Barred Tiger Salamander as a Water Contamination Bioindicator

Introduction

The relationship between eco-toxicological factors and amphibians has long been of interest to both environmentalists and herpetologists alike. For both, the informational needs are germane to those ecological factors pertaining to the decline of species as well as efforts to avert any weakening in the reproductive cycle of amphibians. In fact, to many scientists, the amphibian — especially the salamander — is considered the "canary of the coal mine" for water pollutant investigations. These amphibious creatures are being adversely affected not only by the reduction of wetlands due to commercial development, but also by the unchecked use of pesticides, the introduction of exotic predators, and industrial pollutants that cause excessive ultraviolet light bombardment through the thinning of the ozone layer.

As amphibians play an important role in ecosystem dynamics, it is crucial to understand how these creatures can shed light on environmental factors affecting the environment and mankind in general. Reasons for species decline might well provide answers indicating ways conservationists and environmentalists could improve the environment, especially the wetlands of the Texas Panhandle. To this end, research is necessary for setting regulatory standards to protect not only the species, but the environment as well. Studying the effect of water pollutants within the Texas Panhandle aquatic areas on selected amphibians is an important step in the preservation of wetlands and in safeguarding one of their natural inhabitants.

Studying the effects of pollutants on environmental problems has long made use of an area's indigenous inhabitants. Within the Texas Panhandle wetlands, the favored subject has been the tiger salamander. This particular species has afforded investigators the opportunity to determine whether or not wetland pollutants are producing adverse effects on selected developmental characteristics of the salamander — namely, length, weight, and gill length. Descriptive statistical results concerning the sensitivity of the Barred tiger salamander to contaminant exposure would permit the investigator to draw conclusions with respect to the effects of those contaminants on the species (and to potentially extrapolate results to other species), as well as to develop corrective procedures to eliminate the environmental contaminants.

Throughout the Texas Panhandle, there are a significant number of natural ponds, lakes, and lagoons in which pollutants can be studied to determine their effect on aquatic inhabitants. In turn, conclusions can be drawn concerning the effect of man and the environment on those inhabitants. Observing groups of the Barred tiger salamander in aquatic environments of the Texas Panhandle with a potential for contamination would provide evidence as to the value of the species as an indicator of contaminant accumulation and effect. In order to investigate particular variables that may be relevant to the development of the Barred tiger salamander, the following problem was explored in depth: To what extent did pre-selected water contaminants, effluent discharges, chemicals, and pollutants influence the Barred tiger salamander across six various aquatic environments?

The recorded results would supply data referencing observed and measured differences in the growth patterns of those amphibians exposed to contaminants compared to those that were not exposed. Net results would also relate the degree to which the bioaccumulation of contaminants of concern (COC) within the food chain potentially affects the entire Texas Panhandle ecosystem habitat. Through exposure to — or directly as a result of — food chain characteristics, potential changes in the growth rate and general development of the Barred tiger salamander were observed and recorded.

For convenience and expediency, the independent variables of the present study were designated according to the following symbols:

LC: Lake Cove aquatic environment.
POTW: Sewage runoff aquatic environment.
PL: Playa Lake aquatic environment.
EST: Earthen Stock Tank aquatic environment.
IW1: Industrial Wastewater #1 aquatic environment.
IW2: Industrial Wastewater #2 aquatic environment.

The dependent variables are abbreviated as follows:

L: Body length of Barred tiger salamander.
GL: Gill length of Barred tiger salamander.
W: Weight of Barred tiger salamander.

