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Doctoral Dissertations 1896 - February 2014
1-1-1984
Physiological short-term indicators of chronic stress on the dragonfly Somatochlora cingulata (de Selys) (Odonata:Anisoptera).
Manuel Correa Cruz University of Massachusetts Amherst
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Recommended Citation Cruz, Manuel Correa, "Physiological short-term indicators of chronic stress on the dragonfly Somatochlora cingulata (de Selys) (Odonata:Anisoptera)." (1984). Doctoral Dissertations 1896 - February 2014. 6211. https://scholarworks.umass.edu/dissertations_1/6211
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PHYSIOLOGICAL SHORT-TERM INDICATORS. OF CHRONIC STRESS
ON THE DRAGONFLY Somatochlora cingulata
(de Selys) (Odonata:Anisoptera)
A Dissertation Presented
By
MANUEL CORREA CRUZ
Submitted to the Graduate School of the University of Massachusetts in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
MAY 1984
Environmental Sciences (C) Manuel Correa Cruz 1984
All Rights Reserved
Dissertation Research Supported in part by Massachusetts Agricultural Experiment Station University of Massachusetts Project No. Hatch 511 PHYSIOLOGICAL SHORT-TERM INDICATORS OF CHRONIC STRESS ON THE DRAGONFLY Somatochlora cingulata (de Selys) (Odonata:Anisoptera)
A Dissertation Presented
By
MANUEL CORREA CRUZ
Approved as to style and content by:
Dr. Robert W. Walker, Member
Dr. Chih-Ming Yin, Member
Dr. Allen V. Barker, Head Department of Plant & Soil Sciences To my wife Alicia and my sons Alejandro and Javier
Money will buy a bed but not sleep; books but not brains; food but not appetite; finery but not beauty; a house but not a home; medicine but not health; luxuries but not culture; amusements but not happiness; religion but not salvation; a passport to every¬ where but heaven.
Anonymous ACKNOWLEDGEMENTS
I would like to thank Dr. Robert A. Coler, for his continued encouragement, assistance and thoughtful criticism from the early proposal stage to the final manuscript.
Special recognition and appreciation are extended to my committee members Dr. Robert W. Walker and Dr. Chih-Ming Yin for their encouragement and suggestions to my research.
To my wife, Alicia, I owe a special debt of gratitude for her unflagging understanding and encouragement during my graduate years. I wish to thank, CONICIT, for support through its scholarship program. Grateful acknowledgement is extended as well to Mr. Simon Zatyrka for his technical assistance and personal concern.
This research was supported, in part, by Massachusetts
Experiment Station. University of Massachusetts.
Project No. Hatch 511.
v ABSTRACT
PHYSIOLOGICAL SHORT-TERM INDICATORS OF CHRONIC STRESS ON THE DRAGONFLY Somatochlora cingulata (de Selys) (Odonata:Anisoptera)
(April 1984)
Manuel Correa Cruz, B.S., Instituto Universitario Pedagogico de Caracas, M.S., Instituto Oceanografico (Universidad de Oriente), Ph.D., University of Massachusetts/Amherst
Directed by Dr. Robert A. Coler
The problem of pollutant toxicity and subsequent physio¬ logical impact on aquatic organisms is often reflected in the oxygen-uptake and ammonia excretion rate either through dis¬ rupted metabolism or in the mobilization of a compensatory homeostatic mechanism. Consequently, the respiration and excretion rates provide a critical index of environmental suitability and the cost for survival.
Accordingly, the objective of this research was to de¬ termine the feasibility of interpreting the graded physiologi¬ cal response to stress as an index of environmental quality.
Hence, the oxygen (0) consumption, ammonia nitrogen (N) excretion, 0:N ratios, of four nymphal growth stages of the dragonfly Somatochlora cingulata (de Selys)were determined, relative particularly to elevated aluminum and hydrogen ion concentrations, and to naphthalene and trichloroacetic acid as well. vi An increase in respiration and ammonia excretion rates resulted in an increase in 0:N ratios for all nymphal stages, upon exposure to low pH and sublethal aluminum concentrations plus low pH. The earlier growth stages, however, were the most sensitive. The ratios obtained may be indicative of a decreased dependence on protein reserves and increased utilization of carbohydrate or lipid reserves. The same responses were observed in animals exposed to naphthalene and trichloroacetic acid. The ratios obtained were indica¬ tive that these organic compounds, also, decreased depend¬ ence on protein reserves. However, these 0:N ratios were less pronounced than animals exposed to low pH and aluminum plus low pH concentrations.
I found that nitrogen excretion and oxygen consumption do not always vary in the same direction, nor to the same extent, in response to changes in the environment.
I believe that the balance in catabolism between the different nutrient reserves in the tissues is useful in assessing the physiological responses of dragonfly naiads to various stress¬ ful environments.
Because the physiological changes reported in this research are easy to analyze, I suggest considering their incorporation into a routine screening bioassay procedure for chronic toxicity.
Vll TABLE OF CONTENTS
Page
DEDICATION. iv
ACKNOWLEDGEMENTS . v
ABSTRACT. vi
LIST OF TABLES. xi
LIST OF FIGURES.xiii
I. INTRODUCTION . 1
1.1 Justification . 2 1.2 Objectives. 3
II. LITERATURE REVIEW . 6
2.1 Historical Perspective . 6 2.2.1 Acid Rain. 6 2.1.2 Polycyclic Aromatic Hydrocarbons ... 10 2.1.3 Chlorinated Aliphatic Compounds ... 11 2.2 Impact on Aquatic Life. 12 2.2.1 Acid Rain . 12 2.2.2 Naphthalene. 16 2.2.3 Trichloracetic Acid. 17
III. MATERIALS AND METHODS. 19
3.1 Sample Site. 19 3.1.2 Specie Description . 19
3.2 Laboratory Procedure . 19 3.2.1 Experimental Protocol . 19
3.3 Bioassay Procedure . 23 3.4 Analytical Techniques (Chemical Assay) 25 3.4.1 Azide Modification of the Winkler Method. 25 3.4.2 Ammonia in Water (Nesslerization Method). 25 3.4.3 Ammonia in Tissues. 26 3.4.4 L-glutamate in Tissues. 26
vm Page
3.5 Physiological Procedure . 28 3.6 Physiological Procedure For Organic Contaminants . 30
3.7 Data Treatment.32
IV. RESULTS.37
4.1 Bioassay.37 4.4.1 Effect of pH on Mortality.37 4.1.2 Effect of Aluminum on Mortality .... 39
4.2 Physiological Data.39 4.2.1 Experimental Protocol . 39 4.2.2 Respiration .48 4.2.3 Histological Examination of the Tracheal Gills . 50 4.2.4 Ammonia Excretion Rate.5 3 4.2.5 Ammonia Accumulation . 53 4.2.6 L-glutamate Accumulation . 56 4.2.7 Oxygen-Nitrogen Ratios . 56 4.2.8 pH of Haemolymph.64 4.3 Physiological Results Using Organic Pollutants.64 4.3.1 Respiration and Ammonia Excretion Rates Using Naphthalene.64 4.3.2 Respiration and Ammonia Excretion Rates Using TCA.65
V. DISCUSSION .70
5.1 Bioassay.70 5.1.1 Effect of pH on Mortality.70 5.1.2 Effect of Aluminum on Mortality .... 71 5.2 Physiological Data.72 5.2.1 Respiration .72 5.2.2 Mechanisms of Toxicity on Respiration . 75 5.2.3 Ammonia Excretion Rate.75 5.2.4 Ammonia Excretion in Feeding Naiads . . 77 5.2.5 Oxygen-Nitrogen 'Ratios . 78 5.2.6 Ammonia Accumulation in Tissues .... 81 5.2.7 Glutamate Accumulation in Tissues ... 82 5.2.8 pH of the Haemolymph.8 3 5.3 Physiological Responses As Early Warning Indicators of Chronic Toxicity to Organic Contaminants . . 85
IX LIST OF TABLES
TABLE Page
1. Mean body wet weight-respiration of dragonfly (Somatochlora cingulata) at 21 + 1° C, static and flow-through systems and Gilson Differ¬ ential Respirometer . 41
2. Relationship of wet weight-respiration for the dragonfly Somatochlora cingulata exposed to low pH at 21 + 1° C. Each value is the mean and + SD of 10 observations. 46
3. Relationship of wet weight-respiration for the dragonfly Somatochlora cingulata exposed to A1 plus low pH at 21 + lu C. Each value is the mean and + SD of 10 observations. 47
4. Relationship of wet weight-ammonia excretion for the dragonfly Somatochlora cingulata exposed to low pH at 21 + 1° C. Each value is mean and + SD of 10 observations. 54
5. Relationship of wet weight-ammonia excretion for the dragonfly Somatochlora cingulata exposed to different aluminum concentration + low pH, at 21 + 1° C. Each value is mean and + SD of 10 observations . 55
6. Changes in levels of ammonia in the tissues of dragonfly nymphs exposed to low pH (3.59) and sublethal aluminum concentration (30 mg/1) + low pH (4.20). Values are expressed in yg/g wet weight of tissue. Each value is mean and + SD of 4 observations. 57
7. Changes in levels of L-glutamate in the tissues of dragonfly nymphs exposed to low pH (3.59) and sublethal aluminum concentration (30 ppm) + low pH (4.20). Values are expressed in yg/g wet weight of tissue. Each value is mean + SD of 4 observations. 58
8. 0:N ratios of the dragonfly Somatochlora cingulata exposed to low pH and different A1 concentration of 21 + 1° C. 59
xi Page
9. Relationship between wet weight-respiration (yg C>2 h”l g~l ) and excretory rates (yg NH2~N+h ^g ) for the fed dragonfly Somatochlora cingulata exposed to indicated concentrations of A1 + low pH at 21 + 1° C, during 9 days.60
10. Changes in levels of pH in the haemolymph of dragonfly nymphs exposed to sublethal concen¬ tration of Aluminum plus low pH (30 mg/1, 4.20) and low pH alone (3.50). Each value is mean of 4 observations .61
11. Wet weight - respiration relationships of the dragonfly Somatochlora cingulata to indicated concentrations of naphthalene during 24 hours at 21 + 1° C.62
12. Relationship between wet weight-respiration
(yg O^h ^g "*") and excretory rate (yg NH_-N -1 -1 J h g ) for the dragonfly Somatochlora cingulata exposed to indicated concentrations of naphthalene (mg/1) at 21 + 1° C. 0:N ratios were also incorporate .6 7
13. Relationship between wet weight-respiration
(yg 09h ^"g ■*") and excretory rates (yg NH.-
-1-1 4 h g ) for the dragonfly Somatochlora cingulata exposed to indicated concentrations of Trichloroacetic Acid (mg/1) at 21 + 1° C. Each value is the mean and + SD of 7 observa¬ tions 68 LIST OF FIGURES
FIGURE Page
1. Location of Cranberry Pond, Leverett, Massachusetts . 20
2. A gravity feed flow-through assembly to measure oxygen uptake in macroinvertebrates . . 22
3. Body wet weight-respiration of dragonfly (Somatochlora cingulata), using flow-through system at 21 + 1 C at different flow velocities .24
4. Percent survival of the dragonfly Somatochlora cingulata after 96 hours' exposure to low pH. . 38
5. Survival of Somatochlora cingulata naiads as affected by exposure to aluminum contamination for 96 h.40
6. Comparison between the coefficients of regression expressed as function of weight and oxygen consumption using the three different procedures.43
7. Comparison between the coefficients of regression expressed as function of weight and oxygen consumption per gram wet weight using the three different procedures . 44
8. The average (7 replicates) respiration rate
(yg C>2 h ^g ^ wet weight) of the dragonfly Somatochlora cingulata, fed daily during 96 h, exposed to a pH of 3.59 and low pH (4.20) plus aluminum (30 mg/1).49
9. Mean percent of lamellar length in branchial chamber with dark staining granules in Somatochlora cingulata naiads exposed to 100 ppm aluminum contamination for 96 h (•----•) and for controls (•———•).51
xm Page
10. Mean percent of lamellar length in branchial chamber with dark staining granules in Somatochlora cingulata naiads exposed to 100 ppm aluminum concentration for 96 h (•.•) and for controls (•-#).52
11. Oxygen consumption pigC^/h/g by the dragonfly
Somatochlora cingulata exposed to 0; 0.01 and 0.1 mg/1 of naphthalene during 24 hours. ... 63
12. Body wet weight-respiration relationship of dragonfly Somatochlora cingulata exposed to different concentrations of naphthalene during 24 h.66
xiv INTRODUCTION
The need for valid criteria in establishing biological water quality standards is critical to the conservation of aquatic life. Cairns (1981) believes, however, that even extended acute toxicity testing, does not contribute sig¬ nificantly to an understanding of the test organism's tolerance profile let alone the entire biocoenosis. Though, long term chronic toxicity tests can indicate the physiolog¬ ical cost of survival, they are costly in time and money.
They require more sophisticated facilities for longer periods of time, and are predicated on a higher degree of professional competence both for obtaining and interpreting results.
It is clear that a sensitive, high resolution screening protocol to quickly ( < 96 h ) expedite the early recognition of trauma is urgently needed. Further, the method should be well within the budgetary constraints of state and local governments, if the protocol is to be applied to the rapid identification of emerging crises. One such crises, perhaps the most critical, impacting New England is acid rain.
Because of the geological vulnerability afforded by
New England granitic soils (low buffering capacity), more of our lakes and streams biota are showing signs of disruption acid precipitation than any other state. The severity of 2 the problem is synergized by the linkages with the watershed.
The enhanced solubility and consequent mobilization of not only aluminum and heavy metals, but a myriad of toxicants leaching from our dumps and hazardous waste sites contribute to the trauma. It has been reported as well that high levels of polycyclic aromatic hydrocarbons (PAH), of which naphtha¬ lene and its derivatives constitute the most toxic fraction are absorbed by aquatic organisms (Anderson et al. 1974b;
Neff et al. 1976 and Correa and Coler, 1983). Besides (PAH), it has been recognized since early 1980's that chlorination of water rich in organic matter results in the formation of chlorinated short chain non-volatile aliphatics (Uden and
Miller, 1983). Conspicuous among these is the herbicide trichloroacetic acid (TCA). In light of the limited data base associated with the effects of these pollutants on animal models, especially at environmentally relevant levels,
I decided to assess their physiological effects on the dragonfly naiad Somatochlora cingulata (de Selys), as an
indicator organism for the assessment of stress,
(Correa and Coler, 1983) .
