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Shenandoah National Park
Terrestrial Amphibian Monitoring Protocols and Initial Results

Below, we present details on objectives, hypotheses being tested, methods, and some initial results of the various terrestrial salamander studies.


Capture-Recapture using Visible Implant Fluorescent Elastomer (VIE) Marking
Effects of a Prescribed Burn on Terrestrial Salamanders
Artificial Cover Object Study
Large-Scale Terrestrial Salamander Project
Elevational Transect and Contaminant Levels in Plethodon cinereus
Plethodon shenandoah Monitoring and Population Genetics

Environmental Variables Measured at the Terrestrial Plots
Soil Measurements in the Laboratory

Terrestrial Salamanders

In 1998, thirty square plots ranging in size from 15 m to 35 m were established in the park to estimate terrestrial salamander population sizes over time and in relation to various environmental factors.  The figure below shows the locations of these plots in the park.  We used capture-recapture methods to estimate the population size of terrestrial salamanders during the day under natural cover objects.  In 1999, several new studies will be initiated, which are described below.

Figure 1.

Shenandoah Terrestrial Plots.jpg (57904 bytes)

Capture-Recapture using Visible Implant Fluorescent Elastomer (VIE) Marking

Redback salamanders (Plethodon cinereus) and white-spotted slimy salamanders (Plethodon cylindraceus) greater than 38 mm in total length are marked using visible implant fluorescent elastomer (VIE) (Northwest Marine Technology, Inc.;  VIE tagging was initially developed for marking fish (Bonneau et al. 1995), but has recently been used successfully to mark amphibians (

Elastomer is a latex which, after mixing with a hardener, is a liquid at cold temperatures but cures into a pliable, biocompatible solid at higher temperatures.   The elastomer-hardener liquid is placed into a syringe and injected just underneath the skin.  We use three colors (C; red, orange, yellow) singly or in combination at any of four locations (L) on the right or left sides of the salamander behind the forelimbs or in front of the hindlimbs (Fig. 1).  This marking scheme allows for 255 individual marks ({[C + 1]^L} - 1).

Mixing instructions:
For proper curing to occur, the elastomer and curing agent must be properly mixed at a 10:1 ratio.  The following directions are for 1 cc of elastomer and 0.1 cc of curing agent, but smaller quantities can be made if syringes delivering smaller quantities (e.g., 0.5 cc or 0.1 cc) are used.  When working with elastomer that has been (e.g., 0.5 cc elastomer and 0.05 cc curing agent) been mixed with hardener, it is best to use syringes and mixing cups that are cold to delay curing.  Place syringes and mixing cups in refrigerator an hour before making new elastomer.
1)  Dispense 1 cc of elastomer from elastomer tube into the back of a syringe.
2)  Dispense 1 cc of the colored elastomer into the bottom of a mixing cup.  Keep this cup on ice.
3)  Use a 1 cc (or smaller) syringe to draw up a small amount of curing agent.  Remove any air pockets by pushing all the material out and then drawing more up.  Dispense 0.1 cc into the cup with the elastomer.  Discard this 1cc syringe.
4)  MIX THOROUGHLY!  We recommend stirring and scraping the mixing cup walls and bottom for one full minute to ensure complete mixing.
Warnings:  (a)  Never allow the colored elastomer material (unmixed or mixed with the curing agent) to enter the "sampling bottle" that contains the curing agent.  Even a very small amount of colored elastomer will contaminate the curing agent and preclude its future use.  (b)  Contact of either the curing agent or elastomer with the rubber plunger tip of the 1 cc syringe for an extended time may inhibit curing of the elastomer.  Discard each of these syringes immediately after use!
5)  Carefully remove the white and orange caps from a 0.3 cc injecting syringe.   Remove the plunger.
6)  Use a new 1 cc syringe to slowly draw up a small amount of the mixed elastomer material, then wipe off the tip.
7)  The tip of the 1 cc syringe will fit tightly into the opening in the injecting syringe.  Fill each of several injecting syringes about 1/3 full.   For best results, make sure that no air pockets form between the plunger and elastomer.
8)  Replace the plunger, pushing it forward till the air is displaced and elastomer appears at the needle tip.
9)  Always keep syringes in the freezer or on ice when in field.  These syringes can be used for up to two weeks prior to the Elastomer becoming hard. 
10)  Prior to injecting salamander, always wipe needle onto a sterile alcohol wipe pad.
11)  Place salamander in zip-lock bag, and position the salamander's snout against the corner edge of the bag and line up the salamander's body along the bottom of the bag.
12)  Inject salamander through the zip-lock bag with the unique individual color and position code (Fig. 2).
13)  To validate the elastomer mark, one person marks the salamander and the second person double checks the mark to make sure it is visible under black light.  The checker reads back the marking scheme (e.g. XRXX) to the marker to verify that the correct mark has been used and recorded correctly on the data sheet.
14)  Mark-recapture on a weekly basis.  Use program CAPTURE (scroll down to Capture) to estimate the population size in the study area.

