Research Paper Doctorate 1,300 words

Dependent Variable and Environment

Last reviewed: October 13, 2005 ~7 min read

Barred tiger salamander, etc.

In order to monitor ecological developments in forest areas of the United States, the salamander is often used as an indicator of ecological sustainability because of their high site tenacity and environmental sensitivity. As such the salamander has come to represent an essential element of the many balanced and healthy North American ecosystems. In recent years, there have been many studies using salamanders to measure the overall health status of the environment and a readily used procedure as salamanders act as indicators of environmental stress areas because their delicate systems are extremely sensitive to synthetic and manmade pollutants.

The Barred tiger salamander, i.e., Ambystomia tigrinum, and the topic of this thesis, is distributed throughout the Texas panhandle and is a nocturnal and water seeking amphibian that can adjust to environmental conditions -- a process known as neoteny. Results of the present investigative research project indicated that the Barred tiger salamander is an excellent dependent variable against which surface water contamination can be measured within selected aquatic regions of the Texas Panhandle. The conducted research sought to determine whether or not differences and/or relationships existed between and amongst six varied aquatic environments (independent variables) on the developmental of the Barred tiger salamander developmental characteristics (dependent variable). The identified six independent variables included the following environments: a Public Owned Treatment Work (POTW) sewage effluent site, a lake cove, a playa lake, a subsurface earthen stock tank, and two industrial wastewater storm water ponds. The first procedural step taken was to record differences of the sample salamanders' overall length, gill length, and weight. Thereafter, and on a weekly basis for 55 days, each participating sample group was observed as to any sign of metamorphosis, contamination injury, and rate of anatomical development (dependent variable). The resulting data, subjected to statistical analysis (ANOVA, t-test, descriptive statistics), supported the argument that the Barred tiger salamander is an acceptable dependent variable upon which to observe the effects of the independent variables (i.e., contaminant water).

Table of Contents

Chapter One: Introduction

Purpose of study and problem statement

Hypotheses

Definition of terms

Rationale for the study of the selected species

Chapter Two: Physiology, morphology and range

Taxonomic Hierarchy

Chapter Three: Method of study

Sampling

Measurement data reliability and validity

Chapter Four: Results

Chapter Five: Discussion and conclusion

Study Limitations and Sources of Possible Error

Implications for Future Research

Table 1 (?)

Table 2: Locations

Table 3: Beginning and Ending Sample Distribution Chart

Table 4: Means and Standard Deviations for Sample Type -- 5x3

Table 5: t Values for Mean Difference Comparisons between Playa Lake and the Other Groups.

Table 6a: Comparison of the Results of the ANOVA and t-Test for Length

Table 6b: Mean value comparisons to reject null hypothesis

Table 7: Mean value comparisons to accept null hypothesis

Table 8: Comparison of t-test and F-values for Significance Determination for Weight.

Table 9: Comparison of Values for the Gill Length

Table 10: Comparison of t Test Values for Water Analysis Data

Table 11: Analytical Results of the Body Burden Analysis for Metals

and Pesticides. All data is Dry Weight Basis.

List of figures

Figure 2.1. Barred tiger salamander's inhabited territory, U.S.

Figure 2.2. Barred tiger salamander, mature adult, terrestrial phase

Figure 2.3. Neolonic Barred tiger salamander

Figure 3.1 Sampling

Figure 3.2 Sampling II

Figure 3.3. Containment vessels

Figure 3.4 Observing and recording

Figure 3.5 Flow chart of study progress

Figure 3.6 Topographic maps of study area

CHAPTER I

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 such 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. Not only are these amphibious creatures being adversely affected by the reduction of wetlands due to commercial development but through the unchecked use of pesticides, the introduction of exotic predators, and industrial pollutants causing excessive ultraviolet light bombardments through the thinning of the ozone layer.

As amphibians play an important role in ecosystem dynamics it is crucial to gain an understanding as to how these creatures can shed light on environmental factors affecting the environment and mankind in general. Reasons for of the species might well provide answers indicating ways conservationists and environmentalists might be able to improve the environment, especially the wetlands of the Texas Panhandle, and avoid without further disruption. To this end, research is necessary in terms of setting regulatory standards necessary 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 crucial and an important step in the preservation of the wetlands as well as in safeguarding one of its natural creatures.

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 dweller has been the tiger salamander. This particular species has afforded investigators opportunities to determine whether or not wetland pollutants are producing adverse effects on selected developmental characteristics of the salamander, namely, on 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 the contaminants on that species (and to potentially extrapolate the results to other species), as well as to ascertain or develop corrective procedures to eliminate the environmental contaminants.

Purpose of Study and Problem Statement

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 effect of man and the environment on the aquatic inhabitant. 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 for suitable use 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 more extensively explored: To what extent did pre-selected water contaminants, effluent discharges, chemicals, and pollutants, influence the Barred tiger salamander in six various aquatic environments? The recorded results would supply data with references to observed and measured differences in the growth patterns of those amphibians exposed to the contaminants compared to those who were not exposed. Net results would also relate the degree to which the bioaccumulation of the contaminants (COC) existing within the food chain potentially affects the entire habitat of the Texas Panhandle ecosystem. Through exposure to, or directly as a result of, the characteristics of the food chain, potential changes in the gestation, birth rate, growth rate and general development of the Barred tiger salamander were observed and recordedtigersalamander.

For the 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 Waste Water #1 aquatic environment.

IW2: Industrial Waste Water #2 aquatic environment.

The dependent variable are abbreviated also, as shown below:

L: Body length of Barred tiger salamander

GL: Gill length of Barred tiger salamander.

W: Weight of Barred tiger salamander.

Hypotheses

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 ?

2. There is no statistically significant effect of POTW water on the length, weight, and gill length of the Barred tiger salamander at the ?

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 ?

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 ?

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 ?

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 ?

In addition to the null hypotheses listed 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 ?

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 Lake Cove water environments. Ho12: PL = LC

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 exist 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

Definition of Terms

Throughout this report the following list of terms and concepts are used most extensively; therefore, in order to avoid any misunderstanding, they are operationally defined as follows:

Barred tiger salamander: A small lizard-like amphibian. For the study the Barred tiger salamander (Ambystoma tigrinum) was selected due to the amphibians abundance of number and its 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. (33CFR § 323.2, and 40 CFR § 230.3, The Environmental Law and Compliance Handbook by James F. Berry, J.D., Ph.D., Mark S. Dennison, J.D.) 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 the water, which has the effect of rendering water toxic or otherwise harmful (The Condensed Chemical Dictionary, 9th edition, 1977, Gessner G. Hawley, Van Nostrand Reinhold, Company).

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

Weight Measurement: Measurement of the mass in grams of the 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 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: Landbased stage of the Barred tiger salamander. Usually the salamander will undergo metamorphosis, become terrestrial and develop to a mature adult living outside the aquatic habitat.

