriptive rather than quantitative pitfall sampling.

Plate 17. Dipnetting is a “quick and dirty” way of

identifying amphibian species in wetlands. Both adults

and larvae of slow-moving species can readily be netted.

© Klaus O. Richter. Used with permission of the photographer.
20

The first and simpler “transect” method takes many

forms but essentially is a line of pitfalls with or without drift fences. Pitfalls, which may be made with

two #10 tin cans duct-taped together, plastic buckets, or other suitable containers, are buried level to

the ground. Drift fences are typically constructed

from plastic silt fencing and corrugated or smooth

sheeting. In one configuration (Figure 6), pitfalls

can be placed without drift fences at 10-, 25-, or

50 m intervals along a 100 m transect running parallel to shore. In another design they are placed at

either end and in the center of a 25-50 m linear drift

fence. In a third variation, drift fences with pitfalls

spaced at 25-50 m intervals can completely surround small wetlands. Once installed, pitfalls are

economical to operate and may last for several years

in wet environments. Corrosion-resistant cans

manufactured for the seafood industry are highly

recommended for hydric soils and long-term studies. Pitfalls without drift fencing are suggested for

wetlands with narrow (less than 10 m) buffers. Pitfalls without drift fences are also less conspicuous

and may be useful in areas with heavy human visitation (and interference); however, they only collect

animals that venture across the narrow gap of the

trap opening. With drift fences, the effective trapping area can be increased considerably. In either

case, linear transects only partially sample animals

as they move into or out of a wetland, and a majority of animals may avoid being caught. Drift fences

that completely surround a wetland have a greater

probability of sampling more species than linear

transects. In all cases, some species may have lower

or higher probability of detection by remaining sedentary (e.g., some ranids), using tunnels under fences

(newts), or climbing over the drift fence (treefrogs)

(see Dodd 1991). Modifications of the tops of traps

with plastic barriers can reduce even treefrog escapes (Murphy 1993). Clearly, the particular design will depend on study objectives and species of

interest.

The second “array” method (Figure 7) captures

amphibians within pitfall clusters or arrays located

at randomly selected points along a transect. These

may require more effort to install than linear transects

because their placement and structure are more

complex. The principal advantage of this design is

that it samples mobile amphibians traveling in several directions rather than into or out of a wetland.

Like linear formations, this method presents some

bias in that the probability of detection is proportional to the degree of mobility displayed by a species; migratory salamanders and toads may have a

greater probability of being sampled than more sedentary ranids (Dodd and Scott 1994).

When using pitfalls or drift fences, one should follow certain precautions: (1) The bottom of the drift

fence must be directly contacting, even slightly buried below, the ground to prevent animals from crawling underneath the fence (Figure 8). In wet areas,

pitfalls need to be secured in place with long stakes

such as reinforcing rods hammered into the ground

to prevent them from floating up. (2) The bucket

or can pitfall should be topped with an overhanging

lid, 6-10 cm above the top of the pitfall, to shield

the interior from direct sunlight or rain. (3) A moistened, floatable sponge in the pitfall will provide

moisture under most conditions and act as a raft

should the pitfall flood. (4) A string suspended from

the center of the lid provides an escape route for

trapped mammals but also facilitates the escape of

treefrogs. (5) The pitfalls must be checked regularly—daily whenever possible but certainly no

longer than every 3 days (Corn 1994) to avoid

dehydradation and excessive mortality. (6) The pitfalls must be sealed or inverted during periods of

nonuse. (7) Pitfall transects and drift fences can be

used for several years so they are most economical

when studies call for repeated sampling of wetlands

across time (Heyer et al. 1994).

Install pitfalls and drift fences in summer above

the mean winter flood zone outside the hyporheic

zone. For the transect method, lay out two 100-m

transects across habitat types at each wetland and,

if relevant, on opposite sides of the long axis of each

wetland paralleling the shoreline. If drift fences are
21

not used, excavate and install 10 pitfalls along the

100-m transect with their upper edges flush with

the ground surface. Bury them within 2 m of each

station, preferably next to logs, dense vegetation,

and other locations likely to be catching amphibians. For short transects (50 m), pitfalls should be

placed at either end and in the center of the drift

fence. For encircling designs, pitfalls can be placed

in pairs every 25 m on both sides of the drift fence.