1.1 JUSTIFICATION
The chronic physiological effects of pH, aluminum, and naphthalene as well as trichloracetic acid remain undescribed.
This omission may be an oversight in the search for rapid, 3 early warning indices to sublethal stresses.
Recently, the use of insects, and particularly the odonates, in toxicity testing and monitoring has become more
frequent (Julin and Sanders, 1977; Greff and Van Dyk, 1978;
Muirhead-Thomson, 1978; and Correa and Coler, 1983). This is in part explained by the ability of dragonfly naiads to adapt and to tolerate a wide range of habitats and to sus¬ tain long period of starvation (Gaufin and Tazwell, 1952; 1956 ; and Corbet, 1962). Because they possess a well developed capacity for ionic regulation, they are capable of surviving to emerge from salt solutions (Sutcliffe, 1962; Moens, 1975; and Nicholls, 1983). Further, they have a metabolic rate sufficiently high to reflect mobilization of compensatory homeostatic adjustments (Hiestand, 1931). This is particu¬ larly pertinent because research in pollution biology has evolved beyond assessing the quantum response to acute toxicity. Concern is rather with chronic toxicity and identifying organisms with a sufficiently wide tolerance range of activity to mobilize a gradient response. Dragon¬ fly naiads may well provide the tool to obviate the need for costly long-term growth studies.
1.2 OBJECTIVES
The principal objective of this study is to examine selected physiological responses of dragonfly naiads to low 4 pH and aluminum concentrations. Respiration rate, ammonia excretion and tissue accumulation in different nymphal
stages will be assessed as indices of stress to acid rain.
Accordingly, six experimental procedures will be implemented
1. The dose-response survival relationship will be
estimated for each pollutant. This procedure will
provide both insights into the physiological mech¬
anisms involved adaption to aluminum and low pH
exposures and the rationale for the selection of
appropriate sublethal concentrations.
2. Respiration rates in dragonfly naiads (in four
different weight stages) exposed to low pHs and
aluminum concentrations plus low pH will be esti¬
mated. Also, an histological examination of the
tracheal gills of naiads exposed to a sublethal
concentration of aluminum and low pH will be under¬
taken to explore the mode of action.
3. Ammonia excretion rates v/ill be estimated in four
different weight classes as possible short-term
indicators of stress.
4. Oxygen-nitrogen (0:N) ratios will be estimated
because disrupted metabolism are often reflected
in the relative oxygen consumption and ammonia
excretion rates. 5
5. An examination of ammonia accumulation levels will
be made in naiad tissues exposed to low pH and sub-
lethal aluminum concentrations, because ammonia is
the main end-product of carabolism in dragonfly
naiads (Staddon, 1959) .
6. An analysis of glutamate levels will be made in
naiad tissues to determine if ammonia also accounts
for the increase of glutamate in dragonfly
tissues.
A secondary objective of this research is to determine the feasibility of adapting the dragonfly to water quality monitoring . Variations in the respiration and excretion rates as well as changes in the 0:N ratios of S. cingulata will be examined as indications of stress. These deviations from the norm will provide as well insights into mechanisms of toxicity and accommodation. For this reason, these physio¬ logical responses to low pH and aluminum toxicity of dragon¬ fly naiads will be compared to those obtained upon exposure to a polic\dic aromatic hydrocarbon (naphthalene) and a short chain chlorinated non-volatile alephatic compound
(TCA). 6
LITERATURE REVIEW
2.1 HISTORICAL PERSPECTIVE
2.1.1 Acid Rain
Acid precipitation has become a major concern in north¬ eastern Europe, eastern Canada and northeastern United States
(Beamish and Harvey, 1972; Hendrey and Wright, 1976 ;
Schofield, 1976; and Davis et al., 1978). The acidity of rainfall in regions of eastern United States and Canada appears to have increased as much as 50-fold. Further, the impacted areas have grown ever larger in succeeding years.
This phenomenon has been attributed to increasing emissions of sulphur and nitrogen oxides from the combustion of fossil fuels, principally coal.
Ironically, our shift to greater dependence on coal for heavy manufacturing, metal smelting, and the generation of electricity comes at a time when the negative impacts of its combustion are also being recognized. The faltering economy has made it less feasible to introduce emission controls that would limit sulphur dioxide release and consequently its subsequent oxidation to sulphuric acid.
In the atmosphere sulphur compounds are mostly in the inorganic form, and range in their degree of oxidation from hydrogen sulphide to various inorganic sulphates. Acid precipitation is a consequence of the oxidation of sulphur 7 dioxide to sulphuric acid. The effect on the acidity of the rain depends on what others cations and anions (NH4+, CO^ ,
PO^ , etc.) are also present.
Deposition of substances from the atmosphere into eco¬ systems is accomplished by one or more of three processes:
(1) wet fallout of substances dissolved or suspended in rain¬ drops or adsorbed to snowflakes; (2) gravitational settling and impaction of coarse particles and fine aerosols; and
(3) absorption or adsorption of gases. Process (1) includes all forms of precipitation, rain, snow, fog, etc. Processes
(2) and (3) are components of so-called "dry deposition"
(Garland, 1978; and Cowling and Linthurst, 1981). On an annual basis, estimates of the contributions of dry vs. wet processes of atmospheric sulphur deposition range from a ratio of 60:40 percent to 40:60 percent (NAS, 1978).
The NO component of fossil fuel emissions poses essentially the same impact as the sulphur compounds since it too is oxidized to acid. The United States contributed to world wide man-made totals during 1977 with approximately
23 million metric tons. There is some disagreement about whether the major NO sources are stationary (industrial and utilities) or mobile (cars and trucks). The best available figures, however, show that of the U.S. total, approximately
56% of NO emissions result from stationary fuel combustion, 8
one half of which is from the generation of electricity.
The balance resulted from the exhausts of cars and trucks.
The ratio of sulphate to nitrate ions in precipitation
varies in time and place. In much of eastern North America
the average equivalent ratio of sulphate to nitrate varies
from about 3.3:1 in the summer to about 0.7:1 in winter.
The average annual ratio of sulphuric to nitric acids is
currently about 2:1; but the progressive increase in NO X
release reduced this ratio (Likens et al., 1979).
The regions of greatest susceptibility to the impact
of acid precipitation usually are recently glaciated. The
large areas of exposed granitic and other non-calcareous bedrock are highly resistant to chemical weathering. They
are covered generally by thin, coarse-grained glacial deposits
of similar lithology with limited buffering and cation ex¬
change capacity. These watersheds low in alkalinity and
calcium, are consequently, poorly buffered and vulnerable
to acid inputs.
An important consequence of acidification is the mobil¬
ization of elevated metal concentrations (Al, Zn, Mn, etc.)
from the edaphic to the aquatic environment (Cronan and
Schofield, 1979; Driscoll et al., 1980; Schofield and
Trajnar, 1980; and Ulrich et al., 1980). This increase in
the solubility and mobilization of metals occasioned by acid 9
deposition changes the physicochemical environment, dis¬
rupting the dynamic equilibrium within the soils, the water
and the living organisms. Aluminum solubility is pH-
dependent, and increases with increasing acidity. Several
reports have documented elevated aluminum concentrations in
acid lakes and streams in areas known to be impacted by acid
inputs, and in effluents from lysimeters treated with acid
solution(Abrahansen et al., 1976). While aluminum is typi¬
cally leached from the upper soil horizon of podzol soils by carbonic acid and organic chelation, it is usually de¬
posited in lower horizons.
Aluminum occurs in acidic surface waters primarily
as the free aluminum ion and complexed with hydroxide,
fluoride and organic ligands. Driscoll et al. (1980) con¬
cluded that only inorganic forms of aluminum were toxic to
fish.
Accordingly, the final effect of acid precipitation on
freshwater can be described as the result of a large scale
titration. The sulphuric and nitric acid in precipitation
titrate the carbonate-bicarbonate until neutralized by free
bases such as calcium or magnesium carbonates in the water¬
shed (Henriksen, A. 1982). Where the watershed has no free
bases, but an appreciable cation-exchange capacity, some
hydrogen ions will replace metal cations on ortanic or in¬
organic exchange complexes. Some hydrogen ions will also 10
react: to varying degrees with silicates or other minerals in the watershed, releasing soluble metal cation and silica thus neutralizing some proportion of the acid. Johnson et al.
(19S1' considered the neutralization of the incoming acid rain essentially as a two-step process. Initially, the hydrogen ions were consumed by dissolution of preexisting aluminum hydroxide compounds found in the soil zone and by leaching of bases from biological matter. In the second step both hydrogen ion acidity and aluminum acidity were neutra¬ lized by the chemical weathering of the mineral in the bed¬ rock and glacial till of the watershed.
2.1.2. Polycyclic Aromatic Hydrocarbons
Oil pollution can be lethal to aquatic life (Moles et
al., 19~9), but concentrations high enough to cause death probably seldom occur in the environment except after a large
spill. Sublethal concentrations of petroleum pollutants are more common. In the United States pipelines carry crude oil
and refined oils adjacent to or across many lakes and steams.
A break in a pipeline or a leak in a storage tank could have
a serious long-term effect on downstream populations of fish or any other aquatic organism.
Recently, there has been considerable concern with the possible contamination of the aquatic environment by polycyclic
aromatic hydrocarbons (PAH). The composition of petroleum 11 and coal and their wide-spread uses suggest that these materials could be major contributors to such contamination.
A number of organic chemicals appear in water leached from petroleum (Boylan and Tripp, 1971; Lee et al., 1974; and
Larson and Weston, 1976) or with pulverized coal (Carlson and Caple, 1976). Among these, naphthalene and its deri¬ vatives have been conspicuous in the aqueous extracts. They are considered the most toxic fractions absorbed by aquatic organisms (Anderson et al., 1974b and Neff et al., 1976).
Because little is known about the sublethal effects of naphthalene on the respiration and excretion rates on dragon¬ fly naiads, I decided to assess its physiological stress on
S. cingulata.
2.1.3 Chlorinated Aliphatic Compounds
Recent laboratory chlorination studies of humic and fulvic acids have shown numerous nonvolatile chlorinated carcinogens to be formed during chlorination. Several of these compounds, including dichloroacetic acid (DCA) and tri¬ chloroacetic acid (TCA), are formed at concentrations com¬ parable to, if not exceeding, the concentrations of chloroform produced (Uden and Miller, 1983). Miller et al., (1982) have also revealed that TCA formation was more than double that of chloroform for the same sample. Previously, Norwood et al.,
(1980) reported the formation of chlorinated acids including di-and trichloroacetic acids as a result of chlorinating humic materials. They also found that the formation of chlorinated acids would be favored at high Cl/C ratios.
Perhaps, due to its use as a herbicide, the levels of
(TCA) in drinking water have increased recently (Uden and
Miller, 1983). A contributing factor could be that TCA and
DCA are the powerful metabolites of trichloroethylene,
1,1,2-trichloroethane and chloral (trichloroacetaldehyde).
Metabolic studies have repeatedly shown that TCA is excreted via the kidney and may be excreted conjugated with glucur¬ onic acid (Caperos et al., 1982; and Soleo et al.,1979).
Direct investigations of the toxicological effects of
TCA have focused on its potential mutagenicity (Andersen et al., 1972; Waskell, 1978; and Rapson et al., 1980).
2.2 IMPACT ON AQUATIC LIFE
2.2 Acid Rain
The hydrogen ion content of acid rain and the subsequent mobilization of heavy metals has been correlated with envi¬ ronmental damage in Japan, Norway, Sweden, Canada, northeast¬ ern Europe, and the United States. In eastern Canada and the northeastern United States the effect on susceptible lakes has been the most extensive. In the Adirondacks the pH value in many lakes are depressed to the point where fish no 13 longer reproduce. More than 100 lakes in this area have no fish at all. Ontario has 140 lakes without fish, whereas
Nova Scotia has lost its salmon population in 9 rivers, with
11 more threatened (Harvey, 1981 and Blake, 1981).
Fish are especially vulnerable to changes in acidity because they have several critically sensitive life stages such as spawning, egg development, fry hatch and early de¬ velopment. The greatest damage appears to occur in the spring during times of spawning or hatching (Peterson et al.,
1982). This period often coincides with snowmelt which may rapidly flush out the hydrogen ions accumulating in the snow pack from both atmospheric depositions and soil decomposition.
The result is a sustained pulse of increased acidity in streams and lakes which can exert an acute toxicity on adult fish, apparently by upsetting their salt balance (Leivestad
1982; and Wood et al., 1982). Baker and Schofield (1982), on the other hand, observed complex effects of inorganic aluminum on survival and growth of brook trout and white suckers during developmental periods (eggs, larvae and postlarvae) at pH ranging from 4.2 to 5.6. They determined that the presence of aluminum could be both detrimental and beneficial to fish survival, depending on the stage of the life cycle. 14 The impact of acidic precipitation on molluscan popula¬ tion is dramatic. The calcareous shell of these animals is highly soluble at pH 7.0 and requires the animals to fix fresh calcium carbonate faster that it can dissolve (0kland and Kuiper, (1980). Okland (1980) reported of the 27 species of snails in Norway, only 5 were found below pH 6.0. Snails could tolerate higher hydrogen ion concentrations if the total hardness of the water were higher, indicating that pH may stress snails by reducing the calcium carbonate availability
(J. 0kland, 1979).
There is some variation in sensitivity to pH between crustaceans. Malley (1980) observed that Orconectes virilis
(crayfish) was stressed by pH 5.5. However, Cambarus sp. was reported (Warner, 1971) in a stream receiving acidic mine drainage at pH 4.6. Asellus aquaticus (isopod) populations were reduced at sites below pH 5.2 and absent below a pH of
4.8, though Gammarus lacustris (amphipod) was inhibited at pH 6.0 (K. A. 0kland, 1980c).
Studies of benthic insects exposed to acid stress include surveys and some laboratory experimentation. Survey work in¬ volves present or absent type data from which tolerances have been assumed. The general conclusion drawn from many surveys of lakes and streams (Conroy et al., 1975; and Leivestad et al.,
1976) is that species richness, diversity, and biomass are reduced with increasing acidity. 15
The response of benthic insects to low stress varies considerably between different taxa and among different life cycle stages (Gaufin, 1973; Raddum and Steigen, 1981; and Correa et al., 1984). Female mayfly adults (Baetis) did not lay eggs on otherwise suitable substrates in water below a pH 6.0. The adult presumably can detect levels of acid by the dipping motion of her abdomen into the water as she flies.