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

"t" Test: Statistical test used for statistical comparison of two means to determine whether or not they are identical. Two sets of data may: one from a new experiment and one for control; two results of split samples from two laboratories, or; a new analytical and existing analytical method. (Lee, C.C., editor in chief; Shun, Dar Lin, associate editor; 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, John L., How to Think About Statistics, 6th Edition, W.H. Freeman and Company, New York, 2001).

Dependent Variable: A variable whose values are determined by those of the independent variables or measurement. (Phillips, John L., How to Think About Statistics, 6th Edition, W.H. Freeman and Company, New York, 2001).

Statistical Significance: Experimental results that are not likely to have occurred by chance alone: Results that are unlikely to have occurred by chance. (Koosis, Donald J., Statistics A Self-Teaching Guide, fourth edition, John Wiley & Sons, Inc., 1997).

Rationale for the Study of the Selected Species

Regardless of the research investigative situation, both the independent variable(s) and the dependent variable(s) are chosen according to a pre-determined set of criteria. The investigator of this study established the following selection criteria when choosing the necessary sample group of Barred tiger salamanders:

1. Taxonomic soundness and ease of recognition for the purpose of 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 wherein there exists shared trait commonalties rather than shared relationships. For example, all salamanders share a relationship within six identifiable types of salamanders, yet each grouping has its own identifiable trait catalogue. The chosen species for the present study was the Barred tiger salamander rather than other salamander species because the Barred tiger salamander resides in the Texas Panhandle, although its zoological relatives live in other land areas through out the United States. With reference to the Barred tiger salamander's cosmopolitan distribution, only those Barred tiger salamanders aquatic-bound were studied -- not Barred tiger salamanders that are terrestrial inhabitants. This was necessary as the primary purpose was to investigate the effects on, and differences between, aquatic environments on developmental indices of the Barred tiger salamander.

Any investigative study, whether of mammals or other fauna, sedimentary, igneous or metamorphic rock formations, or flora, must provide the investigator a sufficient number of units to be observed, evaluated, and assessed. The Barred tiger salamander provided a sufficient number at this time in the ecological cycle. Had there existed only a selected few tiger units available for investigation, the study would have been greatly limited as to scope and inferential ability. Further, knowing that an acceptable life span of a Barred tiger salamander is upwards to 20 years provided the investigator with more than sufficient time to record any changes aquatic pollutants had on the organism. In addition, knowing the Barred tiger salamander can survive in a controlled environment, one even pet-oriented, provided additional assurances that the Barred tiger salamander could exist in a treatment situation (independent variable effect) without immediate demise.

Necessary to all well-controlled investigative studies is the need for low subject variability. When increased variability occurs there exists the possibility of error control or bias when interpreting study results; thus contaminating the results of the research endeavors. Barred tiger salamanders of the Texas Panhandle, by having limited mobility as well as low genetic and ecological variability, are therefore self-controlled in terms of possible error due to extraneous influences that cannot be controlled or measured1.

Although the Barred tiger salamander has been used in the past in environmental exposure studies for pesticides, herbicides, and ecological risk assessments, there exists limited use of the aquatic species in continued research for contaminant accumulation and its effects2. Amphibians are considered to be extremely sensitive to ecosystem changes: Therefore, the health of the salamander is expected to mirror the health of the inhabitants as an ecological population. Using the Barred tiger salamander as an indicator species would, therefore, provide a study organism that is sensitive to environmental change so that developmental changes or alterations could be recorded and assessed. Even with naturally occurring anomalies, such as droughts, floods_, and dust storms, the species' population numbers are sufficient enough to permit a determination of any correlation to contaminants other than "normal" variations.

CHAPTER II

PHYSIOLOGY, MORPHOLOGY, and RANGE

Taxonomic Hierarchy

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).

Range. The Barred tiger salamander, Ambystoma tigrinum, ranges from Nebraska to the Mexican Plateau (Miller, 2000). They can also be found in prairie and forested areas in which moisture is adequate (Collins, 1982). Presented in Figure 2.1 is a cartographic representation of the Barred tiger salamander's inhabited area.

INSERT FIGURE 2.1 HERE

Description. 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 and bars or blotches on a dark background. The body is robust, with 14 or fewer vertical grooves on each side of the body between the front and hind limbs. The color of the belly of this particular salamander 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 of the body. These markings might or might not travel down and extend on to the belly. Generally the belly is black or gray-black and usually mottled with yellow. Two tubercles are present on the sole of each foot. There are no paratoid glands 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 length. The head is round and short with a pronounced snout. The eyes are small, bulging, and widely spaced (Collins, 1982). A mature adult terrestrial form of the Barred tiger salamander can be seen in Figure 2.2.

INSERT FIGURE 2.2 HERE

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. Thus the Barred tiger salamander also uses it skin as an oxygen receptor source in addition to its saclike lungs. Because of the Barred tiger salamander's somewhat unique makeup, examination of the developmental characteristics may well provide information concerning another route of entry for surface water contamination (Bishop, 1967; Larson, McDonald, Fivissani, Newton, & Hamilton, 1998).

After the egg hatches, the Barred tiger salamander goes through an aquatic larval stage prior to becoming an adult. While in the larval reproductive 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 of this species are adapted to be pond inhabitants and consequently have certain larval characteristics such as external gills and a much wider fin along the tail which permits balance; they lack the two small glands on the side of the head for balance possessed by larvae of other salamander species. With gills and an extra long tail, the larvae can easily navigate through water. Further, the neotonone can stop the metamorphosis process in the larval (aquatic, neotenic) stage when it becomes beneficial to the species to remain aquatic bound, or the species can metamorphose to the terrestrial stage when this autonomy is more favored (Bishop, 1967). Figure 2.3 provides an example of the neotenic form of the Barred tiger salamander with the pronounced gills and tail fin.

INSERT FIGURE 2.3 HERE

Reproduction. The Barred tiger salamander, with moist skin to protect the body in dry climatic situations or to regulate the effects of salt on the body in aquatic environs, is one of the largest representations of a group known as mole salamanders. Reclusive throughout the majority of its adult life, this particular salamander species mates from November to June, depending upon moisture and temperature conditions. Even when adults congregate in ponds, springs, stock tanks, lakes, or flooded quarries, sexing 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 push the female to follow, a process in which she will pick up his deposited spermatophore. Within one or two days, the female lays the eggs, but neither adult will attend to the egg cluster. The period of egg development appears to be the same for both terrestrial and neotenic mature forms (reference here) (?). Depending on the water temperature, the eggs will hatch in three to five weeks (Bishop, 1967; Collins, 1982).

CHAPTER III

METHOD OF STUDY

Sampling

The sample group, in an attempt to reduce actual and extraneous sampling error, was selected on the basis of the following: non-dry climatic conditions; from a common water source (Playa Lake); not currently living in a contaminated area, and; having approximately the same overall weight and length, including gill length. The sampling location (Randall County) 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 0.25m to 1.75m over a desirable area or approximately 950m2. Using a 6m manually drawn pocket seine, 9 to 25 neotenic Barred tiger salamanders were captured in each pass through the sample location 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. Figures 3.1 and 3.2 show the process for which the salamanders were captured for the study. Three 18.92-liter containers provided temporary storage in route to an appropriate 3.79 hl holding tank filled with lake water.