Alternatively, a terrestrial funnel trap made from

window screen or other similar material can be

placed on either side of the drift fence. Be sure to

record if the animal was caught from the inside or

outside of the drift fence; most researchers place

the animal on the opposite side of the drift fence

from which it was caught (e.g., Dodd and Scott

1994). For the array design, randomly select the

starting point for each of the first of 10 trapping

stations consisting of 3 arrays. Each of the 10 points

is used for installing the central pitfall trap location

of a 3-trap drift fence array (Corn 1994) (Figure

7). Locate the next two central traps at 10-m intervals along the perimeter baseline. Randomly rotate the “spokes” of each array by spinning a dial or

with some other method. Install 0.5-m-high drift

fences between traps.

Figure 6: Pitfall traps can be placed in various configurations,

with and without drift fences.

A 

B 

C 

Key: = Pitfall 

 = Funnel Trap (optional) 

 = Structure 

 = Drift Fence 
22

Ideally, repeated censuses during spring are recommended to maximize capturing the diversity of

species migrating from upland hibernation habitats

to wetland breeding sites. Drift fences are most

efficient when survey plans call for repeated inventories of specific sites. Because of the labor involved, they may not be economical for one-time

events. Census should commence at similar periods of yearly weather conditions. Amphibian movements are inhibited by freezing night-time temperatures and enhanced by warm temperatures in the

40’s with rainy nights. Therefore, sampling periods

may vary slightly from year to year based on annual

phenology. Late winter censuses should be established for ambystomatid salamanders and early

breeding anurans; early spring for tree frogs,

bufonids, and some ranids; and mid-spring to early

summer for other ranids. For example, in the MidAtlantic States, salamanders begin breeding in late

January and February; grass frogs (R. pipiens complex), pickerel frogs (R. palustris), toads, and chorus frogs in March-April; and gray treefrogs (Hyla

versicolor), bullfrogs, green frogs, and northern

cricket frogs (Acris crepitans) from May into July.

Of course, knowing regional variation in times is

critical for maximizing the efficiency of trapping.

 Figure 7: Array configurations of pitfalls are also flexible and

can be adjusted to particular needs.
23

Traps can be opened and closed as favorable

weather permits for a total of 14 trap nights during

each period. See Appendix B: Amphibian Pitfall

Trapping Data Sheet for an example of how to

record data.

2. Artificial cover. Studies that have evaluated

sampling amphibians with artificial cover such as

cover boards and other devices include Fellers and

Drost (1994), Moulton et al. (1996), Bonin and

Bachand (1997), and Monti et al. (2000). Cover

boards are flat sheets of board such as half sheets

of plywood or fencing that make use of the natural

tendency of most caudates to seek shelter under

flat objects during the day. They are useful in determining the presence and abundance of salamanders in wetland buffer and adjacent habitats,

but are not very suitable for sampling anurans, although occasionally dispersing frogs and toads may

Figure 8: The proper setting of pitfall traps involves burying

the pitfall to its lip, burying the drift fence at least slightly

into the ground to prevent amphibians from crawling

underneath, and careful placement of a lid over the pitfall to

prevent excessive exposure to the elements.
24

also be found by this nondestructive method. A

general procedure is to place cover boards at random locations, but in proportion to habitat types

along transects on a forested or grassland floor, and

periodically lift the covers up and count the numbers and species of amphibians living there. Captures are released at the edge of the board so they

can crawl back under. Monthly surveys in damp,

cool spring and autumn weather adequately determine the presence of terrestrial breeders, provide

indicators of densities, and establish ongoing habitat use. Be aware that snakes also use these artificial covers. Another form of artificial cover that is

attractive to some amphibians is 5 cm diameter,

1-1.5 m high pipes made of polyvinyl chloride

(PVC) pounded into the ground. Sometimes referred to as “canopy traps,” these provide relatively

safe and attractive shelters for tree and chorus frogs.

Field and Laboratory Methods

of Assessing Health

Field methods

Eggs surveyed and animals captured in the field

can be evaluated as to their health by visual observations and by several quick measurements. Abnormal egg mortality or the inability of larvae to hatch

(escape) from eggs should be noted, as such traits

may be indicators of high pH or chemical pollutants, injuries and sores, and abnormal coloration.

Clearly, abnormal growths, multiple limbs and digits (fluctuating asymmetry), and other malformations

are instant indicators that something with the animal

and/or population is not normal and should be carefully recorded and if possible saved for pathological analysis. More importantly, as mentioned previously, if such incidences occur, they should be

carefully documented in a standardized manner

(Palmer 1994) and submitted to the NARCAM

reporting center. Similarly, lethargy, poor or unnatural swimming or jumping ability, and other more

subtle behavioral “abnormalities” can immediately

suggest that something is amiss, should be carefully

noted, and may warrant further assessment.