Mayflies and stoneflies were found to be sensitive to pH stress (Sutchiffe and Carrick, 1973; Borgstrom et al., 1976;
Leivestand et al., 1976; and Nilssen, 1980).
Caddisflies (Trichoptera) have been found at a pH of 4.5 in field surveys (Leivestad et al., 1976; and Raddum, 1976) but not at pH 4.0 (Hall and Likens, 1980a). Bell and Nebeker
(1969) reported two species of caddisflies (Brachycentrus americanus and Hydropsyche betteni) having 96-hour TLm value of 1.5 and 3.15 respectively. Also, dragonflies and damsel- flies are resistant to pH stress (Bell and Neberker, 1969;
Bell, 1971; and Borgstrom et al., 1976). The dragonfly nymph,
Libellula pulchela, can tolerate a pH of 1.0 for several hours
(Stickney, 1922) .
Most other insects are largely unaffected, or slightly favored in low pH habitats. The alderfly, Sialis SR.
(Megaloptera) , increased its emergence rates in an artificially acidified stream (Hall and Likens, 1980a) and was commonly found in the shallow acidic waters (pH 3.9-4.6) of Swedish 16 lakes (Wiederholm and Eriksson, 1977). Collins et al.,
(1981) found that several true flies (Diptera) increased in abundance in acidic waters.
It is likely that other factors besides hydrogen ion concentration contribute to the causal mechanisms inducing stress in aquatic invertebrates. Lamentably, the effects of increased aluminum concentrations on invertebrates have not been as thoroughly documented as they have with fish
(Baker and Schofield, 1980).
2.2.2 Naphthalene
Polycyclic aromatic hydrocarbons (PAH) have long been recognized as hazardous environmental chemicals. Naphthalene, a constituent of the water soluble fraction of crude petro¬ leum and many refined oil products, was shown to be toxic to a variety of marine organisms (Craddock, 1977; Sanborn and
Malins, 1977; and Roubal et al., 1977a). Naphthalene and its derivative have been found to induce physiological and biochemical changes in aquatic organisms (Struhsaker et al.,
1974; Anderson et al., 1974; and Heitz et al., 1974). Its uptake and metabolism have been studied in the mussell,
Mytilus edulis (Lee et al., 1972a), spider crab (Corner et al.,
< 1973); zooplankton (Lee, 1975); oyster Ostrea edulis (Riley and
Schaffer etal. , 1981) ; oyster Crassostrea virginica , clam Rangia 17 cuneata (Neff and Anderson, 1975); marine fish (Gruger et al.,
1981) and Correa and Venables, 1984); houseflies and rats
(Terriere et al., 1961 and Capdevila et al., 1973).
Despite the fact that PAH levels have been increasing in the past decade, the literature describing physiological changes in freshwater organisms to naphthalene have lagged behind marine research. One does not normally think of oil spills as an aquatic problem though the Department of
Environmental Quality Engineering (DEQE) identified petro¬ leum products as one of their priority pollutants.
Correa and Coler (1983) determined that variations in the respiration rates of the dragonfly Somatochlora cingulata could be used as a short-term indicator of stress incurred by exposure to aromatic hydrocarbons.
Physiological changes in Daphnia magna exposed to naphthalene have been studied in aquatic toxicity bioassay.
Results using these crustaceans are often less variable than those using fish (Crider et al., 1982 and Crosby and Turker,
1966) .
2.2.3 Trichloracetic acid
Research exploring the toxicological effects of TCA in aquatic organisms is quite limited. Correa et al., (1984) determined that upon exposure to TCA, S. cingulata increased its oxygen consumption and ammonia excretion rates. It is 18 noteworthy that the physiological changes observed occurred at levels of environmental relevance (10-100 ppb) Cor only an 8 hour exposure. TCA levels in Massachusetts' drinking waters have been found in the range of 100-200 ug/1 (Uden and Miller,
1983). It would seem extremely pertinent at this juncture to measure the TCA content of generating plant cooling waters after a routine chlorination. 19
MATERIALS AND METHODS
3.1 SAMPLE SITE
Somatochlora cingulata (Odonata: Anisoptera) naiads varying between 0.01 and 0.5 g. were captured, with a D-
frame net, at Cranberry Pond, Leverett, Massachusetts
(Figure 1). The experimental animals were returned to the laboratory for subsequent identification and acclimation in pond water at 21 + 1° C for a minimum of seven days on a diet of mayfly nymphs. Feeding was stopped two days prior to the initiation of the experiment.
3.1.2 Specie Description
Some of the most important taxonomic characteristics used in the classification of Somatochlora cingulata have been incorporated in the Appendix.
3.2 LABORATORY PROCEDURES
3.2.1 Experimental Protocol
Prior to the initiation of these experiments, pilot tests were performed to identify which of three different procedures to measure respiration yielded the narrowest con¬
fidence intervals. These three methods were:
1. Gilson Differential Respirometer (Model No. GRP 14).
The procedure for evaluating and calculating O2
consumption followed those outlined by Umbreit 20
Figure 1. Location of Cranberry Pond Leverett, Massachusetts 21
et al (1964). Twenty eight naiads previously
starved 48 hours, were blotted, weighed and placed
in 20 ml reaction vessels (one/vessel). A period
of 90 minutes prior to initiating the experiment
was allowed for equilibration as well as to permit
the naiads to recover from handling stress and
adjust to experimental conditions. Oxygen con¬
sumption values were recorded at 15 minute intervals
throughout a one hour period.
2. Static DO Bottle and Chemical Titration. Each of
the 28 naiads was blotted, weighed and transferred
to a 300 ml DO bottle containing filtered, oxygen
saturated water from Cranberry Pond. After 2, 6,
12 and 24 hours, the dissolved oxygen concentration
in each bottle was determined by the Alsterberg
Azide Modification of the Winkler Method (A.P.H.A..
1980).
3. Flow-Through System and Chemical Titration. Cran¬
berry pond water was delivered from a 20 1 glass
Mariotte bottle (Burrow, 1949) to testing chambers
(50 ml volumetric pipettes fitted with ground glass
stoppers) flanked by 60 ml DO bottle upstream and
downstream from the chamber (Figure 2). Water flow
was regulated with Pasteur pipettes at a rate of
10 + 0.5 ml/min. The dissolved oxygen concentration 22
Figure 2. A gravity feed flow-through assembly to measure oxygen uptake in macroinvertebrates.
Reservoir (20LMariotte Bottle )\
sFlOW / Regulator 23
was determined by the Alsterberg Azide Modifica¬
tion of the Winkler Method (A.P.H.A.). Preliminary
testing concerned with flow rate (Figure 3) indi¬
cated that beyond 5 ml/min an asymptote was attained
in oxygen uptake. In all, sixteen specimens were
used, one per chamber.
3.3 BIOASSAY PROCEDURE
Pilot tests were also performed to determine the level at which low pH and aluminum concentrations ceased to be acutely toxic. On the basis of these data, the naiads were divided into four different weight classes: Group I 0.1 g.,
Group II between 0.1 and 0.2 g., Group III between 0.2 and
0.3 and Group IV 0.3 g. The flow-through system described previously was used for all of these experiments. The water used was filtered through a Whatman 42 filter to remove zooplankton and suspended particles. The various aluminum concentrations were prepared by adding appropriate amounts of A1C1^ to the 20 1 bottles of filtered pond water. To obtain desired pH levels, standards were prepared by adding aliquots of 6.5 parts concentrated sulfuric acid, 3 parts nitric acid and 0.5 parts of distilled water. The pH was determined with an Altex pH meter (Model 0 70).
Ten naiads were placed in each chamber (250 ml) for
96 h., after which the mortality rates, within the different Figure 3. Body wet weight-respiration of dragonfly (Somatochlora cingulata), using flow-through system at 21±1 °C at different flow velocities. 25 weight classes, were recorded. The pH and aluminum concentra¬ tions eliciting 50 percent mortality (LC^q) were derived from a modification of the straight line graphical interpolation method outlined in Standard Methods (1980) .
3.4 ANALYTICAL TECHNIQUES (CHEMICAL ASSAY)
3.4.1 Azide Modification of the Winkler Method
Reagents (see Appendix)
Procedure:
To the sample collected in a 60 ml bottle, add 0.5 ml
MnSO^ solution, followed by 0.5 ml alkali-iodide-azide reagent. Hold pipet tip just beneath liquid surface when adding reagents. Stopper carefully to exclude air bubbles and mix by inverting bottle 20 times. When the precipitate has
settled to approximately half the bottle volume leaving a clear supernant above the manganese hydroxide floe, add 0.5 ml cone. H2S04. Restopper and mix by inverting 20 times until dissolution is complete. Titrate a volume corresponding
to 50 ml original sample with 0.0250N Na2S2C>2 solution to a pale straw color. Add a few drops of starch solution and
continue titration to first disappearance of blue color.
3.4.2 Ammonia in Water (Nesslerization Method)
Reagents (see Appendix)
Procedure:
Use 50.0 ml sample and add 1.0 ml Nessler reagent 26
(potassium iodide, mercuric iodide and potassium hydroxide).
Mix samples by capping Nessler tubes with clean rubber stop- pers and then inverting tubes at least six times. Keep such
conditions as temperature and reaction time the same in blank, samples, and standards. Let reaction proceed for at
least 10 min. after adding Nessler reagent. Measure absorb¬
ance or transmittance with a spectrophotometer at 425 nm.
3.4.3 Ammonia in Tissues
Dissolve 10 mg tissue in 1.0 ml 0.03N methanolic HC1
and precipitate at once with 2 ml ethyl ether. Separate the protein from the solution by centrifugation and repeat the extraction twice (Colowick and Kaplan, 1967). After centri¬
fugation, aliquots of this solution were taken for estimation of ammonia by the Nesslerization method described previously.
3.4.4 L-glutamate in Tissues
Reagents (see Appendix)
Procedure:
Homogenize the tissue with 4 parts by weight of per¬
chloric acid and centrifuge for 10 min. at 3000 g. Adjust
3.00 ml of the supernatant to an alkaline pH (ca. 9) with
1.80 ml phosphate solution. Allow to stand for 10 min. in
an ice bath and then filter through fluted filter paper.
Warm to room temperature and analyse 1.00 ml of the filtrate.
Pipette into 1 cm. cuvettes: 3 ml glycine-hydrazine 27 buffer; 0.20 ml sample (doubly distilled water in blank) and 0.20 ml DPN (Diphosphopyridine nucleotide). Mix in a plastic spatula and read the initial optical density. Add
0.05 ml. GIDH suspension (Glutamic dehydrogenase). Allow the experimental and blank cuvettes to stand for ca. 30 min. and then measure the final optical density. For each cuvette there must be the same time interval between adding the DPN solution and measuring the final optical density. Calculate the differences netween the final and initial optical densi¬ ties for the experimental and blank cuvettes ( AE and A E, ,, ). c sam blk A E = AE - AE,,, is used for the calculations, xam blk
Calculations: A E X 3.45 For measurements at 340 my :__ A E X 2.77 6.22 X 0.2
= y moles L=glutamate/ml sample A E X 3.45 For measurements at 366 my: _ = aE X 5.23 3.30 X 0.2 = y moles L-glutamate/ml sample where
3.45 = assay volume (ml)
0.2 = volume of the sample in the assay mixture (ml) 2 6.22 = extinction coefficient pf DPNH at 340 mu (cm /y mole) 2 3.30 = extinction coefficient of DPNH at 366 mu (cm /y mole)
To convert to yg. it is necessary to multiply the values by
the molecular weight of glutamic acid (146). 28
3.5 PHYSIOLOGICAL PROCEDURE
The respiration rates, ammonia excretion rates and 0:N
ratios of each nymph stage were determined during exposure to
low pH (6.75; 4.20 and 3.59) and sublethal aluminum concentra¬
tions (10; 20 and 30 mg/1) at a pH of 4.20. The flow through
system described by Burrow and modified by Correa et al. (1983) was used (Fig. 2). The differential dissolved oxygen (DO)
concentrations between DO bottles were determined by the
Alsterberg Azide Modification of the Winkler Method (A.P.H.A.,
1980). Ten replicates per pH and aluminum concentrations were performed.
A histological examination of the tracheal gills of naiads
exposed to a sublethal concentration of aluminum (100 mg/1)
plus low pH (3.5) was conducted. Ten naiads were exposed to a
100 mg/1 concentration of aluminum for 96 h (approximating the
LC^q) in a flow through system following the methods described
previously. Ten naiads treated in a similar manner but not
exposed to Al were used as controls.
Fixation of abdominal tissues from surviving animals (10
controls and 6 treated naiads) was achieved by injecting a
small amount (ca. 0.5 ml) of Carnoy's a fixative (Barbosa,
1974) into the abdomen of live naiads using a hypodermic
syringe. The posterior two thirds of the abdomen was then
excised and placed in a vial containing the fixative and left 29 overnight to complete fixation. Samples were then dehydrated and embedded following standard histological procedures as described by Humason (1967) . Abdominal sections (lOym thick) were then prepared and placed on microscope slides (ca. 10-14 sections per slide). All sections were stained with Mallory-
Heidenbain stain following the procedures described by
Humason (1967).
All slides were initially examined to identify those con¬ taining sections from the area of the abdomen which included the branchial chamber. Further examination was limited to sections taken from this area.
Ammonia excretion rates of individual naiads were moni¬ tored in a static system. Several sets of DO bottles were filled with filtered pond water saturated with at different pH's and aluminum concentrations. Each individual naiad was blotted, weighed and placed in 300 ml DO bottle. Ammonia excretion rate from which 0:N ratios were derived were mea¬ sured according to the Nesslerization Method (A.P.H.A., 1980).
To determine tissue ammonia and glutamate levels, the specimens were exposed during 96 hours to low pH (3.59) and sublethal aluminum concentration (30 mg/1) using a flow¬ through system. Every 24 hours a set of naiads was analyzed for ammonia and another for glutamate levels. Each naiad was blotted, weighed and then homogenized for 1-2 minutes in a mixer with cold distilled water and 15% perchloric acid 30
and centrifuged at 750 g for 10 min. The supernatants were decanted for determination of ammonia and glutamate levels wfyich were determined by Nesslerization, A.P.H.A. (1980) and the procedure of Begmeyer (1965) respectively.