INSERT FIGURE 3.1 HERE

INSERT FIGURE 3.2 HERE

The container in the foreground was used for transporting the salamanders to the large holding tank for sorting into the sub-groups

Controlled Environment Sources. Once the sample was safely transported to the base holding tank, six contaminant cages measuring 61 cm x 123 cm x 183 cm were constructed of 2 cm PVC pipes overlaid with 2.5 cm mesh chicken wire. The wire was installed in order to contain the populations within varied aquatic environments. Upon introducing the salamanders to their respective cages, additional containment modifications were necessary to ensure total species restraint. To this end, minnow seines with a .6 cm gage were installed beneath the chicken wire. By adding additional restraints to the containment system, sufficient space and adequate aquatic flow were gained, allowing for unrestricted exposure to the various types of aquatic environments being studied. Twenty-five salamanders were placed in each of the six containment cages in which the potential exposure to contaminants would be observed. Figure 3.3 provides an illustration of the construction of the containment cages while Figure 3.4 shows placement of the salamanders into the cages.

INSERT FIGURE 3.3 HERE

INSERT FIGURE 3.4 HERE

During the base location containment period, all salamanders were regularly observed and information was properly recorded with respect to each sample unit's length, measured from snout tip to tail tip: Lengths of 9.7 cm to a maximum of 14.5 cm were recorded. The overall length, gill length in terms of centimeters, and weight in grams were also recorded for each unit. Weekly observations were conducted on each individual cage by lifting the containment unit up from the water source: this also allowed the researcher to continually check the health and safety of the captive salamanders. These weekly observations were conducted and recorded with respect to sample numbers, signs of metamorphosis, and the general condition of individual salamanders with respect to possible injury, dryness of skin, and having bright and clear eyes. In addition, during the weekly inspections, each group of contained salamanders was provided enough dog food to provide additional nutrients sufficient to reduce, as well as possible, the indices of cannibalism amongst the caged salamanders. In addition, because salamanders are extremely sensitive to salts and oils contained in human flesh, handling was kept to a bare minimum. Further, in the event that any one salamander required being handled by the researcher to permit closer inspection, the salamander's usefulness to the study was not compromised; in these cases, the salamander was carefully supported by two gloved hands to avoid human contact saline contamination,, and a possible fall, which could result in injury.

Because the containment facilities were located out-of-doors, frequent checking was conducted to ensure that the holding facilities were not conducive to predator accessibility, that prudent and acceptable water depths were maintained, and that each cage continued to be structurally sound. Further, during the weekly observations, data was recorded with respect to favorable aquatic conditions, and those conditions were corrected if necessary. Regular measurements included eight weekly sets including pH level, conductivity, and temperature. As a precautionary measure, three water sample sets were submitted to an independent laboratory for additional and supportive analysis with respect to proper and acceptable element composition. The method for analysis employed for this study was that supported by the U.S. Environmental Protection Agency (environmental monitoring compliance regulation number 600/4-79-020 -- Methods for Chemical Analysis of Waste). The supplementary analysis included 200.7 (metal containment -- trace element analysis), 130 (hardness -- colorimetric/titrimetric analysis), 314A, 415 (organic carbon -- combustion/UV promoted analysis), 300.0 (inorganic anions - ion chromatography analysis), 150.1 (pH -- electrometric analysis), 120.1 (conductance -- specific conductance analysis), 160.1 (residue -- filterable/gravimetric analysis), and 310 (alkalinity -- titrimetric/colormetric analysis).

Upon the termination of the field observation and record-keeping tasks, randomly selected salamanders were preserved for body burden analysis and for specific influences per Toxic Characteristics Leaching Properties (TCLP) metals and U.S. EPA methods 6020A (toxicological contaminants - ****** analysis (this still has to be identified or omitted), 7471A (pesticide levels - ****** analysis (ditto), (?) (hazardous waste levels - ***** analysis), and SM 18th 2540G (total, fixed and volatile solids - standard method of analysis) (ANA-Lab report 2002). (See Appendix A for Lab reports)

Once the total sample group of 214 Barred tiger salamanders was selected and properly contained, monitoring was vigilant for the150 salamanders selected randomly and assigned to six locations in groups of 25 each per location. Relocation sites of the test sub-group of salamanders were chosen on the basis of possible exposure to environmental contaminants. All locations were in Randall County, Texas, and included a Lake Cove (approximate longitude 35° 3' 19.44" North and approximate latitude 101° 46' 37.2" West); POTW effluent (Public Owned Treatment Work) (approximate longitude 35° 3' 11.0" North and approximate latitude 101° 46' 25.6" West); Playa Lake (approximate longitude 35° 5' 28.2" North and approximate latitude 101° 45' 35.9" West); and a Subsurface Earthen Stock Tank approximate longitude 35° 3' 42.7" North and approximate latitude 101° 44' 46.5" West). Sites selected in Gray County, Texas included a Wastewater-Storm Water Pond #1 (approximate longitude 35° 30' 47.0" North and approximate latitude 101° 0' 56.1" West); Wastewater-Storm Water Pond #2 (approximate longitude 35° 30' 37.5" North and approximate latitude 101° 0' 51.6" West). The reader is directed to Figures 3.5 and 3.6 as presented below for a complete cartographic overview of the six selected testing locations.

INSERT FIGURE 3.5 HERE

INSERT FIGURE 3.6 HERE

The primary purpose of statistical processes is to make order out of chaos. By properly applying selected statistical processes to the measurement data developed for the present research investigation, the author was able to determine whether or not differences in the tiger salamander developmental processes existed with respect to controlled contaminated environmental conditions. In addition, the investigator was able to determine whether or not there had been any effect on the developmental process as a result of being placed into the six various aquatic environments (independent variables). The test results were, therefore, subjected to two different parametric statistical processes, namely, the "t" Test and the Analysis of Variance (ANOVA). Modifications were made to the ANOVA to accommodate the unequal cell frequencies (i.e., due to some loss of sample units), 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 x 3 design and the probability level was set at ?

Since an analysis of variance only has the ability to determine whether or not statistical significance occurred within group comparisons, a modified orthogonal analysis would be necessary to determine wherein the differences exist. Should main effects be non-significant and one of the interaction counterparts significant, a test from simple effects would be employed to determine the whereabouts of the interaction. (NOTE: I cannot find any informational data with respect to the overall ANOVA, i.e., 3x6. If this has not been run it will take no time for you to do so and insert the findings at the very beginning of the Results section. All the other analyses are nicely done but should be based, for discussion purposes, on an overall ANOVA. ()NOTE: I did not have this information, and could not run it -- even if we could run it, which we can't -- without the original statistics. However, if you will provide it, I will be happy to put it into the appropriate place.