As the present study was designed to explore the effects of six contaminated Texas Panhandle water areas on the length, weight, and gill length of the tiger salamander, the following null hypotheses were formulated and subjected to statistical data analysis:

1. There is no statistically significant effect of Lake Cove water on the length, weight, and gill length of the Barred tiger salamander at the α < .05 level of statistical significance. Ho (LC)1: L = GL = W

2. There is no statistically significant effect of POTW water on the length, weight, and gill length of the Barred tiger salamander at the α < .05 level of statistical significance. Ho (POTW)2: L = GL = W

3. There is no statistically significant effect of Playa Lake water on the length, weight, and gill length of the Barred tiger salamander at the α < .05 level of statistical significance. Ho (PL)3: L = GL = W

4. There is no statistically significant effect of Earthen Stock Tank water on the length, weight, and gill length of the Barred tiger salamander at the α < .05 level of statistical significance. Ho (EST)4: L = GL = W

5. There is no statistically significant effect of Industrial Wastewater #1 on the length, weight, and gill length of the Barred tiger salamander at the α < .05 level of statistical significance. Ho (IW#1)5: L = GL = W

6. There is no statistically significant effect of Industrial Wastewater #2 on the length, weight, and gill length of the Barred tiger salamander at the α < .05 level of statistical significance. Ho (IW#2)6: L = GL = W

In addition to the null hypotheses above — wherein water contaminant effects were tested for statistical significance — differences between the six contaminant water areas were also tested for statistical significance at the α < .05 level. The resulting secondary null hypotheses were as follows:

1. There exists no statistically significant difference between the Barred tiger salamander's length, weight, and gill length with respect to Playa Lake and Lake Cove water environments. Ho11: PL = LC

2. There exists no statistically significant difference between the Barred tiger salamander's length, weight, and gill length with respect to Playa Lake and POTW water environments. Ho12: PL = POTW

3. There exists no statistically significant difference between the Barred tiger salamander's length, weight, and gill length with respect to the Playa Lake and the Earthen Stock Tank water environments. Ho13: PL = EST

4. There exists no statistically significant difference between the Barred tiger salamanders' length, weight, and gill length with respect to the Playa Lake and the Industrial Wastewater #1 water environment. Ho14: PL = IW#1

5. There exists no statistically significant difference between the Barred tiger salamanders' length, weight, and gill length with respect to the Playa Lake and the Wastewater #2 water environment. Ho15: PL = IW#2

The following terms and concepts are used extensively throughout this report and are operationally defined below to avoid misunderstanding:

Barred tiger salamander: A small lizard-like amphibian. The Barred tiger salamander (Ambystoma tigrinum) was selected for this study due to its abundance and availability in the area.

Texas Wetlands: Areas that are inundated or saturated by surface or groundwater at a frequency and duration sufficient to support, and that under normal circumstances do support, a prevalence of vegetation typically adapted for life in saturated soil conditions. Wetlands generally include swamps, marshes, bogs, and similar areas (33 CFR § 323.2, and 40 CFR § 230.3; Berry & Dennison, The Environmental Law and Compliance Handbook). The Texas Commission on Environmental Quality has incorporated the playa lakes of the Texas Panhandle into the definition of wetlands with specific regulations; Title 13 § 401.004; Title 2 § 11.502; Title 2 § 26.048 (Texas Environmental Laws, 2004 Edition, Thomson West).

Water contaminants: Any substance introduced into water that has the effect of rendering it toxic or otherwise harmful (The Condensed Chemical Dictionary, 9th ed., 1977, Hawley, Van Nostrand Reinhold).

Length Measurement: Measurement of individual salamanders in centimeters from the tip of the tail to the front of the snout to determine overall length.

Weight Measurement: Measurement of the mass in grams of individual salamanders.

Gill Measurement: Length measurement in centimeters of the gill located on the left side of the salamanders' heads, extended fully from the point of attachment to the tip.

Metamorphosis: The ability of the tiger salamander to transform from the aquatic neotenic form with gills to the terrestrial form to live on land. Both forms can develop to complete sexual maturity, but the neotenic form remains aquatic with external gills and a main fin on the tail. It is a marked change in the mode of life and form of the species.

Terrestrial: The land-based stage of the Barred tiger salamander. The salamander typically undergoes metamorphosis, becomes terrestrial, and develops into a mature adult living outside the aquatic habitat.

ANOVA: Method for statistical analysis of more than one variable (Phillips, J. L., How to Think About Statistics, 6th ed., W.H. Freeman and Company, New York, 2001).

"t" Test: Statistical test used to compare two means to determine whether or not they are identical (Lee, C.C., ed.; Shun, Dar Lin, assoc. ed.; Handbook of Environmental Engineering Calculations, McGraw-Hill, New York, 1999).