Figure 2.  Marking locations (ventral surface shown) for salamanders: 1 = left front, 2 = left back, 3 = right front, 4 = right back.  Marking schemes are designated as follows: X = no mark, R = red, Y = yellow, O = orange.  As an example, an individual marked red at position 1 (left front) would be RXXX, and an individual marked yellow at positions 3 and 4 (right front and back) would be XXYY.




Effects of a Prescribed Burn on Terrestrial Salamanders

Objectives:  To assess the effects of a prescribed burn on terrestrial salamander populations using paired control-burn plots.


Ho:  The prescribed burn has no effect on the abundance of redback salamanders (Plethodon cinereus).

Ho:  The prescribed burn has no effect on various environmental variables (soil moisture and temperature, vegetation parameters) which could influence the presence or abundance of Plethodon cinereus.


Six control plots at Shenks Hollow and six burn plots behind the headquarters at Shenandoah National Park were established in 1998 to monitor salamanders prior to a prescribed burn.  Both sites are located on the Thornton Gap USGS Quadrangle Topographic Map.   The prescribed burn at the Park Headquarters was postponed in 1998, and is currently scheduled for either the spring or fall of 1999.

Paired plots range in size from 15 m2 to 35 m2 (3 paired plots are 15 m2, two are 20 m2, and one is 35 m2).  Plot corners are marked with rebar and flagging tape, and a round  aluminum tag denoting the plot number is affixed to the largest central tree in each plot.  In 1998, plots were visited during the day between 1000 and 1800 on four occasions in the spring (8 April-21 May 1998) and once in the fall (October 1998).  One team searches a control plot while the other team searches the burn plot simultaneously and data is recorded on the Shenandoah Terrestrial Plot data sheet.   Plots are split into individual search areas using wire flags (3 m strips) to prevent duplication of search effort.  All surface rocks, logs, large twigs, and bark are overturned to find salamanders, and the number of overturned rock and wood cover objects is recorded using clickers attached to the right and left pant belt loops.  Use the right clicker to count the number of overturned rocks (right rock), and the left clicker to count the number of overturned wood objects (left wood).   When a salamander is captured, place it immediately into a ziploc bag.   Blow air into the ziploc bag and seal it such that the salamander can not escape and is provided with an air cushion to prevent it from getting crushed.  Replace cover objects as carefully as possible, and return leaf litter and other material around the cover objects if these are displaced.   Place a numbered wire flag in the ground next to the cover object, and write the same number using a Sharpie onto the ziploc bag in which the salamander was placed.  This allows investigators to return the salamander to the appropriate cover object after marking.   Salamanders are measured for snout-vent length and total length. Snout-vent length is made from the snout to the posterior end of the vent.  Total length is made from the snout to the tip of the tail.  Record whether the tail is missing or is regenerating.  Also, look for whether females are gravid with eggs visible through the abdomen.  For Plethodon cinereus, record the color morph (red or lead) on the data sheet.  After marking and processing, the salamander is placed next to the cover object such that it can crawl back under the cover object on its own.  Many environmental variables are recorded during each visit to the plot.