In addition to the ANOVA analysis, "t" Tests were run to determine whether or not any statistically significant differences in component variability from location to location existed. Further, descriptive statistics were used to assist the reader in viewing the variability of factors between and amongst the groups. However, the reader is cautioned not to draw premature conclusions from these particular reported statistics as they are for general informational purposes only and their numerical values have been imported to the "t" Test and ANOVA statistical processes for further analysis.

Measurement Data Reliability and Validity

The measurement data was in ratio form wherein absolute measurement values were received in terms of the measurement having an absolute zero value. (NOTE: I don't understand this. Can you clarify?) As such all the selected statistical analysis procedures were permitted. In addition, as ratio data was the selected type of data construct and predictive validity were absolutely assured as well. (NOTE: Ditto.) By having both ratio measurement data and construct and predictive measurement validity, the amount of measurement error becomes insignificant except when human error might possibly, and without notice, affect the results of the present situation. However, all precautions were taken to ensure that human error was kept at an absolute minimum.

CHAPTER IV

RESULTS

In review, and as stated in Chapter III, developmental progress of the salamander sample was observed via placement in six pre-assigned wetland (aqueous) locations (See Table 2, 2a). In addition to recording weekly physical measurements, data with respect to water temperature, pH, conductivity, and head count was also gathered. After completing the field observation program, randomly selected salamanders were obtained for burden tissue analysis. Additionally, meteorological information was collected from the National Oceanic and Atmospheric Administration (reference 15). All accumulated data for the present research investigation has been reported according to the chosen sample locations, the descriptions of which are listed below along with the sample identification:

Table 2. Locations

Sample A:

A lake cove in Randall County.

Sample B:

An effluent discharge site for a POTW facility in Randall County.

Sample C:

The playa lake of origin in Randall County.

Sample D:

A subsurface earthen stock tank in Randall County that also received the same effluent discharge as did Sample B. from the POTW facility.

Sample E:

An industrial wastewater pond in Gray County.

Sample F:

A second industrial wastewater pond in Gray County.

It is important to mention at this juncture of the research investigation that Sample E. was lost toward the end of the assessment procedure. Unfortunately, the cage containing Sample E, Industrial Wastewater 1, became deeply submerged due to excessive storm water runoff into the pond and, as a result, the salamanders contained in that sample cage escaped. The loss of Sample E, did not, however, drastically alter the results of the study; modifications to the data analysis were applied to correct any possible undue sample loss bias. Further, sufficient data had been collected prior to the loss for the sample to remain in the study as a suitable indicator organism. Table 3, presented below, is a matrix of the sample distribution from initial placement to final assessment with respect to a specimen number count.

Table 3: Beginning and Ending Sample Distribution Chart

Sample

Lake Cove

POTW

Playa Lake

Earthen Stock Tank

Industrial wastewater pond #1

Industrial wastewater pond #2

Initial Count

Final Count

Total loss

% Loss

48.0

92.0

44.0

72.0

36.0

On the basis of length, weight, and gill length, changes in four sample specimens were compared with changes in the group that remained in the Playa Lake throughout the observation period. Although one complete sample, Industrial Wastewater 1, was lost, the remaining five samples produced enough data so that comparisons could be made with respect to the study's independent and dependent variables. In addition, a nested variable, metamorphosis, was also accounted for in the data analysis. The effects of surface water contamination and its effect on the metamorphic process of the salamander were felt to be important, as metamorphic indicators would produce important information; however, The study was unable to adequately measure indicators of metamorphosis.

The first step in analyzing the data involved the construction of a matrix, which revealed the overall mean of the data with respect to length, weight, and gill length as seen in Table 4. Total length, weight, and gill length was greater for salamanders placed in the Industrial Wastewater 2 sample than for the other four selected samples and the respective means were proportional to this sampling.

Table 4: Means and Standard Deviations for Sample Type -- 5x3

Sample

Length

Mean, s.d.

Weight Mean, s.d.

Gill Length

Mean, s.d.

Lake Cove

12.43

0.8713

27.18

7.0625

2.15

0.35522

POTW

12.01

09249

22.10

5.6529

2.00

0.30941

Playa Lake

13.02

1.0039

27.29

7.5329

2.08

0.57261

Earthen Stock Tank

12.77

1.1553

27.01

7.7981

2.00

0.44654

Industrial Wastewater 2

13.44

1.8618

39.35

23.0880

2.15

0.59734

Group Mean

12.74

1.1634

28.59

8.7203

2.08

0.45622

On the basis of the calculated mean values and standard deviations, the F test and "t" test for determining significant differences and effects were used to interpret the obtained results. Thereafter, the remainder of the study tested the hypothesis that the mean values of length, weight, and gill length of the five salamander samples were equal to one another. A probability level of p

Table 5: t Values for Mean Difference Comparisons between Playa Lake and the Other Groups.

Source of Difference

Hypothesized

Mean Difference

"t" Value

Playa Lake x Lake Cove

0.00

1.981371

0.001182

Playa Lake x POTW

0.00

1.983494

8.52E-07

Playa Lake x Earthen Stock tank

0.00

1.982385

0.243098

Playa Lake x Industrial Pond #2

0.00

1.98118

0.128057

(NOTE: In the above Table include a column for critical "t" values. Also * those which meet the 0.05 level. Do the same when reporting all F. And "t" values in a table. Also insert the * and ** at the bottom of all tables reporting significance.) (Done)

On the basis of the F test and "t" Test analyses, the following results were reported with reference to differences in length, weight, and gill length between and amongst the five-recorded samples. The probability level for acceptance or rejection of the null hypothesis that differences and/or effects existed between and amongst the groups was set at p

Length. Of the "t" test values, the following mean comparisons were recorded as being statistically significant to reject the null hypothesis

1. Statistically significant results to reject the null hypothesis that there is no statistically significant effect of Lake Cove water on the length, weight and gill length of the Barred tiger salamander at the ?

2. Statistically significant results to reject the null hypothesis that there is no statistically significant effect of POTW water on the length, weight, and gill length of the Barred tiger salamander at the ?

3. Statistically non-Significant results to accept the null hypothesis that there is no statistically significant effect of Playa Lake water on the length, weight, and gill length of the Barred tiger salamander at the ?

4. Statistically non-Significant results to accept the null hypothesis 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 ?

Comparing the mean lengths of the sub-groups resulted in the Lake Cove and the POTW Effluent groups being compared with the Playa Lake group; the means and variance comparison resulted in rejection of the null hypothesis through the t-test and F-test comparisons. The variance ratios of both the Lake Cove and the POTW samples were much larger than required to accept the null hypothesis. The "t" Test verified the ANOVA, yielding similar results. These results are shown in Tables 6a and b.