Changes in levels of the haemolymph pH were measured in animals exposed in a flow-through system for 96 hours to low pH (3.59) and sublethal aluminum concentration (30 mg/1) plus low pH (4.20). Every 0, 2, 4, 8, 12, 24, 48, 72, and 96 h., one set of 4 naiads per contaminant was analyzed. Each naiad was then washed in distilled water and blotted by means of paper. The naiad was next immobilized on its back on a piece of foam-plastic by means of needles. The pH of the haemo¬ lymph was measured directly by inserting needle pH electrode
(MI-408B from Microelectrodes, Inc.) in the pleurite between the 6th and 7th abdominal segment. The pH values were re¬ corded in a digital ionalyzer (Model 501 Orion Research).
3.6 PHYSIOLOGICAL PROCEDURE FOR ORGANIC CONTAMINANTS
The oxygen consumption rate of each naiad (seven repli¬ cates) was monitored during an 8 hour exposure to one of four concentrations (0.00, 0.01, 0.1 and 1 mg/1) of TCA administered in the flow-through assembly described previous¬ ly. Prior to the initiation of a run, each test animal
(seven replicates) was prepared by adding an appropriate amount of reagent grade TCA (98% pure) to 20 liter reservoir 31 bottles of filtered pond water. The differential dissolved oxygen (DO) concentrations were performed on water collected from the downstream and upstream DO bottles flanking the respiratory chamber in accordance with the Alsterberg Azide
Modification of the Winkler Method (A.P.H.A., 1980).
Ammonia excretory rates of individual naiads were moni¬ tored in a static system (Correa et al., 1984). One set of
DO bottles was filled with filtered pond water (control) and 3 other sets containing 0.01, 0.1 and 1 mg/1 of TCA.
The naiads (seven per set) were each blotted, weighed and placed individually in 300 ml DO bottles for 24 hours.
Ammonia concentrations were measured according to the
Nesslerization Method (A.P.H.A., 1980).
Respiration rates in specimens exposed to naphthalene were estimated by the Winkler Method (A.P.H.A., 1980).
DO bottles were filled with filtered pond water saturated with oxygen containing 0, 0.01, or 0.1 mg/1 of naphthalene.
Forty eight naiads were blotted, weighed, and placed into
DO bottles, (one each). DO bottles from each group were analyzed (4 per period of time) at 2, 6, 12, and 24 h after initial exposures.
Ammonia excretory rates of individual naiads were monitored in a static system (Correa et al., 1984). One set of DO bottles was filled with filtered pond water 32
(control) and two other sets containing 0.01 and 0.1 mg/1 of naphthalene. The naiads (seven per set) were each blotted, weighed and placed individually in 300 ml DO bottles for 24 hours. Ammonia concentrations were measured according to the Nesslerization Method (A.P.H.A., 1980).
3.7 DATA TREATMENT
In the experiments designed to compare three different procedures to measure oxygen consumption; regression analyses were carried out in all three treatments where wet weight of the naiads was the independent variable, and oxygen consumption was the dependent variable. An analysis of variance was con¬ ducted to test for the significance of differences among the three treatment groups in oxygen consumption. Duncan's New
Multiple Range Test (Duncan, 1955) was used to separate the means.
The relationship between naiad mortality and low pH as well as aluminum concentration levels was examined among the different weight classes. Quantification of this relationship is necessary to determine the relative sensitivities of early life history stages of Somatochlora cingulata to acidic condi¬ tions; and to measure the relative toxicity and interactions of H and inorganic AT5 .The low pH and aluminum concentration at which 50% of the organisms died (LC^q) were obtained by using a modification of the straight line graphical interpola- 33 tion method as outlined in Standard Method (1980). In this procedure it is important to tabulate observations of mortality for at least one selected exposure time; this time ordinarily 96 h is the longest one used in the test.
To construct the graph in each stage, I plotted percent¬ age mortality on a probability scale as the ordinate against the log of the concentration on the abscissa. Because the probit scale never reaches 0 or 100% it is necessary to plot any such points with an arrow indicating their true position.
The line was fit by eye, giving more consideration to points between 16 and 84% mortality and minimizing total vertical deviations of the line from the points. The concentration causing 50% mortality from the fitted line is the standard
LC^q for the selected exposure time. The LC^-q estimated by graphical procedures is usually sufficiently close to the accuracy obtained by formal probit analysis with a computer
(Dixon, 1970).
Standard deviations and confidence limits of the LC5C and all the physiological data were calculated by a formal probit analysis with a computer. The confidence limits about the LCj-q do not describe variability of the LC5Q under conditions other than those tested. The limits indicated the accuracy of the estimate of replicate tests at the same time under the same conditions. Tests for significant differences between LCc-A,s by examining confidence limits 50 34 for overlap were evaluated. The equation described by-
Litchfield and Wilcoxon (1949) were applied to determine significant difference between the LCca. 5 0 Oxygen consumption, ammonia excretion and accumulation levels of ammonia and glutamate at the designated low pH and Al concentrations were evaluated by regression analysis and performed on both dependent (02 consumption or ammonia levels or glutamate levels) and independent (weight stages) variables. Each regression was estimated the relationship of one variable with another by expressing the one in terms of a linear (or more complex) function of the other. The degree to which two variables vary together were estimated by correlation analysis (Sokal and Rohlf, 1969). If two or more regression lines from similar data are obtained, it is of interest to know whether the functional relationship described by regression equations are the same. The basic design of such a test is that of the analysis of variance.
Two separate analyses of variance are possible, one for each variable, and also a joint one analyzing the covariance between X and Y. Such an analysis in its complete form is called analysis of covariance. An analysis of covariance provides answers to several questions. First, it furnishes the regression coefficients of Y and X for each of the samples and tests whether the slopes of the several regression lines 35
could have come from populations with the same slope. It
tests whether the means of the dependent variable are sig¬
nificantly different among group and whether these differ¬
ences are due to differences on the independent variable
among the groups. An anova fits a common regression line
to the group means of Y and tests whether there is significant
heterogeneity among these means around this regression line.
Finally, it compares this regression to a pooled regression
of all the Y items on the X items. Duncan's New Multiple
Range Test (Duncan, 1955) was then used to separate the
means.
One-way analysis of variance to determine the effects
of low pH (3.59) and sublethal aluminum concentration (30
ppm) on the change haemolymph pH levels was used (Sokal and
Rohlf, 1969).
Oxygen consumption and ammonia excretion rates at the
designed naphthalene concentrations were evaluated by
regression analysis and performed on both dependent (02
consumption) and independent (weight) variables to normalize
the variances. Statistical differences between means were
evaluated using analysis of variance.
Oxygen consumption and ammonia excretion rates at the
designed TCA concentrations were evaluated in the same way.
I also applied a Duncan's Multiple Range Test (Duncan, 1955) 36
to separate and to compare the means between treatments.
This test was used with naphthalene as well as TCA treatment. 37
RESULTS
4.1 BIOASSAY
4.1.1 Effect of pH on Mortality
The naiad surviving response varied markedly at different pH levels within the four different weight classes
(Fig. 4). The LC,_q ranged between 3.45 (Group I) to 2.40
(Group IV) in 96 hours. The lighter weight classes (Group I and Group II) were more sensitive to low pH than their heavier older counterpart (Group III and IV).
Using Duncan's Multiple Range Test (Duncan, 1955), I found that the values for mortality in Groups I and II differ highly significantly (P< 0.05) compared with Groups III and
IV.
In general, aquatic insects tolerate acid conditions, at least for periods of less than one week. Data obtained by Bell and Nebeker (1969) and Bell (1971) showed that aquatic insects differ markedly in pH tolerance. They determined that caddisfly are very tolerant to low pH, the stoneflies and dragonflies are moderately tolerant, and the mayflies are fairly sensitive. They also found that the dragonfly Qphiogomphus rupinsulensis, commonly found in slow, quiet stream reaches has a 96-hour TL,_q of 3.5 and another dragonfly Boyeria vinosa exhibited a 96-hour TL^q 33
Figure 4. Percent survival of the dragonfly Somatochlora cingulata after 96 hours’ exposure to low pH.
ph pH
pH pH 39
of 3.25. In both species the 30 day TL_ 's were 4.30 and 5 0 4.42 respectively. In general, Somatochlora cingulata seems to be less tolerant to lowpH than rupisulensis and vinosa because it cannot tolerate a pH of 4.20 for more than 7 days.
4.1.2 Effects of Aluminum on Mortality
The relationship between aluminum concentrations and naiad mortality is showed in Fig. 5. The concentration of aluminum contamination resulting in the death of 50 percent of naiads was determined to be approximately 140 ppm. No significant differences in size-toxicity relationship were found between groups.
This value (140 ppm) is a good indication of aluminum tolerance by dragonfly naiads, especially, because aluminum has been found to be toxic to fish at concentrations as low as 0.1 to 0.2 ppm (Baker, 1981; Schofield and Trajnar,
1980 and Muniz and Leivestad, 1981). In addition, a concen¬ tration of aluminum of 20 ppm resulted in 100% mortality in daphnia within 20 hours (Havas and Hutchinson, 19 82) .
4.2 PHYSIOLOGICAL DATA RESULTS
4.2.1 Experimental Protocol
The estimate of specimen's mean resting respiratory rate ranged from 0.197 to 0.682 ml h ^g (Table 1).
Of the three systems, the flow-through design generated 40
Figure 5 . Survival of Somatochlora cingulata naiads as affected by exposure to aluminum contamination for 96 h. 100
75
50
25
0
Aluminum Concentration (ppm) 41
O' IT) OJ o o O • • • o o o H + 1 +1 + 1 u O' 04 1—1 i—1 o\° co 00 G\ fd ID 1—1 CD 04 co • • • >1 o -P o o O rH "P G fal -P 0 G td P CD 1—1 o 0 -P 0 CO 04 tn m 4H > • • • fd 4H u 1X5 ro P - •H i—1 13 U Q mo G Q 0 rH O G CO in 0 +1 ro 04 i—1 G + l«H •P i—1 o O o 0 •H ■P 1 • • • •H r—) o o o o -P 04 £ iH fd 13 G 1 + 1 + 1 + 1 P -P G 0 £ •h rd fd G O' 04 1—1 a> 0 04 O'* 00 CO in ^ 0 O O rp CD 04 1 tj> tr> in •H G CD "H jG ■p £ o tr> 43 G tr> -P fd 0 • •P 00 00 ro 0 P P P G 0 — O' 1—1 04 £ 0 ,g 0 td H tr> 00 O' 1—1 i—1 -p -p 0 — 04 04 04 >1rG i 0 S -P • • • 13 O £ g 0 o o o 0 0 0 0 £ rQ +J i—i p fd m -p g e a fd o 13 0 0 CO G 0 m 0 s — fd Ph 0 00 00 CD
(Fig. 3) oxygen consumption values with the narrowest 95% confidence intervals (+ 0.023), and lowest standard devia¬ tion (0.01) and coefficient of variation (3.7). The appli¬ cation of the flow-through delivery system produces the most reproducible measure of respiration with the greatest capacity to resolve differences in rate.
To ascertain the influence of body weight on respira¬ tion, oxygen consumption (ml C^/h) was regressed (Fig. 6) on wet weight (g). The strong positive correlation co¬ efficients obtained for the three methods, however, were not apparent when specific oxygen consumption was regressed against wet weight (Fig. 7). Instead, my findings by the flow-through procedure support the observations of
Isterni (1963), Petitpren and Knight (1970) and Kapoor and
Griffith (1975). No significant correlation exists between specific oxygen consumption (rate per gram) and size of some aquatic insects. The other two methods, however, indicate an inverse relationship.
It is disturbing that these methods which purport to measure the same phenomenon should generate highly statist¬ ically significant differences (by analysis of variance and
Duncan's Multiple Range Test) among the 3 sets of data.
The respirometer method yielded a value twice that of the flow-through unit but it is not clear why. The former CONSUMPTION IN ml On/h Figure 6.Comparisonbetweenthecoefficientsofregression tion usingthethreedifferentprocedures. expressed asfunctionofweightandoxygenconsump¬ WET WEIGHT(g) 43 CONSUMPTION ml O./h/g WET WEIGHT Figure 7.Comparisonbetweenthecoefficientsofregression expressed asfunctionofweightandoxygenconsump- WET WEIGHT(g) 44 45
may be inflated due to insufficient time allowed for recovery from handling and adjustment to the oscillations (84 cycles/ min) of the reaction vessels. Further, the reaction vessels were not lined with a substrate. Rueger et al.,(1969) and
Olson and Rueger (1968) employed a plastic screen, while
Petitpren and Knight (1970) resorted to boiled brick chips.
The decidedly lower specific static DO bottle respiratory value (0.1974 ml 02/g/h) on the other hand may reflect a less disruptive environment, the accumulation of metabolites not withstanding. Alternatively, the more extended exposure
(24 h) may have resulted in respiratory dependence from depressed oxygen concentrations. Whatever the contributing factors may be, the respirometer procedure yielded results that were not consistent with published data. Petitpren and Knight (1970) determined the specific respiratory rate for Anax junius to range between 128.2 and 74.6 yl 02/g/h wet weight. The flow through delivery estimate of specific respiratory rates for Aeslina sp. was 145 yl 02/h/g wet weight (Rueger et al., 1969), half that of our values to
Somatochlora cingulata.