Independent Variable: A variable that is manipulated by the experimenter, or a treatment variable (Phillips, 2001).

Dependent Variable: A variable whose values are determined by those of the independent variables (Phillips, 2001).

Statistical Significance: Experimental results that are not likely to have occurred by chance alone (Koosis, D. J., Statistics: A Self-Teaching Guide, 4th ed., John Wiley & Sons, Inc., 1997).

Both the independent and dependent variables in any research investigation are chosen according to a predetermined set of criteria. The investigator of this study established the following selection criteria when choosing the sample group of Barred tiger salamanders:

1. Taxonomic soundness and ease of recognition for accurate sample selection.
2. Cosmopolitan distribution of the sample.
3. Numerical abundance of the sample.
4. Low genetic and ecological variability of the sample.
5. Large body size of the sample.
6. Long life history and mobility of the sample.
7. Auto-ecological awareness by the sample.
8. Laboratory tolerance by the sample.

Taxonomic categories are best described as those that constitute a sample of likeness in which shared trait commonalities — rather than shared relationships — exist. For example, all salamanders share a relationship within six identifiable types, yet each grouping has its own identifiable trait catalogue. The Barred tiger salamander was chosen rather than other salamander species because it resides in the Texas Panhandle, although its zoological relatives live in other land areas throughout the United States. Only aquatic-bound Barred tiger salamanders were studied — not terrestrial inhabitants — because the primary purpose was to investigate the effects of aquatic environments on the developmental indices of the species.

Any investigative study must provide the investigator with a sufficient number of units to observe, evaluate, and assess. The Barred tiger salamander provided a sufficient number at this point in the ecological cycle. Had only a few individuals been available, the study would have been greatly limited in scope and inferential ability. Additionally, knowing that the Barred tiger salamander has a lifespan of up to 20 years provided ample time to record changes caused by aquatic pollutants. Knowing the species can also survive in a controlled, even pet-oriented, environment provided additional assurance that individuals could exist in a treatment situation without immediate demise.

Low subject variability is essential in well-controlled studies. When variability increases, the possibility of error or bias in interpreting results rises, thereby contaminating research findings. Barred tiger salamanders of the Texas Panhandle, with limited mobility and low genetic and ecological variability, are therefore self-controlled with respect to extraneous influences that cannot be measured or controlled.

Although the Barred tiger salamander has been used in past environmental exposure studies for pesticides, herbicides, and ecological risk assessments, its use in continued research for contaminant accumulation and effects remains limited. Amphibians are considered to be extremely sensitive to ecosystem changes; therefore, the health of the salamander is expected to mirror the health of the ecological population as a whole. Using the Barred tiger salamander as an indicator species provides a study organism sensitive to environmental change so that developmental alterations can be recorded and assessed. Even with naturally occurring anomalies — such as droughts, floods, and dust storms — the species' population numbers are sufficient to permit a determination of any correlation to contaminants beyond "normal" variation.

Physiology, Morphology, and Range

Kingdom: Animalia
Phylum: Chordata
Subclass: Lissamphibia
Order: Caudata
Family: Amystomatidae
Genus: Ambystoma
Species: Ambystoma tigrinum
Subspecies: Ambystoma tigrinum mavortium (Baird, 1850)

The neotenic form of the Barred tiger salamander (Ambystoma tigrinum mavortium) has often been referred to in the literature as a "waterdog" or "mud puppy." Although many individuals use these terms to denote the species studied here, it is an erroneous usage. The true "water dog" is associated with the family Proteidae, genus Necturus. Therefore, the Barred tiger salamander is not the true "water dog" (Miller, 2000).

The Barred tiger salamander, Ambystoma tigrinum, ranges from Nebraska to the Mexican Plateau (Miller, 2000). It can also be found in prairie and forested areas where moisture is adequate (Collins, 1982).