Table 6a: Comparison of the Results of the ANOVA and t-Test for Length

Sample

df t-Test

t-critical

F-Test

F-critical

Playa Lake vs. Lake Cove

3.33

1.98

11.0794

3.93

Playa Lake vs. POTW Effluent

5.24

1.98

27.5052

3.93

Playa Lake vs. Earthen Stock Tank

1.17

1.98

1.38

3.93

Playa Lake vs. Wastewater Pond #2

-1.53

1.98

2.35

3.93

Table 6b: Mean value comparisons to reject null hypothesis

Location

t Stat

t Critical two-tail

Playa Lake vs. Earthen Stock Tank

1.174

1.982

Playa Lake vs. Wastewater No. 2

-1.533

1.981

Simply, the salamanders in the Wastewater No. 2 pond and the Earthen Stock Tank developed more than those in the control group in the Playa Lake. The "t" Stat test for these two groups was less than the "t" Critical two-tail test.

On the other hand, two groups' mean comparisons were recorded as being significant to accept the null hypothesis.

Table 7: Mean value comparisons to accept null hypothesis

Location

t Stat

t Critical two-tail

Playa Lake vs. Lake Cove

3.329

1.981

Playa Lake vs. POTW Effluent

5.245

1.983

Again, the salamanders in the Lake Cove and POTW Effluent ponds developed at the same rate as those in the control group in the Playa Lake. The "t" Stat test for these two groups was greater than the "t" Critical two-tail test.

NOTE: Table 8 and Table 9 from you Nov. 09 revisions is really not necessary as you have adequately supplied the needed information is a condensed form in Table 6a& b and Table 7. If you want to insert a note here that a full analysis sheet is available then place both these expanded tables in the Appendix. The same applies for Gill Length additional data (?)

Weight. Weight was an indicator of the salamanders' overall health as well as development. The following mean comparisons were recorded as being significant to reject the null hypothesis (Table 8). With the "t" Test and ANOVA in comparison of the means for the weight, the results are recorded as follows:

1. Statistically non-significant results to accept the null hypothesis there is no statistically significant effect of Lake Cove water on the length, weight and gill length of the Barred tiger salamander at the ?

2. Statistically significant results to reject the null hypothesis there is no statistically significant effect of POTW water on the length, weight, and gill length of the Barred tiger salamander at the ?

3. Statistically non-significant results to accept the null hypothesis there is no statistically significant effect of Playa Lake water on the length, weight, and gill length of the Barred tiger salamander at the ?

4. Statistically non-significant results to accept the null hypothesis for t-test, but significant for F-test 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 ?

Three of the comparisons indicated that the mean values of the group comparison resulted in equivalent weight gain during the observation period. The F-test resulted in an acceptance of the null hypothesis for the comparison of the Playa Lake and the POTW as well as the Industrial Wastewater Pond #2. The POTW Effluent group did not have sufficient weight gain during the period to result in equivalent mean and variance results. Table 8 provides comparison of the values statistically calculated to determine significant or non-significant results. Both F. values indicate a significant

(NOTE: Insert a df column for both t and F. Also * and ** those values exceeding the required

variance in the weight development of the two sub-groups. The actual Mean values indicated that the POTW Effluent group had a lower rate of weight development while the Industrial Wastewater Pond #2 has a larger weight gain. Again, the salamanders in the Lake Cove and POTW Effluent ponds developed at the same rate as those in the control group in the Playa Lake. The't Stat test for these two groups was greater than the "t" Critical two-tail test.

Table 8: Comparison of t-test and F-values for Significance Determination for Weight.

Sample

df t-stat t-critical

F-stat

F-critical

Playa Lake vs. Lake Cove

0.28

1.98

0.081

3.93

Playa Lake vs. POTW Effluent

3.89

1.98

15.13

3.93

Playa Lake vs. Earthen Stock Tank

0.19

1.98

0.036

3.93

Playa Lake vs. Wastewater Pond #2

-3.78

1.98

14.28

3.93

Note: Omit all references to "one-tailed test" because you need the two-tailed test. Make sure you check everything carefully for this through out the entire thesis in case I missed one. ( Also, nice to see that you ony report the statitically significant test result as that is all a researcher is to do

Gill Length. Like overall specie length, gill length was an important measurement of development as it gave insight into the level of metamorphosis. The following mean comparisons were recorded as being significant to reject the null hypothesis. All sets of data rejected the null hypothesis.

In comparing the gill length with the "t'Test and the ANOVA for the means and variance the following results between the Playa Lake and the other groups are as follows:

1. Statistically non-significant results to accept the null hypothesis that 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. Statistically non-significant results to accept the null hypothesis that 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. Ho12: PL = LC

3. Statistically non-significant results to accept the null hypothesis that 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. Statistically non-significant results accept the null hypothesis that 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

The results of the t-stat values as well as the F-values were all below the critical values for both the "t" Test and the ANOVA. All the gill length measurement data calculated as non-significant, leading to accepting the null hypothesis as the means and variances being equivalent. Table 9 provides a comparison of the values statistically calculated.

Table 9: Comparison of Values for the Gill Length

Sample

df t-stat t-critical

F-stat

F-critical

Playa Lake vs. Lake Cove

-0.77

1.98

0.14

3.93

Playa Lake vs. POTW Effluent

0.79

1.98

1.78

3.93

Playa Lake vs. Earthen Stock Tank

0.84

1.98

1.75

3.93

Playa Lake vs. Wastewater Pond #2

-0.59

1.98

0.07

3.93

(NOTE: Insert a df column for both t and F. Also * and ** those values exceeding the required

The gill length did not have the variance as seen in the overall length and weight during the development of the salamanders.

Water Measurements. In performing a statistical analysis of the water data in order to examine for any variability in the composition between the locations, it was found that among all the components analyzed, none of the comparisons with the Playa Lake water were significant to the t-test analysis. With the null hypothesis being that all means are equal, acceptance of the null hypothesis based on the "t" Test indicates that there was no statistically significant difference in the components from location to location. Table 10 provides a comparison of the t-stat values with the t-critical values.

Table 10: Comparison of t Test Values for Water Analysis Data

Sample

Cond

TDS

Ca++

Mg++

Hard

Cl-

NO2_

PO4

t-crit

PL vs. LC

-11.47

-3.43

-0.86

-1.72

-107

-7.18

-7.88

-7.50

0.21

4.30

PL vs. POTW

-1.14

-3.41

-0.78

-2.03

-15.8

-1.35

-10.7

-12.5

-5.38

4.30

PL vs. EST

-21.47

-0.47

-1.32

-0.15

-3.47

-2.35

-14.8

-12.5

1.20

4.30

PL vs. IWW

-0.05

-1.62

-2.49

-4.25

-4.93

-7.60

-7.68

-1.52

1.19

4.30

In reviewing the actual analytical results, it was initially felt that the higher concentrations of NO2 and PO4 might be contributing factors in the differences in the POTW Effluent group and the Lake Cove group. Both were significant in rejection of the null hypothesis for length, while the POTW group also rejected for weight. . (Done)

Body Burden Analysis. In a final investigation of the salamanders, two individuals from each sub-group were randomly selected for performing body burden analysis for heavy metals. The accumulation of contaminants in surface water should be reflected in the tissues of the salamander as with other biomarkers. The results of the analysis are reported in Table 11. The table shows that there are positive indications of some metals accumulated within the tissues of the individuals; however, none of the chlorinated compounds or pesticide compounds expected were detected. The assumption is that there was no exposure to or use of these compounds in the areas of the test locations.