The data do not provide any insights (other than instuitive) into which protocol most faithfully measures
"normal" metabolic rates. They do indicate, however, that the flow-through regime, as the least variable, most readily 46
4-1 • ro rH VO G U • • • • O cn ro cr> in tnO r—1 rH o o 0 i—1 0 p c CTi + 1 + 1 + l + 1 TJ +1 O in •H • VO in in 00 CL) '—14-4 ro • • • • ,G CN 0 4-> CT> 00 CN in -P > 0 r- CTi rH rH -P P i—i rH CN CN P 0 0 O 0 a 4-i as a 03 a. o G + i o^o •r| O '—I ■P i 0 fd 4-1 tn 0 u o O G ■H 4-4 1 rH ro cn rH P, Q A fd • • • • 0 'O 03 > cn VO r"- 0 0 CN o P 0 + 1 O CN + l + 1 +i + l 0 1 0 Pu • G 4-* tn P1 VO 1—1 VO i—i i—l A X G 3- -P • • • • 0 tn 0 0 -— G rH 00 VO o > •P 0 rH 1—1 CN p1 0 0 G G P CN CN CN CN -P 0 0 0 O 0 0 •H 44 P -P i—I £ ■P 44 ■P 0 G a •H G S tn 0 e T3 O G .G G O 4-1 •H -P 0 O U G 0 0 O 00 00 i—1 o A a. 0 •H O • • • • 4-> •P P cn CN o A O 0 CN * o rH »—1 CN
0 rH G in as G .G iH r- + l + 1 + 1 + 1 O O fd G • T3 •P O > 0 vo in VO W 0 0 PI tn H H H > •rH 0 H H H A § •P H Eh Eh 03 * 47 u ro 00 uo CO • • • • c -p i~H o r~ o c i—I i—1 1—1 o + i Cn 0 0 p o o + 1 +i + 1 + 1 P 7* S3 0 CM ro o Ip • CO 00 CO o •H 4-1 • • • • CD •p •H CO r-~ ro rd id TS 44 CM LO r" 00 > 0 i—1 i—1 ■—i i—1 p -P p a 0 S3 0 >5 0 Qa ^ I m CO CM oo o Q -p • • • • CO CO n~ in oc + o i—i o o o I—I + ! ,—„ 3 < i—1 1—1 o + i + 1 + i + i td 44 1 \ CM p o o tn r-~ f" p1 p1 •H -P i—1 e • • • • Oi Q 1 —' o CM CM U1 T! W 43 ro CO r~ 00 0 0 0 i—1 i—1 i—i 1—1 P 0 +1 CM £3 ! O O 0 -P QjTS •H O 43 X c tr> -P P Cn 0 cd p. fd r*- in 1-1 P> •H — p • • • • c 0 fd c -P 00 CO CO uo o £ -p fd S3 S3 rp i—i O i—I u fd (D 0 0 o •P iH e ■H U ■—l + ! + 1 + 1 + 1 0 0) 3 -P S3 43 £ tn 0 & O CM CO in CM -P £3 a U • • • • 44 •H -P 3 LO o 00 CM 0 o o 0 a 00 00 O fd 0 S3 i—1 1—1 1—1 CM CU fd H 0 £3 TS •H P O ■H 0 43 O 0 a 0 0 .-4 p CM 3 ro 00 1—1 r~~ P S3 43 H O r~H • • • • O 0 ftf * Ch CO t"" r"> 0 •H > S3 i—I td -P -P rd O +1 + 1 + 1 + 1 £ fd fd 43 0 P >h a O -P co 1—1 CO 1—1 o 0 O 0 S3 • • • • CM P3 w w 0 1—I 00 CO o U l—1 1—1 CM p* CM CM CM CM 44 ro o H 0 ffi Pj tn H H H > a CQ fd H H H < -P H tH CO ■K 48 permits indentification of deviations from the norm, the prerequisite for chronic toxicity studies. 4.2.2 Respiration Oxygen consumption in naiads decreased with increasing concentrations of aluminum and hydrogen ions (Table 2 and 3). While a reduction in respiration was associated with all nymphal stages, it was most pronounced in earlier stages. Very significant differences (P< 0.01) were found between controls and experimental animals exposed to low pH and to sublethal aluminum concentrations plus low pH. When comparing reductions of the respiration rate in both treat¬ ments it was noted that sublethal aluminum concentrations at low pH were as toxic as low pH alone. Aluminum did not provoke a significant change in the respiratory rate compared to low pH alone. An increase in oxygen consumption in feeding animals was observed in all treatments. However, in animals exposed to low pH and aluminum plus low pH treatment this increase was less pronounced. Significant differences at (P< 0.01) were found between control and treatments and between treatments. Under feeding condition Aluminum and low pH treatment seems to be more toxic than low pH alone (Fig. 8). Figure 8 . The average ( 7 replicates ) respiration rate (Ug Ov) h g wer weight the dragonfly Somatochlora cingulata Jed daily during 9(> h. exposed to 49 500 J pH of 3.59 and low pH (4.20) plus aluminum ( 30 mg 1 ). TIME (h) 50 4.2.3 Histological Examination of the Tracheal Gills Preliminary observations suggested staining differences in the gill lamellae between the control specimens and those treated with aluminum exposure (100 ppm). Dark staining granules were found in the lamellae of both control and treated animals. However, the quantity and distribution of these granules appeared unequal. In the branchial chamber of the control specimens, there seemed to be dark staining granules in a greater percent of the lamellae, and the granules generally were included along a greater length of each lamella. The distribution of dark staining granules in the lamellae of the branchial chamber of those specimens treated with aluminum exposure seemed to be less extensive. They appeared to be more restricted to the proximal portions of the lamellae, than in the central portion of the branchial chamber lumen. An attempt was made to quantify these apparent dif¬ ferences. Two treated and two control specimens were examined. From each slide containing sections from the branchial chamber 4-10 sections were randomly selected and evaluated using a light microscope at lOOx. For each section examined, two estimates were recorded: (1) the percent of total gill lamellae area present which had dark staining granules, , and (2) the average percent of the overall length of the lamellae with dark staining granules among lamellae Mean Percent of Lamellar Area With Dark Staining Granules Figure 9.-Meanpercentoflamellarlengthinbranchialchamber 51 Mean Percent of Lamellar Length With Dark Staining Granules Figure 10.-Meanpercentoflamellarlengthinbranchialchamber .-.elative LocationinBranchialChamber with darkstaininggranulesinSomatochloracingulata (anterior -4) 52 53 with at least some visible evidence of staining. The estimates for both measures were averaged for the sections examined on each slide. A comparison of the estimates of percent of available gill lamellae with dark staining granules in the branchial chambers of control and aluminum treated naiads is presented in Fig. 9. A similar comparison of the average portion of total lamellar length with granules is presented in Fig. 10. In both of these figures the slide with the highest mean value for each animal depicted is arranged so that they coincide on the horizontal axis. 4.2.4 Ammonia Excretion Rate Ammonia excretion levels decreased very significantly (P< 0.01) with increased exposure to aluminum and hydrogen ions concentrations (Tables 4 and 5). It may be that these pollutants alter the normal deamination pathway of the excretory process. 4.2.5 Ammonia Accumulation Ammonia accumulation in tissues increased signficantly (P<0.01) in both treatments compared with controls. The normal tendency observed in my control was a decrease in ammonia with time, an obvious consequence of the starvation process. However, low pH treatment (3.59) ammonia accumulated • 0 G 0 •H -P •p fd -P (U •H 0 T3 i—1 I-1 o i—l CO *A pH of 4.20 was used as control 56 with time reaching a high value at 48 h (78.25 yg NH^-N/g wet weight) in 72 hours indicating that pH alone seems to increase the rate of ammonia accumulation (Table 6). 4.2.6 L-Glutamate Accumulation Since ammonia is toxic, tissues may reduce it by incorporating it into the formation of glutamate. Indeed, I observed elevated glutamate levels in animals exposed to low pH and aluminum (Table 7). In the present experiments the increase of glutamate levels varied directly with time. The higher value of glutamate found after 96 h (172.93 yg L-glutamate/g) of exposure to A1 and low pH suggested an increase in toxicity occurs when compared to that from low pH alone. My results support, in part, the observations of Seshagiri Rao et al. (1983) , who found that pesticide exposure increased ammonia levels in fish causing a shift in nitrogen metabolism toward synthesis of glutamine. 4.2.7 Oxygen-Nitrogen Ratios No 0:N ratios below 10 were observed during nymphal stages in any of the treatment (Table 8). The average ratio in the different stages ranged between 15.3 (Stage I and 27.4 (Stage IV). This 0:N ratio is indicative of decreased dependence on protein reserves, because protein catabolism leads to the production of ammonia in addition •H POQ) •rl fOHP-P •H GOOP 4-1 p ,P P*H r! rHcnp •rl XPTi 4H o(J> • Changes gonf ly U0 £0 03 U 03 nymphs (0 4(DP 0 -H*H inum \ -PrQ EC concent • U3 re express e is <3* me an an •H (U •H A •H ro VO TS -P co i—1 -P +> £ a p cn 0 P 0-H p +i p cn poQ e £ P P Do P a) P P p. 6 Do P 0 E P EC P l P+ u\ O Do p P (U P — E rH •—1 EC •H 2" EC < '— rH * pi +i u -p Eh cr £ 0 £ 0 *A pH of 6.83 was used as control 58 14-1 g 0 tr> 0 0 r- 0 g 0 cr, 0 0 CO CO P G -H P • • • • • Ti G a in in O'! CO -p 0 0 1—1 CM 1—1 H-l g P G 5 0 G 0 >h • 0 + l + 1 + 1 + 1 + 1 1—1 0 P r-j Q (C W > CM VO CO 1—1 CO 0 0 VO + in VO VO cr> cn • • • • • 0 0 nH 0 Q 1—1 r- 00 CM in p 0 0 tn cn < uo CM in in r-- •H -P > H G 1—1 1—1 1—1 1—1 -p 0 •H +1 1—1 p 0 p • • G -P P 3 ^ 0 T5 Pi ■p 0 O G t7> CM 0 G -H r-- CM CO O'! CTi G ts • 0 0 0 00 1—1 VO VO •iH G ^ ’H ■H £ • • • • • G -P -P CM CO 1—1 r- [■" 0 0 -P p 1—1 1—l CM -4-» -—* GG M-4 H 0 a 0 cr\ Ql, 0 3 £ + 1 + l + 1 + l + 1 g in £ £ 0 • £ -P • G tr> 0 CO CM VO 1—1 CO -P CO 0 P 0 O \ p CO CT> in 0 G «h tn G U 0 • • • • • 1—1 ’H 0 0 -P 1-1 CO CM 00 tG P + 0 -H 0 CO VO O CM 1 a £ -p 0 g 1—1 I—1 1—1 1—1 p ~ 0 -P 0 £ g -p > 0 -P mh 0 a 0 P g 3 O rH a £ 0 0 1—1 0 -P tn 000 tT>P G l 0 VO CM 1—1 CM 1—1 -P CO ^— 0 iH PI in CM 0 ^ tn tn ■X • • • • • > fp p. ^ 1 tr> 1—1 in CO in in CM 0 0 G P ^ 0 1—1 1—1 1—1 1—1 1—1 i-l 0 0 G M-i —• p 0 *H -H 0 G -p + 1 +1 + ! + 1 + 1 G &-P 0 G •H X 0 0 Q 0 0 00 0 VO in CM te-H 0 p 0 m U VO CO 00 00 CM 0 -p 0 • • • • • 0 0 G 0 +1 in cn 1—1 in i^P 0 0 VO VO vo in in G cr O P G 0 g G &1 0 P >1 0 X 0 U G 0 0 g • ___ r- P H PI 0 0 CO CM vo PQ S CM CTl C •H Eh Eh *A pH of 6.83 was used as control 59 Td ■p o 0 G CN 0 0 • 0 P CO OO a • 0 o • • • • X CJ 4-1 4-1 CO r- O CO CN 0 4-1 0 H CN CN CN p •H fd i—1 Td SC -P a fd + ! -P rH fd + 3 i—) tj CN 0 in CN 00 G 0 I—1 o • • • • •H 4-1 •H \ CN t"* in rH 00 O 0 -P tn i—i CN CN I—1 fd e fd G p •—■ p 0 0 *H S • i—i -P • • u ,G td o G u p O 0 -P G u CO o ID -P G fd o • • • • fd 0 0 rH 1—1 in CO 00 CO e O S < rH 1—1 1—1 1—1 o G w 0 O >1 rH i—1 4H < oo CO 00 uo • G uo • • • • u 0 -P • uo VD CN c tP g CO CN CN CN CN o fd 0 0 o p P 0 Td 0 •H 1—\ 4-1 -P sc < 0 4H fd p4 £ •H p + G -P Td IS o r- uo VD CN •H S 0 CN • • • • 4h Td • • iH • CO UO ["• 1-1 rH 0 G o -=r i—i i—1 CN i—1 0 0 fd -p p p 0 G fd -p ■p 0 ffi td G G •H 04 0 0 0 -P *r-< * O o fd £ in ao CO CO oo p 0 • • • • 0 0 rH • VD o r~ uo .G .g S VD i—1 CN ■—i 1—1 -P -p • • 0 o -p 0 0 fd fd • Td Td 00 0 0 0 0 W 0 3 3 Cn H H H > PQ fd H H H sc sc < -P H 04 04 Eh in * + 60 >i H W m G £ o tr> O H tji -p • 0 0 0 g p p >i cx p< o P1 -p (d cx • • • CN c TS CD I—1 00 O 0 o CN u cp CN i—I tn^ M-i0 c .p. O tn 0 O C A ■H C -p T5 P 0 0 2 •H p -P -p O 0 0 4-4 U - g p •H U cx p1 O'! LO •H ^ CX • • • CX i—I c o 00 o 00 0 I *P i—I o r- CN 0 tP i—l i—i PM O +1 I I -P +j d <—i £i + T3 CN tP S 0 •P I 0 -P 0 coO 0 £ EC CX S X EC CN -P 0 CX • 0 tn P1 3. td £ — CD CP LO — +J o • • • C 0 ec 00 00 r- 0 0 rP cx I—1 CN 0 0 2 T5 CN £ -P tr> 0 £ -P 0 C 0 0 0 p -p 1—1 rQ o EC >1 cx CX p 0 •H O p £ C! -P o o 0 0 I—I C P c! O o o + 1—I •P X o 0 00 00 -P 0 -p p • • • 0 0 c -p CN LT) 00 i—I t> g c i—1 CO 0 0 O m 0 ro Pi 0 CO o u (T> 0 O 0 W •P C 0 -P 0 •p m 0 •p -p >1M iH O 00 r* G\ m rH 00 CN 1-1 rH o in CO MH > o VO in CO 00 CO VO VO CP CP rH VO G W U • 0 G H* CN H1 r- CO VO in CO CO CO o H tP 0 pH 1-1 rH 1—1 1—1 I—1 rH 0 *H P -P 13 fd u (U 4-> .G G -P • -p rH G — i—1 0 M i—1 £ \ o rH • W -P tP o • PI 0 £ o o PO 0 — C P Eh Eh Figure 11. Oxygen consumption mg02/h/g by the dragonfly Somatochlora cjngulata exposed to 0; 0.01 and 0.1 mg/1 of naphthalene during 24 hours. o n O Control s/h/2o3ui m o ro o CN NOLLdlMflSNOO o o o o TIME (h) 63 64 to water and CO2. I found a decrease of 0:N ratios in animals exposed for 9 days to low pH (4.20) and A1 concentrations (10 and 20 ppm), under feeding conditions. These ratios were observed in control (8.7) and all treatments (7.5; 8.5 and 8.4; respectively) (Table 9). 4.2.8 pH of Haemolymph Also observed (Table 10), as a consequence of ammonia and glutamate accumulation in the haemolymph were increased pH values (7.66 to 8.27 in 72 h with the pH treatment and 7.65 to 8.16 in 12 h with pH-Al treatment). Probably, the HCO^, PO^ and CC^ usually present in high concentrations in odonate haemolymph (Stobbart and Shaw, 1965) may play an important role in this increase. 4.3 PHYSIOLOGICAL RESULTS USING ORGANIC POLLUTANTS 4.3.1 Respiration and Ammonia Excretion Rates Using Naphthalene Oxygen consumption in naiads increased with the con¬ centration of naphthalene in the water (Table 11). The respiration rate at 0.1 mg/1 was twice that of the controls (Fig. 11). While not as pronounced as the 0.1 treatment, the 0.01 ppm treatment elicited a clearly discernible increase in respiration. The metabolic rate was initially high in all treatments probably as a consequence of stress 65 through handling or through their introductionto a new environment. It is interesting that the extent of this heightened uptake was magnified by naphthalene concentrations. Oxygen consumption, per unit body weight, was constant (Fig. 12) so it would seem that the differences observed had not been compounded by sampling errors in size and age. Ammonia excretion levels increased significantly (P < 0.05) in all treatments as a result of exposure to naphthalene (Table 12). Increased ammonia excretion was related to an increase of napththalene concentrations. The average (0:N) ratio ranged between 14.67 and 18.22. This 0:N ratio is also indicative of an less pronounced increased dependence on protein reserves compared with low pH and A1 concentrations (Table 12). 