Standard counts and measurements often taken

as part of regular field surveys may also suggest

that a given animal and/or population is unhealthy.

Animal lengths and weights that are outside normal

ranges for the species or population may suggest

wetland or upland perturbations, although predation, competition, and other density-dependent factors play an important role. Nevertheless, even these

density-dependent influences can suggest abnormal

environmental conditions. An index of health combining several measurements into one, such as described in Wilbur and Collins (1973), is another

useful way of quickly evaluating the health of amphibians in the field. Total length and snout-to-ventlength of the animal can be measured readily with a

small millimeter ruler or calipers (Plate 18). Weight

can be determined by placing the animal in a small,

thin plastic bag and using a Pesola spring or other

appropriate scale. Standard techniques for such

measurements are provided in Nussbaum et al.

(1983) and other advanced field guides or in

Petranka (1998) for caudates. More complex data

manipulation and summary statistics of length,

weight, and other readily measured data may be

more suitably analyzed back in the laboratory or

office.

Lab-assisted methods

Although several laboratory tools may be useful

in assessing the health of individuals, these usually

involve technical methodology and instrumentation

Plate 18. Measuring lengths of amphibians, such as bullfrog larvae, may provide an indication of yearly growth.

© Klaus O. Richter. Used with permission of the photographer.
25

that can be costly and time-consuming. Some require sacrificing animals, others are invasive but

nonlethal, and a few are noninvasive. Thus most

laboratory investigations should be considered on

a higher order or tier than routine bioassessments.

If, after an initial investigation, it is determined that

an amphibian population or community may be in

poor health, these techniques can be valuable in

determining the cause(s) of the problem so that appropriate remedial actions can be taken. Some of

the methods also may be used to screen amphibian

communities for adverse effects in landscapes where

specific physical or chemical problems have been

identified. The laboratory techniques summarized

below have worked well with other vertebrate

classes such as fish, birds, and mammals, but have

not been used as extensively in amphibians and may

require some adjustments for optimal results. This

review is not intended to be comprehensive; rather

its purpose is to increase awareness of some of the

tools that are available.

The cost of such analyses can be expensive compared with other methods, and we have provided a

rough guide for having the analyses conducted by

contracting labs; obviously, direct costs might be

reduced if one has in-house capabilities. Costs per

sample are: $ = less than $50; $$ = $50-200; $$$

> $200.

Cholinesterase determination—$ to $$. Acetylcholinesterase (AChE) is an enzyme that blocks

the action of acetylcholine at the synapse between

two neurons and helps regulate the central and peripheral nervous systems. Organophosphorus pesticides including organophosphates (e.g. diazinon,

chlorpyrifos, methyl parathion) and carbamates (e.g.

carbofuran, methiocarb, aldicarb) work by binding

with AChE and blocking its inhibition of acetylcholine. In target organisms (i.e., insects) and, unfortunately, in lethally exposed nontarget organisms (e.g.,

amphibians exposed to agricultural runoff or to

broadcast spraying), death usually occurs because

of nervous system failure leading to respiratory failure. Sublethal and recent lethal exposures can be

detected by comparing AChE activities between

suspect animals and those coming from a reference

population. At least in birds, AChE activity depressed by >50% is forensic evidence that animals

have been exposed to organophosphorus pesticides

(Hill and Fleming 1982). There are also methods

that promote reactivation of inhibited AChE and,

by comparing activity levels before and after application of reactivation techniques, help determine if

specific animals have been exposed to pesticides.

Studies that have used cholinesterase inhibition to

determine pesticide exposure in amphibians include

Guzman and Guardia (1978), de Llamas et al.

(1985), and Sparling et al. (2001). The most reliable tissue for AChE determinations is brain, because it shows the least amount of variance among

individuals collected from reference sites. In larger

amphibians, AChE can be measured in plasma without sacrificing animals, but in small amphibians such

as most tadpoles, entire bodies less the gut coil may

have to be used. Spectrophotometric measurement

of AChE is relatively straightforward (Ellman et al.

1961, Hill and Fleming 1982), especially with multiwell spectrophotometers (Hooper 1988).