Table 11: Analytical Results of the Body Burden Analysis for Metals

and Pesticides. All data is Dry Weight Basis.

Unit

Result

Lake

Cove

POTW

Effluent

Playa

Lake

Earthen

Stock Tank

Indust.

WW #2

Total As

mg/kg

8.31

10.3

12.8

6.38

14.2

Total Ba

mg/kg

34.9

55.5

44.6

35.3

13.3

Total Cd

mg/kg

0.841

1.37

0.787

Total Cr

mg/kg

16.6

18.5

13.4

10.9

17.1

Total Pb

mg/kg

1.4

Total Se

mg/kg

3.52

Total Ag

mg/kg

Total Hg

mg/kg

0.0562

0.127

0.0989

Gamma-BHC (Lindane)

ug/kg

Alpha-BHC (hexachlorocyclohexane)

ug/kg

Beta-BHC (hexachlorocyclohexane)

ug/kg

Delta-BHC (hexachlorocyclohexane)

ug/kg

Chlordane

ug/kg

4,4-DDD

ug/kg

4,4-DDE

ug/kg

4,4-DDT

ug/kg

Dieldrin

ug/kg

Endosulfan I (alpha)

ug/kg

Endosulfan II (beta)

ug/kg

Endosulfan sulfate

ug/kg

Endrin

ug/kg

Endrin aldehyde

ug/kg

Heptachlor

ug/kg

Heptachlor epoxide ug/kg

Methoxychlor

ug/kg

Toxaphene

ug/kg

Total Solids

16%

14.5%

15.2%

17.4%

15.5%

Aldrin

ug/kg

The origination of the metals can only be assumed, but the fact that the metals were detected indicates that the salamander is sensitive to the accumulation of pollutants in the aqueous environments. NOTE: This statement belongs in the discussion section. .. NOTE: This statement belongs in the discussion section. (Done)

Additional investigation of the organism in relation to these specific exposures could yield conclusive evidence concerning these substances in the sample locations. However, this study was not designed to produce that determination, but rather was designed to test the sensitivity of and determine the usefulness of the Barred tiger salamander as a biomarker for water contamination and pollutants.

NOTE: Overall you did a fine job here. Congratulations! (

CHAPTER V

DISCUSSION AND CONCLUSION

As man in the twenty first century attempts to reach the far corners of the universe, create yet another mega-corporate merger, or topple a heinous dictator, little is found on the newspaper's front page about the international citizen's plight to sustain humanity and maintain a life-giving environment. Putting natural disasters aside, man's communion and co-existence with nature is becoming more difficult as the decades pass. With the world's population set to increase by two billion more people by the year 2022 (U.S. Census Bureau, 2003), many natural environmental wonders or marvels known today will likely no longer exist. The advent of a continued population increase is not solely a phenomenon of concern to impoverished third-world populations, but a wake-up call to the industrialized world as well. As the population increases, the demand for foodstuffs and habitable locations will increase at the very moment that many natural and pristine environmental areas are "re-purposed" to provide sustenance for the body rather than the soul. A movie produced more than three decades ago -- Soylent Green directed by Richard Fleischer (1973) and based on a novel by Harry Harrison, Make Room! Make Room! (1966) -- is a surrealist narrative of what can and will happen if sustainable development is not practiced. It offers a chilling snapshot of the results if the world's natural, unencumbered topographical resources -- namely those of the wetland, tropical forest, and everglade category -- are lost.

Several factors must be evaluated to ascertain, not to mention maintain, the health of an ecosystem, especially a wetland ecosystem. The most significant factors include ecological indicators such as human induced stresses, as well as water quality, sediment quality and transport, hydrodynamics, biological conditions, and specie assemblage and relative abundance, to name but a few. Maintaining the integrity of a wetland ecosystem is an arduous task. As ecosystems are very complicated interrelated communities of many living organisms, the physical environment within which they all interconnect is a vital component to everyone's well being.

Sustaining or restoring natural systems must be goal-driven and based on a collaboratively developed vision that man and the animal and vegetable kingdoms can live in harmony and benefit one another. In order to be proactive in the preservation of our ecosystem's, health and well-being, man must be ever vigilant in maintaining the natural order of the ecosystem and avoiding contamination of natural areas. To this end, this research study was designed and executed.

The major purpose of the study was to investigate the relationship between the ecosystem health of the Texas Panhandle Wetlands and the Barred tiger salamander. Emphasis was also placed on specific differences found in location-specific wetland areas and the effects various contaminants might or might not have had on the physical development of the Barred tiger salamander in one or more of those areas. Speculation assumes that the nitrite and phosphate may have contributed to the reduced development of the two groups, but specific evidence cannot be provided with this study.

The specific dependent variables under investigation were the length, weight, gill length, and metamorphosis of the Barred tiger salamander with respect to pre-selected wetland contaminants. The following hypotheses were provided in null form to predict statistically non-significant differences and interactions between and amongst the independent variables:

1) There is no statistically significant difference in the amount of length, weight, gill length, and metamorphic change with respect to Barred tiger salamanders coming from the Lake Cove, POTW, Playa Lake, Earthen Stock, Industrial Wastewater #1, and Industrial Wastewater #2 areas.

2) 2) There is no statistically significant difference in length, weight, gill length, and metamorphosis with respect for contaminants pH, Conductance, Total Dissolved Solids, Calcium, Magnesium, Hardness, Chlorides, Nitrites, and Phosphates within and between the six-wetland placement areas.

3) 3) Interaction of the contaminant effects are statistically non-significant with respect to length, weight, gill length, and metamorphosis of Barred tiger salamanders from the six containment areas.

For the present comparative study, 150 Barred tiger salamanders were randomly selected from a sample population of 214 Barred tiger salamanders. A 25-unit sample was placed in one of six wetland areas, namely: Lake Cove, PTOW Effluent, Playa Lake of origin, Earthen Stock, Industrial Wastewater #1, and Industrial Wastewater #2.

All Barred tiger salamander samples were randomly chosen from a larger group to which certain restrictions were applied for length; weight and gill length was ultimately measured, but not for selection purposes. Only neotenic forms of the Barred tiger salamanders were selected in order to observe and gather data concerning potential effects of the environments on development. All individuals appeared to be vibrant, normally developed, and with no evidence of being lethargic when they were selected. All Barred tiger salamanders selected for the study appeared to have been born during the early to middle spring and were not in the larval form; all possessed fully developed limbs and gills and had no visible developmental defects. Each Barred tiger salamander was handled very gently with two hands to prevent any threat of harm from dropping or from excessive physical restraint that could result in injury.