With moist skin and long tails for swimming, the Barred tiger salamander represents one of the largest groups of mole salamanders. Distinguishing markings of the adult include light spots, bars, or blotches on a dark background. The body is robust, with 14 or fewer vertical grooves on each side between the front and hind limbs. The belly is of a mottled light or light-dark shade. The head, limbs, body, and tail are deep brown, dull black, or black with spots, bars, or blotches on the sides. These markings may or may not extend onto the belly, which is generally black or gray-black and mottled with yellow. Two tubercles are present on the sole of each foot, and no paratoid glands are present. Adult Barred tiger salamanders average 15 centimeters in length but have been observed at lengths up to 20 centimeters, with the tail being nearly half the total length. The head is round and short with a pronounced snout, and the eyes are small, bulging, and widely spaced (Collins, 1982).

Like other amphibians, the skin of the Barred tiger salamander is permeable, giving it the ability to absorb and expel water and gases through the skin. The Barred tiger salamander therefore uses its skin as an oxygen receptor in addition to its saclike lungs. Because of this somewhat unique makeup, examination of the species' developmental characteristics may provide information concerning an additional route of entry for surface water contamination (Bishop, 1967; Larson, McDonald, Fivissani, Newton, & Hamilton, 1998).

After hatching, the Barred tiger salamander goes through an aquatic larval stage before becoming an adult. In the larval stage, the species remains uniformly olive brown with small brown and sometimes irregular spots on the back, sides, and tail. Having large gills, a broad body, stout legs, and greatly flattened and pointed toes, the larvae are adapted as pond inhabitants. They possess external gills and a much wider fin along the tail for balance; they lack the two small glands on the side of the head for balance that are possessed by larvae of other salamander species. With gills and an extra-long tail, the larvae can easily navigate through water. The neotenic individual can halt the metamorphosis process in the larval (aquatic, neotenic) stage when it is beneficial to remain aquatic, or it can metamorphose to the terrestrial stage when that transition is more favorable (Bishop, 1967).

Reclusive throughout the majority of its adult life, the Barred tiger salamander mates from November to June, depending on moisture and temperature conditions. Even when adults congregate in ponds, springs, stock tanks, lakes, or flooded quarries, determining sex is difficult. Courtship begins as the male and female circle on the bottom of a water habitat, nudging each other as the male rubs his chin on the female's head, neck, and back. The male will guide the female to follow and deposit his spermatophore, which she will pick up. Within one or two days, the female lays eggs, but neither adult attends to the egg cluster. Depending on water temperature, the eggs will hatch in three to five weeks (Bishop, 1967; Collins, 1982).

Method of Study

The sample group was selected on the basis of the following criteria to reduce actual and extraneous sampling error: non-dry climatic conditions; origin from a common water source (Playa Lake); no current residence in a contaminated area; and approximately equal overall weight, length, and gill length. The sampling location was chosen because it was likely to provide environmental uniformity; the specific common location was Playa Lake. The depth of the seined area ranged from 0.25 m to 1.75 m over an area of approximately 950 m². Using a 6 m manually drawn pocket seine, 9 to 25 neotenic Barred tiger salamanders were captured per pass for a total collection of 214 salamanders. Considerable waste accumulation from livestock and waterfowl appeared to provide a nutrient base for the aquatic fauna of the playa. Three 18.92-liter containers provided temporary storage in transit to an appropriate 3.79 hl holding tank filled with lake water.

Once the sample was safely transported to the base holding tank, six containment cages measuring 61 cm × 123 cm × 183 cm were constructed of 2 cm PVC pipes overlaid with 2.5 cm mesh chicken wire. The wire was installed to contain the populations within their respective varied aquatic environments. Upon introducing the salamanders to their cages, additional containment modifications were necessary to ensure total restraint. Minnow seines with a 0.6 cm gauge were therefore installed beneath the chicken wire. By adding these additional restraints, sufficient space and adequate aquatic flow were maintained, allowing for unrestricted exposure to the various aquatic environments under study. Twenty-five salamanders were placed in each of the six containment cages.