4.3.2 Respiration and Ammonia Excretion Rates Using TCA Oxygen consumption was markedly and progressively enhanced in Somatochlora cingulata as a result of the TCA treatment (Table 13). Highly significant differences (P <0.01) were found between controls and experimental animals exposed to TCA with the respiration rate at 1 mg/1 being nearly double that of the controls. Ammonia excretion levels increased significantly (P < 0.05) in a dose- dependent manner as a result of exposure to TCA. As with oxygen consumption, the average ammonia excretion rate at 66 WET WEIGHT (g) to exposed eingulata Somatochlora dragonfly of relationship -respiration wet w sight 12, Body Figure V o MI NOIXdWaSNOO 0 G 0 H 0 0 •H i—i Cn >i +J fd 90* H i—1 0 MH P 0 H1 ,G G -P p o CND G 0 O Cn 0 £ + 1 + i (d 0 Cn P G 0 o CN 0 0 • LO CN —■ O •H o • • 0 -P o 00 G .G TS fd ro 1—1 1—1 0 -P 0 p -H -P -P P fd S cd o a • • p m ■H o ■H TS JO — G OVH •H • CD 1 u P Cn 0 l * I -PO -P I 1—1 jg ,g T3 Cn 0 + 1 ■H 3 0 0 1 0 rH £ roa cn ffi X -P S 0 -p (D fd £ Cn fd 3- -P ,—v G 0 i—i 0 1-1 \ 0 0 G Cn £ -P Cn £ -P rd G ■— 0 p -rH & u 0 >i G a p fd 0 •H o p i—1 0 .G -P 0 0 -P 0 0 i~H .G (d G P 4d -P p 0 U u 4d o •P X 0 CU Q* -p 0 -p 0 p 0 G o rH TS £ o 0 G 0 ip G fd cn 0 •H CN G w 0 0 -H G 0 CQ -P 0 •H < •H -p Eh p -p fd •H 0 p 04 p 0 O S 0 X • • Oh w O 68 r-' u r- ro ■H • • • + LO oo ro •H ** O LO 0 rH • (L) Q) O + 1 + 1 04 > CO a) 0 LO H1 iH P-i N G O in 00 0 I-1 0 0 • • • si LO +> 1 ■H G CN o 1-1 +> O 0 tr» t>1 -P rH CM CM r—1 r—1 fd 0 + O >i 1 + P > 0 iH JG G ■P -P CM 0 G .G 0 to G O &> 0 0 0 0 fd O 0 i—1 G 0 0 tN p G W O O •H ,Cj *H G TS O • • 0 + + — 0 CO CM > •H 0 • ro ■P G G rG TS U 0 0 to 0 -p 0 Si •H •H -P 0 + 1 + 1 +> P 0 -P p 0 rH o 0 fd 0 0 • 00 r- ,G P P cm •H + 10 o CM i—i -P 0 •H 05 G • • • LO 0 + G rH 0 1—1 H1 CO LO + CO i—1 •H CN] -M ro i—1 CM CN Si -H 0 1 +> ro 1—1 0 0d P tn 0 -P 0 0 I r H -p 0 > G 0 0 ■P 1 P 0 G Si Si 'O — 0 0 £ *H Cn P 0 ■H 0 CN G 0 H •H 1 0 \,Q 00 i—1 G P P (D 0 tn 0 • • Q + 0 £ W a £ CN ro —' ra s X —' ro P G ■P 0 +> 0 G 0) tn 0S + + 1 + 1 0 + £ G 0 •H 0 0+10 ■— -P 0 o CO CM -p •H jG G fd >< Q ro o CM 05 4J CD 0 rH CO • • • 0 CD 0 G 0 o ro CM tn+> >i £ -P tn ■H +1 CN T—\ CM G o si -P fd G +> CM 0 G 0 P •H 00i P 05 rQ 0 0 G 0 0 >i 0 0 0 05 -P Pl, p G 0 1—1 0 •H 0 P P G CM spp 0 ,g -P 0 0 0 r- ro •H 0 G CO 0 1—1 rH 0 • • -P G G G P si si £ 00 CM i—1 •H 0 0 u 0 0 I—I 1—1 G H 0 •H X 0 •H 0 0 £ P -p 0 -p PrG 5-1 + 1 +1 0 +> fd 0 Eh +> -P 0 id o i—1 TS E G CM 00 mm G G 0 G 0 + 0 0 LO ro 1—1 G G Oh 0 CO 0 -H U • • • 0 0 00 1—1 o 0 0 0 CM I—1 CM G ■P 0 CM G G ,G ro Q 0 -P £ tn+> 0 W G G 0 i—1 PJ 0 0 •H 0 -H CQ •H G 0 in P rG < •P 0 *H p +> ^ Eh 0 *H -P p -P td •H 0 P a P CD 0 0 S ■P 1 mg/1 was twice that of the controls. High 0:N ratios were found using this contaminant. Organic pollutants tend to increase* 0:N ratios, especially when compared with low pH or aluminum plus low pH treatments. 70 DISCUSSION 5.1 BIOASSAY 5.1.1 Effect of pH on Mortality Somatochlora cingulata naiads are apparently more tolerant of acid conditions than fish, crustaceans and most other insects. Dragonflies were among the most numerous organisms in a stream whose pH ranged from 4.0 to 6.8 (Bick et al., 1953). Warner (1968) and Bick et al. (1953), collected the dragonfly Boyeria vinosa from streams with pH values of around 4.0, indicating it is at least tolerant of low pH values, for short periods. Another specie, Ophiogomphus rupinsulensis, which Bell and Neberker (1969) reported to be only moderately tolerant, has not been recorded in areas where acid pollution is a problem. Like Boyeria vinosa it could probably tolerate low pH values if they were of short duration. My data (Fig. 6) showed that the tolerance of Somatochlora cingulata to low pH's varied markedly at different weight classes (3.45 Group I to 2.40 Group IV). Little has been published regarding the mech¬ anisms contributing to the death of earlier stages in acidic waters. The physiological results, however, indicated that respiratory and excretory systems are implicated in the failure. Borgstrcm and Hendre-y (1976) and Shaw (1960) 71 2+ + speculated that low pH's may block the Ca , Na uptake mechanism of crustacean during moulting, while Leivestand (1982) found that gill damage is the immediate cause of failure in fish. 5.1.2 Effects of Aluminum on Mortality Metals, in general, are readily solubilized and sub¬ sequently mobilized in soils by low pH's. Predictably, this has been observed as well as a consequence of acidic deposi¬ tion. Of these, aluminum has received the greatest attention as being potentially toxic to aquatic biota. Aluminum con¬ centrations found in acidic lakes of the northeastern United States are five to ten times higher than levels (0.01 mg/1) in circumneutral waters in the same area (Schofield and Trajnar, 1980). Aluminum has been found to be toxic to fish at concentrations as low as 0.1 to 0.2 mg/1 (Baker, 1981; Schofield and Trajnar, 1980; and Muniz and Leivestad, 1981), a level well within the range of elevated concentrations measured in acidic surface waters (Schofield and Trojnar, 1980) . Nehring (1976) found that the aquatic insects appear to be excellent biological monitors of heavy metal pollution because they are more tolerant of metal than fish. Under experimental conditions, the dragonfly Somatochlora cingulata was able to tolerate high aluminum concentration (LC5Q=140 mg/1) 72 for 96 h. The effect of aluminum on the survival of naiads at different weight stages was the same. The earlier stages were as sensitive as the older stages. The presence of aluminum at a low pH was shown to be as toxic as low pH alone. 5.2 PHYSIOLOGICAL DATA 5.2.1 Respiration The toxic mode of action of low pH and aluminum plus low pH on the respiratory system of aquatic organisms is not well understood. One theory suggests that mucous normally present on the gills of fish is denatured in solutions of high acidity or heavy metals, and is thus no longer capable of protecting the respiratory epithelium from the corrosive impact of low pH's. The permeability of the gill membrane system is pre¬ sumably impaired and the fish eventually succumb to anoxia (Vaale and Mitchell, 1970; Skidmore, 1970; Nichols and Bulow, 1973; and Ultsch, 1978). A similar mechanism has been sug¬ gested for some benthic organisms (Nichols and Bulow, 1973). An alternate theory of toxicity involves displacement of functional cations from active sites of enzymes by low pH and metal ions, resulting in failure of a life process such as respiration (Eichorn, 1975) . My results indicate a different theory may be applicable depending on the species under con¬ sideration . Low pH and sublethal aluminum concentrations plus low pH 73 decreased the oxygen consumption in S. cingulata in all nympha1 stages (Table 2). The earlier stages, however, were the most sensitive. In Group I, the average oxygen consump¬ tion of controls and pH treatments (3.59) respectively were 265.5 and 179.6 yg C^/h/g. In Group IV, the resoective oxygen consumption rates were 26 3.7 and 215.8 yg C>2/h/g. It is interesting that in Group I at a pH of 3.59 a reduction of 32% in the oxygen consumption rate was observed compared with a reduction of 18% in Group IV. While not as pronounced as the pH of 3.59 treatment, the 4.2 pH treatment elicited a clearly discernible decrease in respiration. A similar response was observed for the aluminum plus low pH treatment. The decrease of oxygen consumption at the lowest concentra¬ tion (30 mg/1 and pH of 4.2) average 211.6 as compared to 126.6 yg 02/h/g in their group I counterparts. In Group IV at the same aluminum plus low pH concentration the oxygen consumption decreased from 240.1 to 183.0 yg 02/h/g (Table 3). A reduction of 40% in oxygen consumption rate was observed in Group I compared with 23.7% in Group IV. The decrease of oxygen consumption at the highest concentration (30 mg/1 plus a pH of 4.2) was more pronounced than the 10 and 20 mg/ and 20 mg/1 plus a pH of 4.2 treatments. An analysis of variance failed to demonstrate, however, that the differences in percent oxygen consumption reduction 74 consistently observed between animals exposed to low pH's and to aluminum plus low pH were statistically significant. Low pH alone proved to be as toxic as aluminum plus low pH. In the feeding animal treatments exposed to (pH and aluminum plus pH), the large initial increase observed in the metabolic rate was due to food intake, independently of changes in locomotor activity. This increase (Fig. 11) was observed in controls (365.7 y g C>2/h/g) and in treatments (200.3 yg 02/h/g and 237.8 yg O^/h/g for aluminum plus low pH respectively). Significant differences (P< 0.01) were found between control and treatments and also, between treat¬ ments. This disruption in the efficiency to use the energy im¬ plicate less energy was available for somatic maintenance (repair, replacement secretions, etc.). A reduction of approximately 50-60% in the specific dynamic action (increase in metabolic rate of an animal occasioned by food consumption) was observed between control and treatments. This decrease in SDA reflect a low and saving energy demand in organisms exposure to exper¬ imental treatments. Lamentably the reason why this process occurs is not well understood. If a dragonfly naiad metabolically compensates without proportionally increasing food consumption, an increase in hydrogen ions and aluminum concentrations in the environment will result in the diversion of energy from growth, moulting and the emerging process. 75 5.2.2 Mechanisms of Toxicity on Respiration Observations of individual gill lamellae failed to substantiate a quantifiable difference in the general histology between control specimens and those treated by exposure to aluminum concentration plus low pH. The histological pro¬ cedure and observational criteria used provided no support for the hypothesis that aluminum plus low pH contamination specifically affects the tracheal system. Adelstenad and Valee (1962) determined that metals can alter the respira¬ tory process by activating or inhibiting those enzymes responsible for the synthesis, destruction or release of metabolites. They determined that slight shift in the balance of trace metals or changes in the ionic equilibrium can affect a metabolic process. Due to the fact that absortion of ions would take place through the body surface and gills, perhaps anoxia is due to ionic interference occasioned by aluminum or low pH through osmoregulation and is the major cause of the failure of the respiratory system. 5.2.3 Ammonia Excretion Rate While terrestrial insects excrete waste protein nitrogen mainly in the form of urea and uric acid, among aquatic insects ammonia is excreted as the main end-product of pro¬ tein catabolism (Staddon, 1955). Ammonia has great ecological significance because though it is an important plant nutrient, 76 it is toxic in its undissociated form. Karlson (1975) determined the ammonia levels that inhibit the activity of the mitochondrial enzyme (GDH) in fish. Seshasgiri Rao et al. (1983) found that pesticide exposure increased acetyl choline (ACh) levels in the tissues affecting the diffusion of ammonia through the gill by increasing the vascular resistance. The results presented in this research confirmed that ammonia is the main end-product of protein metabolism. The normal tendency consistently observed among the starved controls was a decrease in ammonia excretion with time. Also, this decrease was observed in response to all pH treatments (Table 4) and aluminum plus pH treatments (Table 5). The lowest ammonia excretion rate averaged from 15.63 as compared to 6.92 yg NH~,-N/h/g and from 11.94 to 7.28 yg NH^-N/h/g were found in individuals belonging to the earlier stages exposed to low pH (3.59) and aluminum (30 mg/1) plus a pH of 4.2 respectively. Obviously, the normal deamination pathway of the excretory process has been markedly reduced. Seshasgiri Rao et al. (1982) attributed the similarly observed decrease in fish ammonia levels to decreased deam¬ ination of the free amino acids during pesticide exposure. Further, Karlson et al. (1976) observed that increased ammonia and lactate levels in fish tissues inhibit the activity of the mitochondrial enzyme (glutamate dehydro- 77 genase) and increase the acetyl choline levels. The lowered respiration rate in dragonfly naiads may be due not only to uptake of aluminum and exposure to low pH, but also to the accumulation of ammonia and/or ammonia salts. In animal tissue, deamination of amino acids can be liberated of ammonia via a number of enzymatic pathways. These pathways can be subdivided into two groups. Animals in the first group are able to produce ammonia from a large number of amino acids by the transfer of amino groups to a common acceptor ( a-ketoglutarate) which serve as the ultimate substrate for deamination. Indirect production of ammonia through a transdeamination process can also be carried out. In this pathway, the amino group of amino acids is transferred to a-ketoglutarate by a number of pyridoxal phosphate requiring enzymes, called amino transferases. The resulting glutamate is deaminated by mitochondrial glutamate dehydrogenase. The second group contains a number of specific enzymes, which produce ammonia from a single type of amino acid by direct deamination (Waarde, 1982). I determined that a decrease in glutamate dehydrogen¬ ase (GDH) activity may be the major alteration in the normal deamination pathway naiads exposed to aluminum concentrations and low pH toxicities. 5.2.4 Ammonia Excretion in Feeding Naiads It has been estimated in the dragonfly Aeshna cyanea 78 that during the 24-48 h period after feeding a quantity of nitrogen is excreted equivalent in amount to the greater part of the food nitrogen absorbed during that period (Staddon, 1955). A similar relationship was observed in S. cingulata controls. Feeding was followed by a large temporary increase in the amount of ammonia excreted in control; low pH and aluminum plus low pH treatments (Table 9). Aluminum plus low pH, however, seems to elicit more of a disruptive response than low pH alone. In the controls, the excretory rate reached a value of 35.8 as compared with 28.9 and 20.9 and 21.0 yg NH^-N/h/g obtained from exposure to pH (4.2) alone and the same low pH combined with aluminum (10 and 20 mg/1) respectively. Significant differences (P< 0.05) were found between the controls and the treatment and between low pH and low pH plus aluminum treatments as well. There were not significant differences, however, between the two acid plus aluminum treatments. These data indicated that the threshold of the excretory response is below 10 mg/1. 