In spring and fall 1999, we will continue the capture-recapture work at the plots during the day, and additionally conduct wet night surface surveys in an effort to improve recapture rates.  The two survey methods will be conducted on a biweekly basis, allowing us to compare wet night surface surveys to diurnal natural cover object checks.  To better record the locations of individual salamanders, we will divide the plot into equal 2 or 3 m squares with wire flags and map out all cover objects within each grid.  During each visit, the locations of all salamanders will be recorded on this grid map xeroxed onto Rite-in-the-Rain paper.

This table shows the population estimate data for redback salamanders in 1998 from the control (C) and burn (B) plots based on Program Capture (scroll down to Capture) analyses.

Mark-Recapture Population Estimates
Terrestrial Salamanders at Shenandoah National Park

Plot Size
(m x m)
# Marked Adjusted
Pop. Size (N)
SE Proportion Detected (P) Model
C1 35 x 35 138 646 171 0.06 TH
C2 20 x 20 73 165 16 0.12 H
C3 15 x 15 42 199 94 0.06 O
C4 20 x 20 34 - - - -
C5 15 x 15 33 42 9 0.31 B
C6 15 x 15 68 99 21 0.25 B
B1 35 x 35 170 279 52 0.21 B
B2 15 x 15 37 - - - -
B3 15 x 15 63 220 67 0.08 O
B4 15 x 15 42 199 94 0.06 O
B5 20 x 20 33 301 241 0.03 O
B6 20 x 20 26 282 288 0.03 TH

Artificial Cover Object Study

Artificial cover objects (ACOs), or coverboards, are being used as a monitoring technique for terrestrial salamanders (   However, little published information is available comparing capture indices and capture-recapture population estimates under ACOs versus natural cover objects, and how the addition of ACOs to plots might influence salamander density and visibility.


To compare the use of natural cover objects to ACOs as a monitoring technique for terrestrial salamanders and to examine the potential influence of adding ACOs to plots on salamander abundance and visibility.


Ho:  The addition of ACOs has no influence on the number of salamanders found in a plot.

Ho:  There is no difference in the density of salamanders (number of salamanders/surface area of cover object) under ACOs as compared to natural cover objects in the plots.

Ho:  The use of ACOs does not influence the recapture rate of salamanders in a plot.


In the fall of 1998, the amphibian crew established eighteen 15 m2 plots with six plots in each of 3 areas, Tanner’s Ridge, Pocosin Gap, and Fisher’s Gap, which represent different ages and types of forest stands and previous land use disturbance histories.  The plots are placed a minimum of 50 m apart.

Tanner’s Ridge (Big Meadows USGS Quadrangle Topographic Map) currently supports a mixed deciduous forest with a few 70 year old and older trees.  This area was cleared for pastureland in 1928 by the early settlers, and a fire occurred here in 1930.   In 1941, this area was classified as open grassland with the exception of one small locality of mixed age red oak (Quercus rubra).

Fisher’s Gap (Big Meadows, USGS Quadrangle Topographic Map) was logged prior to 1920 but was never used as pastureland.  From 1940's records, the area was described as open yet surrounded by 1-20 year old red oaks.  The area borders an area in which a major fire occurred around 1930.

Pocosin Ridge (Fletcher USGS Quadrangle Topographic Map) located just south of the Pocosin Fire Road is an older predominately red oak forest (21-40 years old in 1940) that is relatively undisturbed.

Salamanders were surveyed using diurnal natural cover object searches.  During each check, the same natural cover objects are overturned.  Replace cover objects as carefully as possible, and return leaf litter and other material around the cover objects if these are displaced.  The size of the rock or wood cover objects (length, width, depth) is measured, and a decay and moisture score is assigned to wood cover objects.

Plots were surveyed a total of five times between 23 September and 31 October 1998.   At these plots, we use batch marking, using only one color at one position on a salamander for each visit.  On subsequent visits, if a batch-marked individual is recaptured, we batch mark it again using the new batch mark to indicate that the individual was recaptured on the new occasion.

At the end of October 1998, 25 oak cookie ACOs were placed 3 m apart in a 5 x 5 grid in three of the six plots at each site.  A numbered wire flag was placed next to each oak cookie and the number was also written on top of the cookie using a Sharpie.  The oak cookies were cut from trees that had fallen in Shenandoah National Park as a result of the March 1998 ice storm.  In 1999, to better record the locations of individual salamanders, we will divide the plot into equal 2 or 3 m squares with wire flags and map out all cover objects within each grid.  During each visit, the location of all salamanders will be recorded on the data sheet in addition to the grid map xeroxed onto Rite-in-the-Rain paper.