Fluctuating asymmetry—$. Under optimal conditions an animal may be expected to develop with

minimal defects or teratogenic effects. As conditions deteriorate, for any of a number of reasons,

developmental processes may be affected, resulting in quantifiable abnormalities. In many cases these

abnormalities may affect the two halves of bilaterally symmetrical organisms unequally. In extreme

cases, these abnormalities may be readily apparent, such as the widely publicized amphibian malformations. The concept of fluctuating asymmetry

(FA) proposes that the quality of habitats can be

assessed by comparing bilaterally paired morphological parameters. Greater differences in morphology indicate greater stress on developmental processes (Palmer and Strobeck 1986, Bailey and
26

Byrnes 1990, Leung and Forbes 1996). However,

FA may respond to many different kinds of stressors, and the method may not identify specific causes

for the difference in morphology. The method relies on very careful measurements of paired body

parts such as length of hind feet or distance from

one joint to another. Any difference due to injury

or predation must be discarded. In theory, FA can

be used on live animals, but if the subjects are very

active, they may have to be quieted through sedation to achieve precise measurements. Palmer

(1994) provides instructions for performing this

analysis.

Flow cytometry—$ to $$. Exposure to certain

clastogenic (i.e., mutagen-inducing) contaminants

such as polycyclic aromatic hydrocarbons (PAH,

e.g., benzene, anthracene) can be detected by using flow cytometry. Its principle is based on the

observation that exposure to such chemicals can

cause chromosomal damage, which causes variation in DNA content among cells. A flow cytometer (Otto and Oldiges 1980) is an automated, precise method of quantifying this variation in DNA

content. The method has been used on birds and

fish (Easton et al. 1997), turtles (Lamb et al. 1991,

Bickham et al. 1988), and mammals (McBee and

Bickham 1988), and has been proposed for amphibians (Bishop and Martinovic 2000). The technique uses a small piece of tissue, perhaps the tip of

a salamander or tadpole tail or a digit from a frog

taken during toe-clipping, and thus does not sacrifice animals. The method will not identify the specific cause for DNA variation, but can facilitate forensic investigations. Methodology is explained in

Deaven (1982) and Otto and Oldiges (1980).

Blood elements and chemistry—$ to $$$ depending on analyses. The list of diagnostic factors that

can be obtained from a sample of blood is extensive. They include the standard battery of enzymes

(e.g., lactate dehydrogenase, alanine aminotransferase, hematocrit, and hemoglobin) that are often

measured in routine health examinations for humans.

In general, variations in these factors suggest similar health conditions such as impaired liver or kidney function or anemia. Although these factors have

been used to assess health status of other vertebrates (see, e.g., Ritchie et al. 1994, Sparling et al.

1998), they have not been widely applied to amphibians. Experimental work may have to be conducted to obtain baselines and verify interpretations.

Some other plasma measurements are quite specific and point to certain conditions. For example,

(-aminolevulinic acid dehydratase (ALAD) and

porphyrins can be used to assess exposure to lead

in amphibians (Vogiatzis and Loumbourdis 1999,

Steele et al. 1999). Elevated metallothionein activities can indicate exposure to other metals such

as cadmium or zinc (Vogiatzis and Loumbourdis

1998). A high frequency of red blood cells with

small nuclei can indicate exposure to clastogens

(Fernandez et al. 1989). For descriptions of other

bioindicators, see Huggett et al. (1992).

General Comments on Care

and Preservation

All appropriate care and attention should be considered whenever handling live amphibians. Specific care recommendations are provided in the joint

animal care and use committees of the major herpetological societies (Anonymous 1987). If tadpoles are to be examined in the field or laboratory

and then released, they may be transported with

pond water in 4 L or larger containers that have

been rinsed with commercial bleach and distilled

water to deter transmission of disease. If possible,

carry ice in a cooler and use it to keep the water

from becoming too warm. Containers should always be kept in a cool, shaded spot. For adults,

soaked paper towels or sponges in a bucket with a

tight-fitting lid are preferable to standing water because adults can drown. Handle the animals as little

as possible, place inspected animals in a clean

bucket, and return to the source as quickly as possible to minimize stress.
27

If animals are to be euthanized for purposes other

than enzyme determinations, the preferred method is to overanesthetize them in tricaine

methanesulfonate (MS 222) (0.5 g/L of water).

The effects of MS 222 on physiological, especially

neurological, processes in amphibians are unclear. When physiological measurements are prescribed, the preferred method is flash freezing in

liquid nitrogen.

For most histological purposes including examination for parasite cysts, amphibians should be

euthanized in MS 222 and fixed in 70% ethyl alcohol (10:1 ratio of alcohol to specimen volume). A

small slit in the abdominal wall facilitates complete

and rapid fixation.

Data Analysis

General Comments

We have mentioned that rigorous statistical precision is not as critical in surveys for IBI development as it might be for experimentation and

hypothesis testing (see Module #4: Study Design

for Monitoring Wetlands and Module #6: Developing Metrics and Indexes of Biological Integrity).