5.2.5 The Oxygen-Nitrogen Ratios The oxygen-nitrogen (0:N) ratio has been used in several studies (Conover and Corner, 1968 ; Corner and Conway, 1968) as an index of substrate utilization of energy production and has been shown to vary with stage of development, diet, 79 and degree of physiological stress. Bayne and Scullard (1977) found that nitrogen excretion and oxygen consumption do not always vary similarly in response to changes in the environ¬ ment. This fact is of significance, not only for understand¬ ing the rates of protein carabolism in general, but also, for our interpretation of the balance in catabolism between the different nutrient reserves in the tissues. They concluded that the 0:N ratio has proved useful in assessing the physio¬ logical response of bivalves to various stressful environments. Pandian (1970) suggested that the principal source of energy during embryonic development of the American lobster was from lipid oxidation which might be carried over to some degree in the early stages of post-embryonic development. The observed decrease in lipid content of the final larval stages was an indication that utilization of lipid reserves provided an additional source of energy during larval and early postlarval development. Using Cancer irroratus larvae, exposed to Cu+ and Ca++, Johns and Miller (1982) noted that the ratio of oxygen con¬ sumed to nitrogen excreted (0:N), fluctuated during the nymphial stages between 28.3 and 35.4. They concluded that oxygen to nitrogen ratios of approximately 7 indicate that protein is the sole substrate used for energy production and that increasing values can be interpreted as an indication 80 of an increased reliance on carbohydrates and/or lipids. Upon exposing larval lobster to crude oil, Cappuzzo and Lancaster (1982) , observed delayed molting and reduced respiration rates and 0:N ratios as a result of inhibition of lipid utilization. This is consistent with their earlier thesis (1981) and Johns and Miller (1982) conclusions. Changes in metabolic activity and energy utilization during those intense morphological and behavioral changes associ¬ ated with metamorphosis are interesting phenomena to consider as indication of stress. No 0:N ratios below 10 were observed in starved animals. The average ratio among the different weight stages ranged between 15.3 and 27.4. The highest 0:N ratios ranged be¬ tween 22.5 to 25.9 were found at the lowest pH (3.59). Also, high 0:N ratios ranging between 17.3 to 22.4 were found at aluminum concentration of 30 mg/1 and a pH of 4.20. These results are indicative of decreased dependence on protein reserves. When feeding dragonflies were exposed to low pH (4.2) and sublethal aluminum concentrations (10 and 20 mg/1) plus a pH of 4.2 for 9 days, the average of 0:N ratios decreased from 8.7 (controls) to 7.5 at low pH and 8.5 and 8.4 at 10 and 20 mg/1 aluminum concentrations respectively. In feeding dragonflies a low 0:N ratios was observed in all treatments compared with those results obtained with 81 starved animals. These ratios indicate that protein is the major substrate used for energy production. Perhaps their high nutritive protein diet, facilitated protein as the major substrate. 5.2.6 Ammonia Accumulation in Tissues I observed that the decreased rate of ammonia excretion was reflected in elevated tissue levels of ammonia in all treatments (Table 6). Thus in starved controls the amount of ammonia in tissues changed from 46.68 to 27.20 yg NH^-N/g wet weight in 96 h. In starved low pH (3.59) and aluminum (30 mg/1) plus low pH (4.2 treatment, however, the amount of ammonia in tissues increased from 50.08 to 69.45 and from 56.73 to 66.81 yg NH^-N/g wet weight respectively. In the low pH treatment the high ammonia level was reached in 48 h (78.25 yg NH^-N/g) while the pH (4.2), A1 (30 mg/1) treatment elicited the same response (78.45 y.g NH^-N/g) in 72 h. The data indicated that aluminum seems to reduce the rate of ammonia rate accumulation but not the final concentration. I believe that the increase in tissue ammonia levels can be attributed, functionally, to damage of the excretory system. The increase of tissues in the ammonia was produced by a deamination of the free amino acids during aluminum and low pH exposures. 82 -5.2.7 Glutamate Accumulation in Tissues Since ammonia is a toxic metabolite the tissues attempt to eliminate it either by excretion or by synthesizing glutamate and glutamine respectively. This is evident from the increased levels of glutamate in dragonfly tissues found in this study. L-glutamic acid is oxidized by a specific enzyme, L-glutamic acid dehydrogenase, which is one of the few enzymes oxidizing amino acids that require NAD+. The oxidation of glutamate by glutamic acid dehydrogenase also provides a means of regenerating a -ketoglutarate which normally would serve as an amino acceptor for amino groups during transamination. Since a -ketoglutarate is also oxidized by the citric acid cycle the dehydrogenase serves as a link between two important metabolic systems (Chefurka, 1965). Another pathway for the metabolism of glutamate is by decarboxylation to y -amino butyric acid. Glutamic acid is also converted to glutamine by glutamine synthetase. 2 + + This enzyme requires ATP, Mg , and NH^ to function. If any one of these is limiting glutamine synthesis is re¬ stricted . Levenbook (1962) suggested glutamine synthesis as a possible trapping mechanism for traces of metabolic NH^ • Its activity in the fat-body may, in part, account for the high levels of glutamine in tissues. 83 Perhaps, the high levels of glutamate found in naiad tissues suggests an adaptative mechanism to reduce ammonia toxicity by minimizing the addition of further ammonia to the existing elevated ammonia levels. During this experiment irregular spasmodic contractions were observed in legs of specimens exposed to aluminum and low pH. Muscles are stimulated to contract by the arrival of a nerve impulse at the nerve/muscle junctions where L- glutamate is the chemical transmitter across the synaptic gap (Miller, 1975). Perhaps, the amount of glutamate in the haemolymph and muscles produced an increase of transmitter substance in the synaptic gap changing the permeability of the postsynaptic membrane on the muscle surface. Consequently, changes in the permeability lead to polarization by influx of sodium ions in the muscle membrane potential and the un¬ controlled movement of muscle. 5.2.8 pH of the Haemolymph In most insects the blood has a slightly acid reaction, pH (6.0 - 7.0), but in some, such as the dragonfly it is distinctly alkaline, pH 7.59 - 7.66 (Correa et al., 1984). During normal activity there is a tendency for the blood to become markedly acid due to the liberation of acid metabolites, including carbon dioxide. This tendency to change is countered by the buffering actions of bicarbonates and phosphates. 84 ’Buffering on the acid side of this range relief on the carboxyl groups of organic acids such as citric acid are important while on the alkaline side the amino groups are the most important. Duchatean and Florkin (1958) identified fifteen amino acids in the blood of the dragonfly larvae Aeshna whose concentration was 399 mg/100 ml plasma. Wyatt (1961) has already pointed out, that the free amino acids in insect haemolymph frequently make a net cation contribution rather than anion. Free aspartic and glutamic acids usually occurs in low concentrations (280 mg/1) in Aeshna (in contrast to their amides) whereas arginine, lysine and histidine are generally more abundant (550 mg/1). The likelihood that the respiratory function of the cuticle of the rectal gills confer a high permeability suggest that changes in the haemolymph composition readily reflect changes in the external medium. Accordingly, measurements of haemolymph pH in starved controls did not change in 96 h. Those specimens exposed to low pH (3.59) and A1 (30 mg/1) plus low pH (4.2) showed a significant increase at 96 h. from 7.66 to 8.27 and from 7.65 to 8.17 respectively. Statistical analysis showed a significant difference at P < 0.05 between control and experimental animals. Sutcliffe(1962a) and Moens (1975) working with Aeshna cyanea and Libellula depressa respectively, determined that 85 no changes occurred in the haemolymph composition of starved organisms maintained for 240 h in tap water. Since changes in haemolymph composition would be reflected in pH. I assume that my controls as well, did not undergo changes in haemo¬ lymph composition. As the products of nitrogenous excretion are, in most insects, eliminated from the haemolymph via the Malpighian tubules, the increase of ammonia and glutamate levels in the haemolymph of experimental animals could be the cause of the observed increase in pH. Because dragonfly naiads are capable of regulating the ionic composition of the haemolymph, a large part of their ability to tolerate low pH and aluminum plus low pH reside in their capacity to maintain a constant haemolymph composition. 5.3 PHYSIOLOGICAL RESPONSES AS EARLY WARNING INDICATORS OF CHRONIC TOXICITY TO ORGANIC CONTAMINANTS 5.3.1 Introduction Protracted testing of a toxicant to all stages of the life history of a species are likely to provide more reliable estimates of a safe concentration than toxicity tests. Unfortunately, long-term tests, using more subtle parameters such as growth, reproductive success, physiological condition and the like are comparatively expensive. They require more sophisticated facilities for longer periods of time, and also 86 a higher degree of professional competence both for obtaining results and interpreting them (Cairns, J., 1981). There is a need then for a method to extrapolate from short-term testing the effects of chronic exposures (Mount and Stephan, 1969; McKin et al., 1970). Because changes occurred in respiration and excretion rates as well as oxygen-nitrogen ratios at low pH and sub- lethal aluminum concentration plus low pH, each of these responses showed promise as such a short-term predictor. Each has advantages and disadvantages, depending upon the type of information required. Oxygen consumption and nitro¬ gen excretion were the best of all physiological indicators for showing stress with regard to low pH and aluminum con¬ centration. My intent, as well, was to explore the feasi¬ bility of identifying a family of toxicants (Naphthalene and trichloroacetic acid) from the range and amplitude of physio¬ logical responses observed. Formulation and standardization of this procedure, based on the physiological response of the dragonfly Somatochlora cingulata could rapidly infer disequilibration from low level exposures to selected xenobiotics in water. 5.3.2 Naphthalene toxicity Respiration rate was determined to be directly propor¬ tional to the levels of naphthalene in the water. Statistical 87 differences at P< 0.01 were found between controls and treat¬ ments. The highest respiration rate (408.2 pg C^/h/g) was found in animals exposed to 0.1 mg/1 of naphthalene for 2 h, though the 0.01 mg/1 treatment also elicited a clearly dis¬ cernible increase in respiration (Table 11). Correa and Coler (1983) determined that an increase in energy expendi¬ ture is a consequence of naphthalene uptake. Naphthalene increased the oxygen uptake about 50% at 0.1 mg/1 levels during 24 h (Table 11). Perhaps, as observed with other aquatic organisms (Harmon,etal. , 1981 and Crider et al., 1982), the inhibition of NADH oxidase and NADH-cytochrome c reduct¬ ase could be the major cause of increased respiration rate. Ammonia excretion levels also increased with naphtha¬ lene concentrations. Significant differences at P 0.05 were found between controls and treatments. The highest value (16.50 y.g NH^-N/h/g) was found in 0.1 mg/1 treatment compared with 12.06 y.g NH^-N/h/g found in controls. The 0:N ratios varied between 14.67 and 18.22. It is interesting that these values were lower than the 0:N ratios obtained with low pH and aluminum plus low pH exposures. It seems to be that naiads exposed to naphthalene have a less pronounced tendency to use carbohydrate and/or lipids. The mechanism(s) responsible for the energetic deviation are not well understood. It is evident that the successful 88 development of naiads are dependent on the balance and efficient utilization of energy reserves. Perhaps, this increase in lipid reserves may be a defense mechanism against the incorporation of naphthalene which has lipophilic properties. 5.3.3 Trichloroacetic acid toxicity Oxygen consumption increased as a result of TCA treat¬ ment in experimental animals. The respiration rate (449 ug C^/h/g) found at the highest concentration (1 mg/1) was nearly double that of the controls. Coincidently, the ammonia excretion rate at the same concentration was also doubled (22.22 yg NH^-N/h/g). Significant differences at P <0.05 were found between controls and treatments in respi¬ ration and excretion rates changes. The 0:N ratios found in all treatments were higher than the controls ranging beweeen 21.34 to 23.17 (Table 13). When comparing all the 0:N ratios obtained in this study, I found that the highest 0:N ratios were found to a pH of 3.59; while the lowest 0:N values were obtained in animals exposed to naphthalene. The 0:N value obtained for TCA was close to the 0:N value obtained with aluminum treatment. The possible mechanisms underlying alterations in meta¬ bolic activity following pollutant exposure are not well understood. However, increased in 0:N ratios by increasing 89 respiration and excretion rates are indicative of a decreased dependence on protein carabolism for immediate energy needs. This tendency was observed in naiads exposed to organic con¬ taminants (Naphthalene and TCA). The higher 0:N ratio found in dragonfly naiads exposed to low pH are indicative of a greater dependence on carbohydrate and/or lipid compared with the 0:N ratios obtained from organic pollutants. The results of my research illustrate the contribution of the physiological chronic tests to assess pollutant effects. This type of analyses does not provide a definitive explanation of the mode of action for a pollutant. It does, however, provide a means by which the energetic costs can be applied to observed changes at the system level and can establish the importance of these changes. 