The coefficient of variation (CV = standard deviation/mean) of counts at the plots are shown below (C = control, B = Burn, F = Fisher's Gap, P = Pocosin, T = Tanner's Ridge).   Fall 1998 was very dry and counts were more variable in the fall than in the spring.

Variation in Counts of Plethodon cinereus
Daytime Cover Searches at Shenandoah National Park

Spring 1998 Fall 1998
Plot CV Plot CV
C1 0.26 F1 0.55
C2 0.26 F2 0.75
C3 0.29 F3 0.75
C4 0.38 F4 1.17
C5 0.33 F5 0.39
C6 0.66 F6 0.25
B1 0.51 P1 0.38
B2 0.31 P2 0.65
B3 0.52 P3 0.62
B4 0.64 P4 0.94
B5 0.60 P5 1.24
B6 0.38 P6 0.73
    T1 0.71
    T2 0.82
    T3 1.05
    T4 0.87
    T5 1.00
    T6 0.43
OVERALL 0.43   0.74

Figure 3.  Recapture rates were extremely low on these plots, and we only recaptured one individual each from plots F5, T1, and T4, and two individuals each from plots P1 and P6.  These low recapture rates may have resulted from the very dry conditions in the fall of 1998.

cover_graph.gif (8642 bytes)

Large-Scale Terrestrial Salamander Project

Objectives: To determine relationships between habitat variables and salamander distribution and abundance throughout Shenandoah National Park.


Ho:  There is no relationship between numbers of salamanders in a plot and soil pH, moisture, or temperature, or other habitat variables (e.g., forest type and age).

In the spring of 1999, a stratified random sample of 200 terrestrial plots (5 m2) will be surveyed for salamanders.  Stratification will be based on elevation (low vs. high) and bedrock type (granitic, siliciclastic, basaltic).  We will use the same technique of turning over cover objects within the plot during the day and record the same environmental variables as used in the other terrestrial salamander studies.

Elevational Transect and Contaminant Levels in Plethodon cinereus

Terrestrial salamanders live in close association with soils and are sensitive to soil pH levels (Wyman and Hawksley-Lescault 1992).  Low soil pH can increase the bioavailability of aluminum levels in soils, which can be toxic to salamanders (Albers and Prouty 1987, Miller et al. 1992).  Contaminants in soils can persist for long periods of time (Jordan 1975).  Salamanders can bioaccumulate contaminants through dermal contact (absorption of contaminated soil pore water) and dietary ingestion (soil arthropods). 

Previous studies have shown increasing metal concentrations in salamanders at sites closer to zinc smelter sites (Storm et al. 1994), and persistence of organochlorines and their metabolites in salamanders long after applications of these compounds (Dimond et al. 1968).  These studies were concerned with point sources or single applications of toxicants.  We are interested in determining whether the distribution and abundance of salamanders in Acadia National Park is influenced by general nonpoint atmospheric deposition patterns of acid rain and contaminants, and whether salamanders in "hotspots" of contaminant deposition show higher bioaccumulation of metals and organochlorines.  Recent evidence shows that higher elevations are subjected to a greater deposition of contaminants because of increased wet, dry, "cold condensation," and cloud deposition (Lovett 1994, Blais et al. 1998).  We would like to determine whether salamanders at higher elevations have higher contaminant body burden levels, and whether this might influence distribution and abundance.

In 1999, we will compare the levels of metals and organics in Plethodon cinereus along an elevational transect along the east and west sides of the Blue Ridge Mountains in Shenandoah National Park. We will collect five Plethodon cinereus at each of 5 elevations on the west side and 5 elevations on the east side of the ridge (separated by 500 feet in elevation) within a 50 m distance from a stream.   The collections will be made either along one transect in a low pH area of the park (e.g., near Paine Run), or from several locations at each of the 5 elevations.  These salamanders will be analyzed for metals (aluminum, mercury, etc.), and organics (e.g., chlorinated organics such as p,p’-DDE, dieldrin, toxaphene, etc.).