Where time, expertise, and resources allow, the

higher the quality of the data, the more certain are

the interpretations, and it is ill-advised to cut corners if it is not absolutely necessary. We do not

have the space to thoroughly cover statistical design and analysis here. Rather, we refer interested

readers to sources such as Donnelly and Guyer

(1994) or Hayek (1994). Consultations with good

statisticians prior to collecting data are always recommended.

Possible Metrics

We are unaware of any study that has published IBIs using characteristics of amphibian communities in metrics, but there are several

studies are in progress. A thorough examination of

the ecological literature on amphibians, such as an

upcoming literature survey by Paul Adamus of Oregon State University, would undoubtedly provide

excellent leads for metric development in certain

species. For example, Boward et al. (1999) reported that some amphibian species were not found

along streams in areas with more than 3% impervious surface in the watershed basin, others disappeared at levels greater than 10%, and still others

survived up to 25% impervious cover. Richter and

Azous (1995, 2001) and Thom et al. (2001) found

that species richness correlated to increasing water

level fluctuations, which were attributable to greater

runoff into wetlands because of increasing proportions of impervious surfaces in urban habitats.

Pechmann et al. (1989) found a relationship between wetland hydroperiod and the species diversity and abundance of metamorphs. In a review of

the literature, Sparling (1994) showed a clear relationship between pH and the tolerance of 26 species of amphibians. Many more examples can be

gleaned from research publications. The following

should be considered as potential metrics or attributes based on amphibian ecology, distribution,

and sensitivity to anthropogenic stressors.

 Suggested Attributes

1. Species richness. The diversity of amphibians

in a given wetland is often much smaller than that of

other taxa traditionally used in IBI development,

such as plants, fish, or invertebrates. A very diverse region may have 20 endemic amphibian species (McDiarmid and Mitchell 2000), with only a

fraction expected to inhabit any given wetland. In

comparison, there may be far more than 20 inver-
28

tebrate or macrophyte species inhabiting wetlands

in even fair condition. Thus, species richness may

not be as useful a metric for amphibians as it is for

other assemblages. Because species distributions

show marked regional differences, the choice of

specific metrics also may be regional. What works

for New England, for example, may not apply to

the Mid-Atlantic States.

2. Comparison of species presence/absence

with regional taxonomic lists. There is an abundance of published State, regional, and national taxonomic lists of amphibians for many parts of the country. These lists can provide information on the species that might be expected to inhabit a wetland.

One attribute could be a ratio of the number of species actually found to the number possible. A depauperate fauna would yield a low score whereas a

diverse community relative to regional distributions

would have a higher score. Also, the presence of

rare species, especially ones that are listed for a

State or the Nation, should be positive indicators

of wetland health, whereas the absence of a ubiquitous species may signal problems. Because of the

large annual variations in amphibian populations, an

absence one year might not mean an absence in

subsequent visits.

3. Proportion of nonindigenous species.

Nonindigenous or invasive species generally are

characteristic of impaired wetland conditions. In

the South, the cane toad (Bufo marinus) may be a

pest species, and there is some concern that introduced bullfrogs cause problems for indigenous species of anurans in the West (Lawler et al. 1999).

4. Frequency of malformations. Malformations

in anurans take many forms. In addition to missing

or extra hindlimbs, there are malformations of the

mouth, extra webbing, body coloration, digits, and

unabsorbed tails. Information on what to look for

can be obtained at the website for the North American Reporting Center for Amphibian Malformations

(NARCAM) http://www.npwrc.usgs.gov/

narcam/. A 2% rate of deformities among metamorphosing frogs is considered acceptable; rates

much higher than that may indicate serious problems in wetland health.

5. Evidence of mass mortality. Certainly, observations of large numbers of dead and dying frogs

or tadpoles should be taken seriously. Such mortality demands further investigation.

6. Number and condition of egg masses. The

number of egg masses and some indication of their

status (i.e., percent of eggs with fungus, percent

showing no embryonic development) should provide good estimates of amphibian breeding conditions. However, each wetland may have to be visited repeatedly to capture data across the breeding

periods of several species, or to record data from

species such as green frogs or bullfrogs that have

extended breeding seasons.

7. Ratios of the relative abundances of different life stages. By repeatedly visiting a site and

using different data collection techniques, an assessment team may be able to obtain information on the

relative abundances of breeding adults, egg masses,

tadpoles, and metamorphs. Ratios of these life

stages may reveal information on population status.

For example, wetlands with abundant breeding

adults but very low abundances of subsequent stages

could indicate a population sink.