Prior to placement of each sample unit in the six designated wetland units, the entire sample was observed for a period of 48 hours to ensure that the entire sample group was viable and did not exhibit potential containment anxieties or threats to each other. Once the six sample units were properly placed in the six designated wetland areas, the units were observed initially after 24 hours, then weekly for eight weeks to record attrition, signs of cannibalism, general condition of the salamanders, signs of metamorphosis, and the physical characteristics of the aquatic environments for assurance that there was not any detriment to the individuals; data was also recorded with minimal contact. Because, as noted earlier, one sample (Industrial Wastewater #1) escaped the submerged container,, the final statistical analysis was conducted on the remaining five locations.

The use of amphibians (Barred tiger salamander) as a front line of possible contaminants to the environment has long been employed with respect to ecosystem health. By identifying amphibian exposure pathways, environmentalists will be able to identify those exposures that are having a seriously negative impact on the health of the wetland ecosystem. As salamanders make up a great portion of wetland biomass, they are, therefore, excellent indicators of a wetland's ecological integrity. Being indigenous to the area, as well as available in sufficient in numbers, the Barred tiger salamander is an excellent choice for field and laboratory research. The species itself is easily recognized and characterized, and physical characteristics are stable within and throughout the population, thereby affording researchers a standard species from which to draw samples. As the Barred tiger salamander has a skin that permits it to breathe underwater, it is very susceptible to air and water pollution that makes it an excellent indicator of wetland health. In addition, this particular species, the Barred tiger salamander, is highly susceptible to change with respect to the effects of commercial collecting, invasive species, pesticide usage, acid rain, and global warming. Focusing on the synergistic effects of anthropogenic disturbances on the Barred tiger salamander of the Texas Wetland Panhandle was of primary importance to this research study. Even though Barred tiger salamanders have previously been used in ecological studies and assessments, those studies have oftentimes been limited, as samples were often observed and analyzed without knowledge of length of contaminant exposure. This study strove to obviate that lack of control and to develop replicable results.

Thirty-two statistical analyses were derived from the hypothesis that changes in length, weight, and gill length of the Barred tiger salamander would be occur and be observable a result of eleven identifiable contaminants. In addition, the theory hypothesized that the dependent variables under investigation would be not be dissimilar with respect to the six (reduced to five) Texas Panhandle Wetland areas.

Hypothesis one predicted that the length between the means and variances of the groups when compared to the group of the Playa Lake were significant for the Lake Cove and the POTW Effluent groups, rejecting the null hypothesis that the means and variances were equal. Both these groups did not develop in an equivalent as the Playa Lake group as well as did the Earthen Stock Tank and Industrial Waste Water #2 groups. (NOTE: This is confusing. Can you explain more clearly?) The latter two results being non-significant, the null hypothesis that the means and variances are equal was maintained.

Hypothesis two predicted that the weight between the means and variances of the groups would be equivalent compared to the Playa Lake group. The weight comparisons yielded only one group in comparison that was significant. In comparing with the Playa Lake group, the POTW Effluent group was significantly below an equivalent weight gain, thereby rejecting the null hypothesis. The other three comparisons showed equal or greater weight gain than the Playa Lake group, thus accepting the null hypothesis.

Of the metals listed in the analytical results, barium is recognized as having a slightly elevated concentration throughout the region. Again, the concentrations seemed to be slightly higher except for arsenic in the POTW Effluent. In addition, lead was found, as well as mercury

In conjunction with the nitrite and phosphate levels these may have contributed to the lack of length and weight development in the POTW group as well.

Hypothesis three predicted the gill length of all three groups being equivalent. The results from both the "t" Test and ANOVA were all non-significant, indicating that the gill length may not be an indicator as to the development or inhibited development of the species in contaminated environments.

It is suggested that additional investigation be performed to assure the hypothesis is true. Additional water analysis may need to be implemented to examine for components that were not feasible for this study.

With the reduced development being seen in the POTW Effluent group, and in the length of the Lake Cove group, additional study may be needed for these areas as well. Although the water analysis produced higher nitrite and phosphate concentrations in the effluent a direct correlation between the small size and lower weight for that particular group could not be verified. A specific conclusion linking the two compounds with the salamanders' reduced development could not be conclusively supported. This study concerned the use of the Barred tiger salamander as a viable indicator species; it did not address any increase of morphology rates nor investigate why the groups did better or worse compared to the Playa Lake group.

As an indicator species, it appears the Barred tiger salamander would be a viable organism to determine the presence of pollution in the aquatic habitats of the Texas Panhandle. Although the specific reasons for length and weight differences are not determined in this, the organism indicated a variance from location to location. Salamander. With the species having a large population that covers the continental Midwest, the Barred tiger salamander can be a bio-indicator organism that would be most suitable for use throughout the Texas Panhandle region in determining potential water contamination, and in other regions in their habitat range, as well.

Study Limitations and Sources of Possible Error

As in any study of organisms in an environment, it is not possible to test for all possible parameters, not to investigate all the effects of every possible combination and permutation of factors. In the case of this study of the Barred tiger salamander and the effects of various wetlands environments on it, with the intention of ascertaining the salamander's bio-indicator suitability, several factors were necessarily eliminated from consideration. In effect, because the main intention of the study was to determine whether changes in the salamander were sufficiently significant to permit its use as a bio-indicator, the specific causes of specific changes was thought to be beyond the scope of the study.

In addition, the Barred tiger salamander was the only organism considered for this study, both because of its prevalence and hardiness. In addition, it had been used previously in other more limited studies -- studies that did not impose the environmental controls included in the current study -- so that it was already clear that the organism was relatively convenient to work with. While there may be other organisms that offer significant potential as bio-indicators, the dual nature of the Barred tiger salamander's acquisition of oxygen, though the respiratory system and the skin, make it potentially more useful for fine-tuning any investigation of environmental effects. On the other hand, there may be researchers who view this as a detriment; they may insist on separating pollutant acquisition by gill and by exterior membranes.

Seasonality was also not considered here; it may be that the Barred tiger salamander acquires pollutants and exhibit physical changes at different rates depending on the season, a factor that could not be assessed in the limited time frame of the current study, or without some means of correlating four or more different populations collected for four seasonal studies.

Implications for Future Research

Some of the limitations of this study are indicators of additional research that could prove valuable to those who need bio-indicators to assess various environmental effects. For example, individual studies based on the prevalence of various metals (mercury, lead, etc.) of particular environmental concern and assumed impact could be conducted using the Barred tiger salamander, which was shown, in this study, to be sensitive to those metals. Other metals and compounds commonly found in wetland areas affected by human populations might also be studied specifically over time, using changes in the Barred tiger salamander, to indicate increasing or decreasing pollutant severity and to extrapolate likely effects on other zoological populations and on humans.

In addition, the consequences of the pollutants under study in Barred tiger salamanders might be useful to medical/environmental research; a study of the magnitude of changes, vis-a-vis the amount and character of one or more compounds, might form a useful study.

Do not forget to include the two Appendices if you decide to do so. (?)