Ho:  There is no difference in levels of contaminants in Plethodon cinereus along an elevational gradient.

Ho:  There is no difference in levels of contaminants in Plethodon cinereus along the western slope as compared to the eastern slope of the Blue Ridge Mountains.

Plethodon shenandoah Monitoring and Population Genetics

The Shenandoah salamander (Plethodon shenandoah), which occurs only in Shenandoah National Park, was listed as an endangered species by the Commonwealth of Virginia in 1987 and was listed as federally endangered in 1989.  Transects will be conducted along trails on rainy nights beginning one hour after sunset.  Transects will be run once a month in April and May, and again in September and October.  The principal threats to the salamander are severely restricted distribution and competition from the closely related red-backed salamander (Plethodon cinereus).  The principle concerns associated with the restricted range of the species include the potential effects of acid deposition and forest defoliation die to exotic insect species.  A new recovery plan written in 1994 recommended research objectives for Plethodon shenandoah, including standardized long-term monitoring of populations (U.S. Fish and Wildlife Service.  1994.   Shenandoah salamander (Plethodon shenandoah) Recovery Plan.  Hadley, Massachusetts.  36 pp.).  A monitoring program for Plethodon shenandoah will be initiated in spring 1999 and tail tips will be collected for a population genetics study of the three populations (Hawksbill, Pinnacles, Stony Man) conducted by Dr. Jack Sites (Brigham Young University, Provo, UT).  Surveys will be conducted at the Hawksbill and Stony Man sites in collaboration with Bill Witt, Lester Via, and Mary Willesford-Bair.   Three transects will be run at each site.  Transects will be 25 m long and will cover (1 m on both sides of the trail).  Searching will be done by looking for salamanders on the forest floor and on vegetation.  UTM coordinates for locations of salamanders will be recorded.  Air temperature and soil temperature will be measured at the location where the salamander was found.  All salamanders will be photographed (both dorsal and ventral), scored for various attributes (dorsal back color, width and length of back stripe) as well as measured for snout-vent length, total length, and body mass.  Other features such as broken or regenerating tails and whether females are gravid will also be recorded.

Environmental Variables Measured at the Terrestrial Plots

Many variables were recorded during each visit to the terrestrial salamander survey plots.

Plot #
Begin Time
End Time
Break Time:
If a break is taken during the survey, record length of break time such that this period can be subtracted from the total search effort.
Begin Time
Air Temperature:
In 1998, air temperature was measured at the center of the plot at breast height.  In 1999, we will measure air temperature at three locations near the center of the plot: 1 cm above ground in the shade, 1 m above ground in the shade, and under 5 natural cover objects and 5 wood and rock ACOs in each plot.

Wind Speed Codes (Beaufort wind scale):

0 = < 1 mph, calm, smoke rises vertically
1 = 2-3 mph, light air movement, smoke drifts
2 = 4-7 mph, light breeze, wind felt on face, leaves rustle
3 = 8-12 mph, gentle breeze, leaves and twigs in constant motion
4 = 13-18 mph, moderate breeze, small branches move
5 = 19-24 mph, fresh breeze, small trees begin to sway
6 = 25-31 mph, strong breeze, large branches in motion

Sky Codes:

0 = clear or few clouds
1 = partly cloudy or variable
2 = cloudy or overcast
3 = fog
4 = drizzle
5 = showers

Weather History:

Describe the previous 48 hours of weather.  Include details regarding temperature, precipitation (fog, mist, light or heavy rain, snow), cloud cover (clear, partly cloudy, overcast), and wind (none, moderate, high).

Rain Gauge:

A rain gauge is placed at each of the sites in an open area to determine the amount of rainfall between sampling periods.  The level of water in the rain gauge should be recorded during each visit.  Empty the rain gauge after recording water level.

Relative Humidity:

Record using Oakton WD-35612-00 Digital Thermohygrometer.  Follow Operating Instructions.

Dominant Plant Species:

Identify and record the dominant plants in the overstory and understory.  In 1999, we will record tree species and their dbh (diameter at breast height) for all individuals greater than 5 cm dbh in each plot.