8. Percent tolerant and intolerant species.

What defines a tolerant or intolerant species depends on the stressor involved. Species with small

tadpoles (e.g., northern cricket frog) may be less

tolerant of predacious fish than those with tadpoles

that can grow larger than the mouth gape of these

fish (e.g.,bullfrog). Sensitivity to contaminants varies

across species and type of contaminant (Sparling

et al. 2000). Some amphibian species are much

more tolerant of habitat destruction than others. Sensitive species often have a very narrow set of habitat requirements whereas tolerant species tend to

be generalists. Thus, a list of tolerant and intolerant
29

species should be developed by region based on

the likelihood of encountering specific stressors.

Wetlands in good condition would reasonably have

a greater preponderance of intolerant species than

highly impaired wetlands.

9. Mensural characteristics. In general, larger

body weights relative to body lengths indicate good

health. Thus, snout vent length (SVL) divided by

body mass (or the square root of body mass because SVL is linear whereas mass is related to volume) by developmental stage and sex of adults may

be useful information. Of particular interest is the

ratio for recently metamorphosed individuals, because body size of metamorphs is related to survival (Wilbur and Collins 1973).

10. Proportion of neotenic and metamorphosed adult salamanders—Some metapopulations of salamanders show variation in the

ratios of neotenic and metamorphosed adults. These

forms are readily distinguished based on the presence of external gills in neotenes and sometimes

color differences.

Some attributes can be very informative but may

lie outside of routine bioassessments owing to the

level of technical expertise or cost required. These

include:

11. Percent of individuals parasitized or diseased by trematodes, chytrid fungus, or

iridoviruses

12. Presence and concentration of contaminant residues in bodies

13. Specific bioindicators (ALAD, hematocrit,

etc.)

See discussion under field and laboratory techniques.

Limitations of Current

Knowledge and

Research Needs

 Current Knowledge

A few species of amphibians, especially northern leopard frogs and the African-clawed frog

(Xenopus laevis), have been extremely well studied in terms of understanding vertebrate physiology. However, less research has been conducted

on the ecology, habitat requirements, and limiting

factors of amphibians than on mammals, birds, or

fishes. Thus, there are many data gaps in our understanding of amphibian ecology. Obviously, we

cannot summarize everything known about amphibian ecology and limiting factors, so here we direct

readers to a few sources of information.

Major sources of information

Excellent compendiums have been written on

amphibian environmental physiology (Feder and

Burggren 1992), general biology and physiology

(Duellman and Trueb 1986), natural history

(Stebbins and Cohen 1995), and ecotoxicology

(with reviews of ecology, physiology, population

declines, diseases, and other issues, Sparling et al.

2000). Some of the information in these sources is

somewhat dated, but they provide good starting

points for understanding amphibians.

An eclectic mix of topics and associated scientific

publications includes research on amphibian ecology has focused on understanding proximate factors driving community structure, including interspecific competition, predation, hydroperiod (Connell

1983, Sih et al. 1985, Hecnar and M’Closkey

1996c, Hecnar and M’Closkey 1997a, Chivers et

al. 1999), presence of predatory fish (Sexton and

Phillips 1986, Chivers et al. 1994, Hecnar and

M’Closkey 1997b), exotic species (Lannoo et al.
30

1994), local environmental toxins such as nitrate or

heavy metals (Berger 1989, Baker and Waights

1994), and water chemistry (Dale et al. 1985,

Glooschenko et al. 1992, Rowe and Dunson 1993).

Studies are typically short-term; emphasize local

processes using seminatural field, laboratory, or

mesocosm experiments; and often target larval

stages (Morin 1983, McAlpine and Dilworth 1989,

Biesterfeldt et al. 1993, Werner 1992, Skelly 1995,

Anholt et al. 1996).

 Limitations of Current

Knowledge

Often information on species distributions is not

complemented with long-term population data and

information on life histories (Mossman et al. 1998).

As a result, virtually no information exists on largescale changes in the incidence of amphibians (Hecnar

and M’Closkey 1996b). The North American

Amphibian Monitoring Program was initiated in

1994 to address this gap.

Amphibian declines and malformations have not

been easily explained. Some declines are tied to

local phenomena whereas others are currently unexplained. Synergistic effects of environmental factors coupled with potential species-specific sensitivities further cloud the understanding of cause and

effect. More research on individual species sensitivities is needed (Hecnar and M’Closkey 1996c).

Lack of information on natural population fluctuations (Pechmann and Wilbur 1994,) coupled with a

difficulty in distinguishing between true population

declines and regional responses to landscape

changes, makes assessment of species status

difficult.