90 l SUMMARY From the data on the respiratory and excretory metabo¬ lism, I observed a decrease in respiration and ammonia ex¬ cretion rates in specimens exposed to low pHs and aluminum concentrations plus low pH. In contrast, an increase in respiration and excretion rates was found in naiads exposed to organic contaminants (naphthalene and TCA). Observations of decreased respiration and ammonia excretion rates in animals treated with low pH and aluminum plus low pH suggested that reductions in the metabolic rate may be related to ionic interference and damage to the ex¬ cretory system. The higher 0:N values (22.5 to 27.4) and (17.3 to 23.7) found in naiads exposed to a pH of 3.59 and aluminum (30 mg/1) plus a pH of 4.20 respectively, suggested that the high energy demand was used to compensate for the disruption of its excretory process. The reduction in the quantity of metabolites is accomplished through: (a) depressed oxygen consumption, (b) restricted utilization of protein as fuel and (c) shifting of intermediate metabolism toward the synthesis of glutamate. The observations of increased respiration in naiads treated with naphthalene and TCA indicated that elevated ■respiratory rates during chronic exposure may be related to 91 interference with mitochondrial enzymes and the uncoupling of oxidative phosphorylation, thereby decreasing the effi¬ ciency of energy utilization and inducing the organism to respire more. Also, high 0:N values (18.2) and (21.8) were observed in test organisms exposed to naphthalene (0.1 mg/1) and TCA (1 mg/1) respectively. These 0:N ratios may reflect an increased reliance on the energy-rich lipid materials to meet elevated maintenance costs and to compensate for the lipophilic properties of these pollutants. The mechanism by which these pollutants alter metabolism remains to be elucidated. It is significant to note that the changes induced occurred at levels of environmental relevance in only a short period of time. It appears that recourse to S. cingulata as a preliminary screening tool to identify chronic levels of toxicity over a short period of exposure offers promise. The metabolic adjustments/fine tuning of such a species would permit resolution of subtle, subclinical metabolic disturbances as reflected in changes in the rates of oxygen consumption and ammonia excretion. This is a departure from the rationale of normal toxicity testing which is predicated on evolving the LC^-q of a sensi¬ tive species. It is my contention that the adaptative response of a tolerant organism would provide a gradient response that would be more sensitive than death as an endpoint. 92 CONCLUSIONS AND RECOMMENDATIONS 6.1 SUMMARY OF CONCLUSIONS 1. When comparing the three methodologies (Gilson, static and flow-through system) for measuring respiration rate, I found the flow-through regime, due to its less variability, most useful in eluci¬ dating deviations from the normal, the prerequisite for chronic toxicity studies. 2. No significant correlation existed between specific oxygen consumption (rate per gram) and size for dragonfly naiads, using a flow-through system. 3. Mortality results have shown that younger stages are more sensitive to low pH than older stages. 4. The effects of aluminum on the survival of Somatochlora cingulata at different weight stages was the same. This species has a high tolerance for aluminum concentrations (LC^q = 140 ppm) . 5. A significant decrease in respiration rate was ob¬ served in dragonfly naiads exposed to low pH and to sublethal aluminum concentrations. 6. Histological examination of the branchial chamber revealed no consistent differences between controls and aluminum plus low pH treatment in dragonfly naiads. 93 7. In feeding animals, a reduction of approximately 50-60% in the specific dynamic action (SDA) was observed between controls and treatments. 8. The normal tendency observed among starved controls was a decrease in ammonia with time. At low pH and A1 treatments this decrease was significantly pro¬ nounced . 9. In feeding animal, an increase in ammonia excretion was observed in all treatments, however, this in¬ crease was less pronounced in experimental animals exposed to low pH and aluminum toxicities. 10. All 0:N ratios showed an increased dependence on carbohydrate or lipids. However, the highest 0:N ratio was found at low pH and the lowest at naphth¬ alene toxicity. 11. Elevated levels of ammonia and glutamate were found in tissues of animals exposed to low pH and aluminum treatments; this increase can be attributed, function ally, to damage or inhibition of the excretory system 12. Somatochlora cingulata partially compensated for this disruption of its excretory processes, induced by exposure to aluminum and low pH, by reducing the quantity of accumulated metabolites. This reduction was accomplished through: (a) depressed oxygen 94 consumption, (b) restricted utilization of protein as fuel and (c) shifted intermmediate metabolism toward the synthesis of glutamate. 13. As the products of nitrogenous excretion are eliminated from the haemolymph via the Malpighian tubules, the increase of ammonia and glutamate levels in the haemolymph could be the cause of this increase of pH levels. 14. An increase in the respiration and excretion rates were observed in naiads exposed to naphthalene an TCA. The 0:N ratios in animals exposed to organic contaminants were lower than those exposed to low pH. These 0:N ratios suggest a possible tendency in dragonfly to use lipid materials to compensate the lipophilic properties of these pollutants.. 6.2 SUMMARY OF RECOMMENDATIONS Based on the accumulated data, I make the following recommendations: 1. Because the flow-through delivery system developed for this study does not require a great investment in time, space, expertise and money, I suggest its incorporation in toxicity studies. 2. Implementation of experiments to measure the 95 accumulation and degradation rates of contaminants in tissue of individuals at different weight stages under laboratory chronic exposure. 3. Underlying histological examination involving the excretory system (Malpighian tubules) in naiads exposed to different concentrations of different pollutants is necessary. 4. 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Sci. 71: 202-215. Waskell, L. 1978. A study of the mutagenicity of anes¬ thetics and their metabolites. Mutation Research. 57: 141-153. Wiederholm, T. and L. Eriksson. 1977. Benthos of an acid lake. Oikos. 29:261-267. Wood, C.M. and D.G. McDonald. 1982. Physiological mechanisms of acid toxicity to fish. In: Acid Rain/Fisheries, pp. 197-226. American Fisheries Society. Maryland. APPENDIX A 107 APPENDIX A The taxonomic description used to classify this aquatic insect was taken directly from Walker, E.N. (1925) North American species of Somatochlora. Toronto Studies. Biological Series No. 26. "Readily known by its large size and blotched colour- pattern. Eyes a little more prominent than usual in the genus. Sides of head behind eyes moderately oblique, passing in a broad curve into the hind margin, which is only barely concave. Hair of head relatively sparse. Labium large, when closely pressed to the body reaching nearly or quite to the posterior limit of the mesocoxae; mentum about as broad as long, extending laterally over the inner edges of the eyes; mental setae usually 10-13, generally the 4th or 5th from the outside longest, the innermost 3 or 4 (sometimes 5 to 7) and sometimes the first much shorter than the others; marginal setae usually extending most of the distance along the mental margin, consisting of a variable distal group and an irregular scattered series extending basad, generally 5 or 6 short setae at the distal mental joints. Lateral lobes with 7 or 8 deep, subangularly rounded crenulations, the first often bifid, most of them bearing 1-3 stoutish setae in a graded series, the hindmost much the longest, but no longer 108 than the depth of the corresponding tooth; lateral setae 6 or 7, rarely fewer. Pronotal processes not prominent, with few and short setae; propleural processes prominent with a noticeable tuft of setae; remainder of thorax sparsely hairy; vaguely blotched with paler and darker brown. Legs pale, the femora with dark, often obscure annuli, viz., an apical, an anteapical and a sub-basal. Hind femora when straightened out reaching from middle to end of 6. Hind wing-pads reach¬ ing from hind margin of 5 to basal fourth of 6. Abdomen ovate, broadest at apex of 5 or base of 6, pale brownish with darker brown blotches, as follows: (1) a pair of large, somewhat ill-defined, dorsal blotches on most of the segments, deepening over a transverse pair of muscle impressions on each; (2) a pair of oblique lateral spots, representing the scars of the tergo-sternal muscles, on the same segments; (3) corresponding pairs of ventral spots, marking the sternal attachments of the same muscles; (4) transverse streaks at the anterior borders of the abdominal segments; and (5) pairs of small, submedian ventral spots. The ventral spots are sometimes quite faint or obscure. Abdomen with minute setae most abundant near the hind tergal margins and on the slight median prominences between the dark submedian blotches, almost absent from the latter 109 and from the lateral blotches. Hind tergal margins with regular rows of short setae with fine long hairs of irregu¬ lar length interspersed at intervals. Lateral fringe com¬ paratively sparse, though fairly well-developed on 8 and 9; a similar fringe also on ventro-caudal margin of 9. Dorsal hooks absent, but low elevations bearing short setae, as above described, take their place. Lateral spines on 8 and 9, slightly bent outwards from the corresponding margins, but not actually divergent, slender; those of 8 one-sixth to one-fifth as long as the remaining segment margin; those of 9 one-fifth (or a little more) of the corresponding margin; margin of 9 straight except at base, where it is rather strongly convex. Anal appendages a little longer than 9 + 10; med. app. of male with a pair of prominent lateral tubercles just beyond the middle, beyond these tapering to a long slender apex, the entire length being considerably greater than the basal breadth. Lat. apps. of male a little shorter than med. app., stout in basal half, rather rapidly contracted beyond, the outer margin and slender-pointed apices being slightly sinuate. Inf. app. about one-seventh longer than med. app., apices slender but stouter than that of the med. app. Apps. of female similar except that the med. app. is simply triangular with a long acuminate apex and the lat. apps. have the outer margins straight, the inner convex. APPENDIX B 110 APPENDIX B Azide Modification of the Winkler Method (Reagents) 1. Manganous sulfate solution: Dissolve 480 g Mn SO^ 4H20, or 364 g Mn SO^ H20 in distilled water, filter, and dilute 1 L. The Mn SO^ solution should not give a color with starch when added to an acidified po- rassium iodide (Kl) solution. 2. Alkali-iodide-azide reagent: Dissolve 10 g Na in 500 mL distilled water. Add 480 g sodium hydroxide (NaOH) and 750 g sodium iodide (Nal), and stir until dissolved. There will be a white turbidity due to sodium carbonate (Na2 CO^), but this will do no harm. 3. Sulfuric acid, H2 SO^, cone: One ml is equivalent to about 3 ml.alkaliodide-azide reagent. 4. Starch: Use either an aqueous solution or soluble starch powder mixtures. To prepare an aqueous solution, dissolve 2 g laboratory-grade soluble starch and 0.2 g salicylic acid, as a preservative in 100 ml hot dis¬ tilled water. 5. Standard sodium thiosulfate titrant: Dissolve 6.205 g Na2S203 5H20 in distilled water. Add 1.5 ml 6N Na OH or 0.4 g soluble NaOH and dilute to 1,000 ml. Standardize with bi-iodate solution. Ill 6. Standard potassium bi-iodate solution, 0.0250N: Dissolve 812.4 mgKHflC^^ in distilled water and dilute to 1,000 ml. APPENDIX C 113 stability for up to a year under normal laboratory conditions. Check reagent to make sure that it yields the characteristic color with 0.1 mg NH^-N/L within 10 min. after addition and does not produce a precipitate with small amounts of ammonia within 2 hr. 4. Stock ammonium solution; Dissolve 3.819 g anhydrous NH^Cl, dried at 100 C in water, and dilute to 1,000 mL: 1.00 mL = 1.00 mg N = 1.22 mg NH^. 5. Standard ammonium solution: Dilute 10.00 mL stock ammonium solution to 1,000 mL with water; 1.00 mL = 10.00 yg N = 12.2 yg NH3. 6. Permanent color solutions: (a) Potassium chloroplatinate solution: Dissolve 2.0 g K2PtCl6 in 300 to 400 mL distilled water; add 100 mL cone HC1 and dilute to 1 L. (b) Cobaltous chloride solution: Dissolve 12.0 g CoCl2 6H20 in 200 mL distilled water. Add 100 mL cone HC1 and dilute to 1 L. APPENDIX D 114 APPENDIX D L-glutamate in Tissues (Reagents) 1. Hydrazine hydrate, ca. 24% (w/v) 2. Glycine, A.R. 3. Sulphuric acid, A.R., 1 N 4. Diphosphopyridine nucleotide, DPN free acid; commercial preparation, see p. 1010 5. Glutamic dehydrogenase, GIDH crystalline, from ox liver; free from ammonium sulphate or Commercial preparation. Additional for the analysis of samples containing protein: 6. Perchloric acid, A. R. , sp. gr. 1.67; ca. 70% (w/v/) 7. Dipotassium hydrogen phosphate, K2HPC>4 8. Tripotassium phosphate, K^PO^ Preparation of Solutions (for ca. 20 determinations) Prepare all solutions with fresh, doubly distilled water. Sterilize containers to prevent bacterial contamination. I. Glycine-hydrazine buffer (0.5 M glycine; 0.4 M hydrazine; pH 9): Dissolve 3.75 g. glycine and 5.50 g. 24% hydrazine hydrate in doubly distilled water, 115 adjust to pH 9 with ca. 14.8 ml. IN H2SO4 and make up to 100 ml. -2 II. Diphosphopyridine nucleotide (ca. 3 x 10 M 3-DPN): Dissolve 100 mg. DPN in 5 ml. doubly distilled water. III. Glutamic dehydrogenase, GIDH (ca. 10 mg. protein/ml) If necessary, dilute the suspension with 0.15 M Na2SO^ solution. Additional for the analysis ofssamples containing protein: IV. Perchloric acid (ca. 0.6 M): Dilute 5.2 ml. 70% HCIO^ (sp. gr. 1.67) to 100 ml. with doubly dis¬ tilled water. V. Phosphate solution (2 M; pH ca. 12): Dissolve 21.2 g. K3PO4 and 18.3 g. K2HPC>4 in doubly distilled water and make up to 100 ml.