Leaf litter depth, % canopy cover, % understory cover, soil collection, and soil T are measured at each of five locations prior to salamander sampling.  One location is in the center of the plot, and the other four locations are halfway between the center and the corners of the plot in the four cardinal directions (Fig. 4).

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Figure 4.  Aerial view of five sampling locations in plot.

Leaf Litter Depth:

Measure by inserting a millimeter ruler through the leaf litter until hard ground is reached and record the millimeter height of the leaf litter above ground.

% Canopy Cover:

Measure using a spherical densiometer.  The spherical densiometer is held level approximately 25 cm in front of the observer at elbow height.  Assume four (4) equispaced dots in each square of the grid.  Count the number of dots equivalent to 1/4 squares which are not covered by canopy, representing the canopy openings.  Multiply the total number of open 1/4 squares by 1.04 to obtain a % overhead area not occupied by canopy.  Subtract this number from 100 to obtain % canopy cover.

% Understory (< 1 m) Cover:

Measure within a m area delineated by laying two meter poles at a 90 degree angle.  Two people assess the % understory cover below one meter in the 1 m plot, and record the average of the two measures.

Soil temperature:

In 1998, soil temperature was measured by inserting a Hanna t-type thermocouple probe into the soil to a depth of 3 - 12 cm.  In 1999, we will record soil temperature at a 5 cm depth.  Record temperature after the temperature reading stabilizes.

Soil collection:

Remove leaf litter from the top of the soil and collect soil using a soil corer (1 inch diameter).  Collect approximately 5 cm of soil from each of the five localities in each plot.  Place soil into a 3" x 5" ziploc bag and place the sample in a cooler.  Back in the laboratory, conduct soil pH and soil moisture tests. 

Cover Objects:

Record the number of rock and wood cover objects overturned in each plot.  When a salamander is found, record the type (rock or wood) and dimensions (length, width, depth) of the cover objects that actually make contact with the ground.  If the salamander is found under wood, record the decay and moisture scores of the wood cover object.

Moisture Score for Wood Cover Objects:

Dry = no moisture or water droplets apparent on squeezed material
Damp = no water droplets on surface, but moisture present in squeezed material
Wet = water droplets appear on surface of squeezed material
Saturated = stream of water expressed from squeezed material

Decay Score for Wood Cover Objects:

Firm = log does not fall apart
Chunky = log falls apart as series of rectangular blocks separated by cracks
Fibrous = log falls apart as loose strands
Crumbly = log falls apart as finely divided particles, sawdust-like


Soil Measurements in the Laboratory

Upon your return to the laboratory, mix the soil thoroughly in the ziploc bag using a clean spatula.  Remove all twigs, rocks, and roots from the soil using clean forceps.  Process for Soil ph, moisture, and % sand, silt, clay and record on Soil data sheet.

Soil Moisture

Label a paper lunch bag with the plot identification and date.  Weigh the bag and record its weight.  Tare the scale to 0.00 g, and add approximately 40 g of soil to the bag using a scoop.  Record the wet soil weight.  Place the bag containing wet soil in a drying oven at 105 C for 24 hours at the Inventory and Monitoring Building.  Record the the dry weight of the bag plus soil and subtract the bag weight to obtain the dry soil weight.  Use the following equation to calculate % soil moisture:
[(Soil Wet Weight - Soil Dry Weight)/Soil Dry Weight] x 100.  To calculate soil water content (g/g) use same calculation but do not multiply by 100.

Soil pH

We use the methods described in

1) To prepare soil for pH measurement, take approximately 30 g of wet soil and place it in a paper cup to air dry overnight.

2) After 24 hours, use a mortal and pestle and grind the soil into a fine powder.   Clean the mortar and pestle with deionized water in between samples.  Dry using Kimwipes.

3) Weigh 20 g of mortar and pestled, air-dried soil into a paper cup.

4) Add 20 ml of deionized water to the sample. Stir vigorously with a clean spatula for 15 seconds and let stand for 30 minutes.

5) Calibrate the pH meter using 4.0 and 7.0 pH standard buffer solutions.