Whereas much current research has led to a

greater understanding of some of the proximate factors that shape amphibian community structure, regional patterns of species diversity may not be understood adequately by reference to local conditions. An increasing number of studies suggest existence of metapopulation structure in amphibian

species (Harrison 1991, Sjögren 1994, Bradford

et al. 1993, Hecnar and M’Closkey 1996c). Thus

the persistence of species within a wetland may be

determined in large part by the dynamics of extinction, colonization, and migration at larger scales

(Gilpin and Hanski 1991). Landscape attributes

such fire, deforestation, and other land-use history

(deMaynadier and Hunter 1995); current land cover

including forests, agricultural fields, and developments (Richter and Azous 1995, Vos and Stumpel

1996, Kolozsvary and Swihart 1999, Skelly 1999,

Werner and Glennemeier 1999); and wetland density and distribution patterns (Gibbs 1993, Semlitsch

and Bodie 1998, Gibbs 2000) are important factors in regional species diversity and distribution

patterns. Therefore, using amphibian species diversity or abundance in any one wetland as a surrogate for wetland health or as a tool for assessing

the status of that species can be misleading without

some knowledge about what is happening in adjacent wetlands. Colonization and extinction events

may be quite frequent in one wetland within a given

suite of wetlands that as a whole is stable for any

given species (Hecnar and M’Closkey 1996a,

Skelly 1999).

 Important Research Gaps

We have enough information to begin using amphibians in bioassessments. However, to improve

our capabilities to diagnose wetland condition

through amphibian communities, we need to increase

our knowledge of certain key areas and learn how

to apply that knowledge to these bioassessments.

Some areas that are most in need of further study

are discussed below.

Expanding from local- to regional-scale assessments of amphibian populations. To assess the

biological integrity of wetlands, researchers and land

managers will have to look beyond the individual
31

wetland using a multimetric approach and a

multiscale approach (Hecnar and M’Closkey

1996b). Specifically, using amphibians as indicators of individual wetland health demands information on regional distribution patterns, life histories,

and species stability based on this expanded perspective. This requires a solid base of reference

wetlands within a region. Gibbs (1993, 2000) also

emphasizes the necessity of evaluating wetland resources as a mosaic rather than as isolated entities.

He found that as human populations shift from rural

to urban landscapes, wetland spatial patterns go

from many clustered wetlands (2-5 wetlands/km2

,

0.2-0.4 km apart) to fewer, more isolated wetlands

(<1 wetland, >0.5 km apart). Gibbs predicted that

wetland mosaics could withstand only modest losses

and still provide wetland densities that are minimally

sufficient to maintain wetland biota. Average dispersal distance is generally <0.3 km for frogs, salamanders, and small mammals (Gibbs 1993,

Semlitsch 1998, Semlitsch and Bodie 1998). In

Gibbs’ model, wetland mosaics characterized by

<1 wetland per km2

 and >0.5 km from other wetlands were not able to sustain metapopulations of

wetland-dependent animals.

Gallego-Fernandez et al. (1999) looked at the

effects of a 60% wetland loss in the past 50 years

in Spain and reported a homogeneous, coarsegrained landscape characterized by low diversity

and high dominance. Hecnar and M’Closkey

(1996a) studied species richness patterns of amphibians in 180 ponds in southwestern Ontario.

They emphasized the importance of a geographic

perspective on the occurrence of amphibians. The

high population turnover within the study area at

the level of individual species showed the transience

and spatial dynamism of amphibian populations even

during a short time period. The researchers concluded that the pattern of species richness is more

of a regional than subregional (or watershed) characteristic and that the most important processes

structuring amphibian communities operate at the

regional scale.

Regional species-specific information on tolerances and adaptability to specific disturbances. More information is needed on the tolerances and adaptability of each species to specific

environmental factors so that their presence/absence

or abundance in wetlands can be interpreted more

reliably (Richter and Azous 1995, Skelly 1996,

Hecnar and M’Closkey 1998, Skelly 1999). Individual species responses are variable depending on

lifestage, geographic region, and type of disturbance

(urbanization, agriculture, forestry). Some information is available that suggests, for example, that

wood frogs and spotted salamanders (Ambystoma

maculatum) rapidly disappear from agricultural

lands (Minton 1992) and are sensitive to percent of

forested habitat surrounding breeding pools

(deMaynadier and Hunter 1995, Lemckert 1999).

Kolozsvary and Swihart (1999) studied the effects

of agriculturally induced fragmentation of forests and

wetlands on amphibian assemblages and their distributions and observed nonrandom patterns, suggesting that amphibians respond to landscape-level

attributes and that species differed substantially in

the nature of their responses to fragmentation (patch

size, isolation, or water regime of wetlands). Additionally, our current information is based largely on

the survey approach (field observations) rather than

a combination of field and experimental approaches.