Do not place anything in Bold Letters (DONE)

Do not forget to fill in those areas I have placed in bold lettering. (As much as possible; please check)

Make sure all tables are properly placed on the pages -- and center all tables. (May change in next round)

Make sure that the beginning of each chapter is 5 lines down from the top of the page. (Save for final round.)

Please not that I had to omit some of the Figure listings, as I could not upload the thesis to the board due to it being too large. Simply insert the figures back into your thesis where I have indicated. (Tof C. dealt with, preliminarily.)

Overall Nice job!! (

Regards,

Dr. O.

ABSTRACT

Table of Contents

Chapter One: Introduction

Purpose of study and problem statement

Hypotheses

Definition of terms

Rationale for the study of the selected species

Chapter Two: Physiology, morphology and range

Taxonomic Hierarchy

Chapter Three: Method of study

Sampling

Measurement data reliability and validity

Chapter Four: Results

Chapter Five: Discussion and conclusion

Study Limitations and Sources of Possible Error

Implications for Future Research

Table 1 (?)

Table 2: Locations

Table 3: Beginning and Ending Sample Distribution Chart

Table 4: Means and Standard Deviations for Sample Type -- 5x3

Table 5: t Values for Mean Difference Comparisons between Playa Lake and the Other Groups.

Table 6a: Comparison of the Results of the ANOVA and t-Test for Length

Table 6b: Mean value comparisons to reject null hypothesis

Table 7: Mean value comparisons to accept null hypothesis

Table 8: Comparison of t-test and F-values for Significance Determination for Weight.

Table 9: Comparison of Values for the Gill Length

Table 10: Comparison of t Test Values for Water Analysis Data

Table 11: Analytical Results of the Body Burden Analysis for Metals

and Pesticides. All data is Dry Weight Basis.

List of figures

Figure 2.1. Barred tiger salamander's inhabited territory, U.S.

Figure 2.2. Barred tiger salamander, mature adult, terrestrial phase

Figure 2.3. Neolonic Barred tiger salamander

Figure 3.1 Sampling

Figure 3.2 Sampling II

Figure 3.3. Containment vessels

Figure 3.4 Observing and recording

Figure 3.5 Flow chart of study progress

Figure 3.6 Topographic maps of study area

CHAPTER I

INTRODUCTION

Purpose of Study and Problem Statement

For the 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 Waste Water #1 aquatic environment.

IW2: Industrial Waste Water #2 aquatic environment.

The dependent variable are abbreviated also, as shown below:

L: Body length of Barred tiger salamander

GL: Gill length of Barred tiger salamander.

W: Weight of Barred tiger salamander.

Hypotheses

7. There is no statistically significant effect of Lake Cove water on the length, weight and gill length of the Barred tiger salamander at the ?

8. There is no statistically significant effect of POTW water on the length, weight, and gill length of the Barred tiger salamander at the ?

9. There is no statistically significant effect of Playa Lake water on the length, weight, and gill length of the Barred tiger salamander at the ?

10. 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 ?

11. There is no statistically significant effect of Industrial Wastewater #1 on the length, weight, and gill length of the Barred tiger salamander at the ?

12. There is no statistically significant effect of Industrial Wastewater #2 on the length, weight, and gill length of the Barred tiger salamander at the ?

6. 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

7. 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. Ho12: PL = LC

8. 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

9. 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

10. There exist 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

Definition of Terms

Throughout this report the following list of terms and concepts are used most extensively; therefore, in order to avoid any misunderstanding, they are operationally defined as follows:

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

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

Terrestrial: Landbased stage of the Barred tiger salamander. Usually the salamander will undergo metamorphosis, become terrestrial and develop to a mature adult living outside the aquatic habitat.

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

Rationale for the Study of the Selected Species

9. Taxonomic soundness and ease of recognition for the purpose of accurate sample selection.

10. Cosmopolitan distribution of the sample.

11. Numerical abundance of the sample.

12. Low genetic and ecological variability of the sample.

13. Large body size of the sample

14. Long life history and mobility of the sample.

15. Auto-ecological awareness by the sample.

16. Laboratory tolerance by the sample.

CHAPTER II

PHYSIOLOGY, MORPHOLOGY, and RANGE

Taxonomic Hierarchy

Kingdom: ANIMALIA

Phylum: CHORDATA

Subclass: LISSAMPHIBIA

Order: CAUDATA

Family: AMYSTOMATIDAE

Genus: AMBYSTOMA

Species: Ambystoma Tigrinum

Subspecies: Ambystoma Tigrinum Mavortium (Baird, 1850)

INSERT FIGURE 2.1 HERE

CHAPTER III

METHOD OF STUDY

Sampling

Figure 3.2 Sampling

Figure 3.2. Sampling II

The container in the foreground was used for transporting the salamanders to the large holding tank for sorting into the sub-groups

Figure 3.3. Containment vessels

Figure 3.4 Observing and recording

Figure 3. Flow chart of study progress

Figure 3.6. Topographic maps of study area

Measurement Data Reliability and Validity

CHAPTER IV

RESULTS

Table 2. Locations

Sample A:

A lake cove in Randall County.

Sample B:

An effluent discharge site for a POTW facility in Randall County.

Sample C:

The playa lake of origin in Randall County.

Sample D:

A subsurface earthen stock tank in Randall County that also received the same effluent discharge as did Sample B. from the POTW facility.

Sample E:

An industrial wastewater pond in Gray County.

Sample F:

A second industrial wastewater pond in Gray County.

Table 3: Beginning and Ending Sample Distribution Chart

Sample

Lake Cove

POTW

Playa Lake

Earthen Stock Tank

Industrial wastewater pond #1

Industrial wastewater pond #2

Initial Count

Final Count

Total loss

% Loss

48.0

92.0

44.0

72.0

36.0

Table 4: Means and Standard Deviations for Sample Type -- 5x3

Sample

Length

Mean, s.d.

Weight Mean, s.d.

Gill Length

Mean, s.d.

Lake Cove

12.43

0.8713

27.18

7.0625

2.15

0.35522

POTW

12.01

09249

22.10

5.6529

2.00

0.30941

Playa Lake

13.02

1.0039

27.29

7.5329

2.08

0.57261

Earthen Stock Tank

12.77

1.1553

27.01

7.7981

2.00

0.44654

Industrial Wastewater 2

13.44

1.8618

39.35

23.0880

2.15

0.59734

Group Mean

12.74

1.1634

28.59

8.7203

2.08

0.45622

Table 5: t Values for Mean Difference Comparisons between Playa Lake and the Other Groups.

Source of Difference

Hypothesized

Mean Difference

"t" Value

Playa Lake x Lake Cove

0.00

1.981371

0.001182

Playa Lake x POTW

0.00

1.983494

8.52E-07

Playa Lake x Earthen Stock tank

0.00

1.982385

0.243098

Playa Lake x Industrial Pond #2

0.00

1.98118

0.128057

Length. Of the "t" test values, the following mean comparisons were recorded as being statistically significant to reject the null hypothesis

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