During the base location containment period, all salamanders were regularly observed and data were recorded for each individual's length (snout tip to tail tip): lengths of 9.7 cm to a maximum of 14.5 cm were recorded. Overall length, gill length in centimeters, and weight in grams were also recorded for each unit. Weekly observations were conducted by lifting each containment unit from the water source, allowing the researcher to check the health and safety of the captive salamanders. These observations were recorded with respect to sample numbers, signs of metamorphosis, and the general condition of individual salamanders concerning possible injury, dryness of skin, and the clarity of the eyes. During weekly inspections, each group was provided sufficient dog food to supply additional nutrients and reduce the incidence of cannibalism among the caged salamanders. Because salamanders are extremely sensitive to the salts and oils in human flesh, handling was kept to a bare minimum. When a salamander required closer inspection, it was carefully supported by two gloved hands to avoid contamination from human contact and to prevent injury from dropping.

Because the containment facilities were located outdoors, frequent checking was conducted to ensure that the holding facilities were not accessible to predators, that appropriate water depths were maintained, and that each cage remained structurally sound. Regular measurements included eight weekly sets covering pH level, conductivity, and temperature. As a precautionary measure, three water sample sets were submitted to an independent laboratory for additional analysis of element composition. The U.S. Environmental Protection Agency analytical methods employed included EPA Method 600/4-79-020 (Methods for Chemical Analysis of Waste), with supplementary analyses covering metal trace elements (200.7), hardness (130), organic carbon (314A, 415), inorganic anions (300.0), pH (150.1), conductance (120.1), residue (160.1), and alkalinity (310).

Upon termination of the field observation tasks, randomly selected salamanders were preserved for body burden analysis per Toxic Characteristics Leaching Properties (TCLP) metals and U.S. EPA methods 6020A (toxicological contaminants), 7471A (pesticide levels), and SM 18th 2540G (total, fixed, and volatile solids — standard method of analysis) (ANA-Lab report, 2002).

Once the total sample group of 214 Barred tiger salamanders was collected and contained, monitoring focused on 150 salamanders randomly selected and assigned to six locations in groups of 25 per location. Relocation sites were chosen based on the potential for exposure to environmental contaminants. All sites in Randall County, Texas, included: a Lake Cove (approximately 35° 3' 19.44" N, 101° 46' 37.2" W); a POTW effluent site (approximately 35° 3' 11.0" N, 101° 46' 25.6" W); Playa Lake (approximately 35° 5' 28.2" N, 101° 45' 35.9" W); and a Subsurface Earthen Stock Tank (approximately 35° 3' 42.7" N, 101° 44' 46.5" W). Sites in Gray County, Texas, included Wastewater-Storm Water Pond #1 (approximately 35° 30' 47.0" N, 101° 0' 56.1" W) and Wastewater-Storm Water Pond #2 (approximately 35° 30' 37.5" N, 101° 0' 51.6" W).

The test results were subjected to two parametric statistical processes: the t-test and the Analysis of Variance (ANOVA). Modifications were made to the ANOVA to accommodate unequal cell frequencies due to some sample loss, repeated measures, and nested variables. Several ANOVA processes were formatted to investigate all possible combinations between the independent variables (aquatic environments) and the dependent variables (length, gill length, weight). The overall ANOVA was a 6 × 3 design and the probability level was set at α < .05 for rejection of the established null hypotheses.

Since an analysis of variance can only determine whether statistical significance occurred within group comparisons, a modified orthogonal analysis was necessary to determine where the differences existed. Should main effects be non-significant and one of the interaction counterparts significant, a test for simple effects would be employed to locate the interaction. In addition to the ANOVA, t-tests were run to determine whether any statistically significant differences in component variability from location to location existed. Descriptive statistics were also used to help the reader view the variability of factors between and among the groups; however, the reader is cautioned not to draw premature conclusions from these statistics alone, as their numerical values were incorporated into the t-test and ANOVA processes for further analysis.

The measurement data were in ratio form, with absolute measurement values that included an absolute zero point. All selected statistical analysis procedures were therefore permissible. By having both ratio measurement data and construct and predictive measurement validity, the amount of measurement error becomes insignificant except when human error might affect the results. All precautions were taken to ensure that human error was kept to an absolute minimum.