Hence it is often unclear what the field data are telling us other than that amphibians are observed and

found under the conditions of our surveys. Such

information is limited, as it demonstrates only the

wide range of conditions under which amphibians

are found but not necessarily their preferred conditions maximizing inclusive fitness or homeostasis.

Moreover, most of our scientific data are correlational, and to improve the benefits of amphibians as

bioindicators we need to establish specific causeand-effect relationships.

Information is needed on amphibian movements (seasonal and dispersal) in different landscape settings. Several researchers (Windmiller

1996, Semlitsch 1998, deMaynadier and Hunter
32

1999) have studied amphibian movements in agricultural, urbanizing, and forest landscapes. More

of this work, however, needs to be done.

Conclusions

 Use of amphibians as indicators of the health

of individual wetlands may be affected by

their population structure. Wetland ecosystems need to be assessed at the landscape scale

if biological integrity is meant to convey functions that support metapopulations of wetlandassociated wildlife. Also, many amphibian species are structured in metapopulations, and rates

of local extinctions and colonization events, over

a short time span, may be high. To overcome

these limitations, the best studies must be broad

in scope, either encompassing a large number

of wetlands in an area or occurring over a span

of several years.

 Some amphibian metrics may prove useful

in assessing wetland health. Several possible

attributes that can be developed from community structure, sensitivity to perturbations, malformations and growth rates, and other aspects

of amphibian physiology and ecology. However, none of these have yet been tested in an

IBI. Thinking regionally rather than universally

will enhance the likelihood of successfully identifying amphibian metrics. An assessment of a

wetland in Oregon that finds that bullfrogs are

dominant would likely conclude that the wetland is degraded, whereas different interpretations could be valid in the Eastern United States

where bullfrogs are native.

 We are still in exploratory stages and should

proceed to investigate amphibians as a useful taxon for monitoring the biological integrity of wetlands with care. However, of

all the vertebrate classes closely associated with

wetlands, amphibians probably give us the best

opportunity to develop bioassessments of landscapes in which wetlands play an important role.

Knowledge of the natural history of the amphibians in an area can lead to judicious selection of attributes and species to monitor that

ultimately may tell the investigator a lot about

the wetlands in a particular landscape.
33

References

Adams MJ, Richter KO, Leonard WP. 1997. Surveying

and monitoring amphibians using aquatic funnel traps.

In: Olson DH, Leonard WP, Bury BR, eds. Sampling

Amphibians in Lentic Habitats. Olympia, WA:

Society for Northwestern Vertebrate Biology, pp. 47-

54.

Altig R, McDiarmid RW, Nichols KA, Utach PC. 1999.

Tadpoles of the United States and Canada: A tutorial

and key. US Geological Survey. http://

www.pwrc.usgs.gov/tadpole.

Anholt BR, Skelly DK, Werner EE. 1996. Factors

modifying antipredator behavior in larval toads.

Herpetologica 52:301-313.

Anonymous. 1987. Guidelines for the use of live

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40

APPENDIX A.

SAMPLE DATA SHEET FOR FUNNEL TRAPS

Trap ID Habitat

Type S e pecies S x tag Se SVL(mm)

TL

(mm)

Mass

(g) Remarks

Wetland Name/ID Number: ______________________________ Survey Date:_______________

Biologists: _____________________________________________________________________

Date and Time Traps Were Removed or Checked:________________________________________

Air Temperature (o

C): _____________________ Water Temperature (oC): ___________________

Weather: Clear Overcast Rain Snow

NOTE: Each individual is recorded on a single line of the table. Trap Ids, Habitat Types, and Species need to

be entered only when they change.
41

APPENDIX B.

AMPHIBIAN PITFALL TRAP FIELD DATA SHEET

Habitat

Type

Habitat

Unit

Transect

Number S x pecies Se

Stage

Neonate

Juvenile

Adult

SVL

(mm)

TL

(mm)

Mass

(g) Injuries

Wetland Name/ID Number: ________________ Survey Date: ____________________________

Habitat Type: ___________________________ Biologists: ______________________________

Habitat Unit: ___________________________________________________________________

Sampling Station: ________________________________________________________________

Replicate Number: _______________________________________________________________

Air Temperature (o

C): _____________________ Water Temperature (oC): ___________________

Weather: Clear Overcast Rain Snow

Wind: Calm Light Strong

Soil Surface Condition: Dry Damp (dew) Saturated