6) Place electrodes in the slurry, swirl carefully, and read the pH immediately. Ensure that the electrode tips are in the slurry and not in the overlying solution.

Soil Texture (% Sand, Silt, Clay)

This procedure is only conducted once for each plot.  Use the LaMotte Soil Texture Unit to determine % sand, silt, and clay in the soil sample.  The amount of time required for soil particles of various sizes to settle in the soil separation tubes forms the basis for this test.  From the amount of material collected in each tube it is possible to determine the approximate percentage of each fraction as represented in the original soil sample.


  1. Place the three Soil Separation Tubes in the rack.
  2. Add the soil sample to Soil Separation Tube "A" until it is even with line 15.   NOTE:  Gently tap the bottom of the tube on a firm surface to pack the soil and eliminate air spaces.
  3. Use the pipette (0372) to add 1 ml of Texture Dispersing Reagent (5644PS) to the sample in Soil Separation Tube "A".  Dilute to line 45 with tap water.
  4. Cap and gently shake for two minutes, making sure all the soil sample is thoroughly mixed with water.

    The sample is now ready for separation. The separation is accomplished by allowing a predetermined time for each fraction to settle out of the solution.  Be sure that you continue to gently shake the separation tube up to the the time of the first separation (Step 5).
  5. Place Soil Separation Tube "A" in the rack.  Allow to stand undisturbed for exactly 30 seconds.
  6. Carefully pour off all the solution into Soil Separation Tube "B".  Return Tube "A" to the rack.  Allow Tube "B" to stand undisturbed for 30 minutes.
  7. Carefully pour off the solution from  Soil Separation Tube "B" into Soil Separation Tube "C".  Return Tube "B" to the rack.
  8. Add 1 ml of Soil Flocculation Reagent (5643PS) to Soil Separation Tube "C".   Cap and gently shake for one minute.
  9. Place the Soil Separation Tube "C" in the rack and allow to stand until all the clay in suspension settles.  This may require up to 24 hours.

    Note:  Unless there is further use of the clay sample for air drying and study as described later, it is not necessary to wait for the suspension to settle.

    Due to the colloidal nature of clay in solution and its tendency to swell and form a gel, the portion of clay remaining in Tube "C" is not used to determine the clay fraction present in the soil.  The clay fraction is calculated by adding the sand and silt fractions and subtracting this total from the initial volume of soil used for the separation.

    Tube "A" Sand


    Initial Volume


    Tube "B" Silt


    Total "A" & "B"


    Total "A" & "B"




  10. Read Soil Separation Tube "A" at top of soil level.  To calculate percentage sand in the soil, divide reading by 15. Multiply by 100.  Record as % sand.
  11. Read Soil Separation Tube "B" at top of soil level.   To calculate percentage silt in the soil, divide reading by 15.  Multiply by 100.  Record as % silt.
  12. Calculate volume of clay as shown above.  To calculate percent clay in the soil, divide value by 15.  Multiply by 100.   Record as % clay.


Soil Separation Tube "A" reads 2.
Soil Separation Tube "B" reads 8.

Percent Sand = (Reading A/Total Vol.) x 100      = (2/15) x 100 = 13%
Percent Silt  = (Reading B/Total Vol.) x 100      = (8/15) x 100 = 53%
Percent Clay = (Calculated Vol./Total Vol.) x 100      = (5/15) x 100 = 33%

Since the scientific basis of the test is the particle size and its mass, as related to its settling time when dispersed in solution, the following table is included for reference.

Soil Particle

Diameter in mm

Very Course Sand 2.0 - 1.0
Course Sand 1.0 - 0.5
Medium Sand 0.5 - 0.25
Fine Sand 0.25 - 0.10
Very Fine Sand 0.10 - 0.05
Silt 0.05 - 0.002
Clay Less than 0.002


Sandy soil is described as soil material that contains 85% or more sand.  The percentage of silt plus 1.5 times the percentage of clay shall not exceed 15.  Silt soil is described as soil material that contains 80% or more of silt and less than 12% clay.  Clay soil is described as soil material that contains 40% or more clay, less than 45% sand and less than 40% silt.


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